Science.gov

Sample records for kinase simultaneous interaction

  1. MAP kinase-interacting kinases--emerging targets against cancer.

    PubMed

    Diab, Sarah; Kumarasiri, Malika; Yu, Mingfeng; Teo, Theodosia; Proud, Christopher; Milne, Robert; Wang, Shudong

    2014-04-24

    Mitogen-activated protein kinase (MAPK)-interacting kinases (Mnks) regulate the initiation of translation through phosphorylation of eukaryotic initiation factor 4E (eIF4E). Mnk-mediated eIF4E activation promotes cancer development and progression. While the phosphorylation of eIF4E is necessary for oncogenic transformation, the kinase activity of Mnks seems dispensable for normal development. For this reason, pharmacological inhibition of Mnks could represent an ideal mechanism-based and nontoxic therapeutic strategy for cancer treatment. In this review, we discuss the current understanding of Mnk biological roles, structures, and functions, as well as clinical implications. Importantly, we propose different strategies for identification of highly selective small molecule inhibitors of Mnks, including exploring a structural feature of their kinase domain, DFD motif, which is unique within the human kinome. We also argue that a combined targeting of Mnks and other pathways should be considered given the complexity of cancer. PMID:24613018

  2. Simultaneous targeting of androgen receptor (AR) and MAPK-interacting kinases (MNKs) by novel retinamides inhibits growth of human prostate cancer cell lines

    PubMed Central

    Ramamurthy, Vidya P.; Ramalingam, Senthilmurugan; Gediya, Lalji; Kwegyir-Afful, Andrew K.; Njar, Vincent C.O.

    2015-01-01

    Androgen receptor (AR) and MNK activated eIF4E signaling promotes the development and progression of prostate cancer (PCa). In this study, we report that our Novel Retinamides (NRs) target both AR signaling and eIF4E translation in androgen sensitive and castration resistant PCa cells via enhancing AR and MNK degradation through ubiquitin-proteasome pathway. Dual blockade of AR and MNK initiated eIF4E activation by NRs in turn induced cell cycle arrest, apoptosis, and inhibited cell proliferation. NRs also inhibited cell migration and invasion in metastatic cells. Importantly, the inhibitory effects of NRs on AR signaling, eIF4E translation initiation and subsequent oncogenic program were more potent than that observed with clinically relevant retinoids, established MNK inhibitors, and the FDA approved PCa drugs. Our findings provide the first preclinical evidence that simultaneous inhibition of AR and eIF4E activation is a novel and efficacious therapeutic approach for PCa, and that NRs hold significant promise for treatment of advanced prostate cancer. PMID:25605250

  3. Simultaneous targeting of androgen receptor (AR) and MAPK-interacting kinases (MNKs) by novel retinamides inhibits growth of human prostate cancer cell lines.

    PubMed

    Ramamurthy, Vidya P; Ramalingam, Senthilmurugan; Gediya, Lalji; Kwegyir-Afful, Andrew K; Njar, Vincent C O

    2015-02-20

    Androgen receptor (AR) and MNK activated eIF4E signaling promotes the development and progression of prostate cancer (PCa). In this study, we report that our Novel Retinamides (NRs) target both AR signaling and eIF4E translation in androgen sensitive and castration resistant PCa cells via enhancing AR and MNK degradation through ubiquitin-proteasome pathway. Dual blockade of AR and MNK initiated eIF4E activation by NRs in turn induced cell cycle arrest, apoptosis, and inhibited cell proliferation. NRs also inhibited cell migration and invasion in metastatic cells. Importantly, the inhibitory effects of NRs on AR signaling, eIF4E translation initiation and subsequent oncogenic program were more potent than that observed with clinically relevant retinoids, established MNK inhibitors, and the FDA approved PCa drugs. Our findings provide the first preclinical evidence that simultaneous inhibition of AR and eIF4E activation is a novel and efficacious therapeutic approach for PCa, and that NRs hold significant promise for treatment of advanced prostate cancer. PMID:25605250

  4. Simultaneous determination of selected tyrosine kinase inhibitors with corticosteroids and antiemetics in rat plasma by solid phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry: Application to pharmacokinetic interaction studies.

    PubMed

    Maher, Hadir M; Alzoman, Nourah Z; Shehata, Shereen M

    2016-05-30

    A sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry method has been developed and validated for the simultaneous analysis of selected tyrosine kinase inhibitors (TKIs)(gefitinib GEF, erlotinib ERL), corticosteroids (dexamethasone DEX, prednisolone PRED), and the antiemetic ondansetron (OND) in rat plasma samples. After the addition of domperidone (DOM) as internal standard (IS), spiked plasma samples were prepared using the solid phase extraction (SPE) C 18 cartridges. Chromatographic separation was performed on a Waters BEH C18 column with an isocratic elution using a mobile phase composed of acetonitrile and water, each with 0.1% formic acid, (80: 20, v/v), at a flow rate of 0.2mL/min. Quantitation of the analytes was performed using the multiple reaction monitoring (MRM) mode with the positive ionization mode at m/z 447.25>128.08 (GEF), m/z 394.20>278.04 (ERL), m/z 393.30>147.04 (DEX), m/z 361.29>147.02 (PRED), m/z 294.18>170.16 (OND), and m/z 426.26>175.07 (DOM). The method was validated over the concentration range of 0.025-100 (GEF, ERL, OND) and 0.05-100ng/mL plasma (PRED, DEX) with very low lower limit of quantification of 0.025 (GEF, ERL, OND) and 0.05ng/mL (DEX, PRED). The intra- and inter-day precision (RSD%) evaluated at four different concentration levels were within the acceptable limits (<15%). The method provided good extraction recovery of all analytes from rat plasma (Er% from -14.05 to -1.08). The validated method was successfully applied to the pharmacokinetic studies following the oral administration of selected combinations of the studied drugs. This study can be readily applied in therapeutic drug monitoring (TDM) in patients receiving these drug combinations as well as investigation of possible drug interactions between TKIs and DEX/PRED/OND. PMID:26966895

  5. The protein interaction landscape of the human CMGC kinase group.

    PubMed

    Varjosalo, Markku; Keskitalo, Salla; Van Drogen, Audrey; Nurkkala, Helka; Vichalkovski, Anton; Aebersold, Ruedi; Gstaiger, Matthias

    2013-04-25

    Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways. PMID:23602568

  6. Kinase-interacting substrate screening is a novel method to identify kinase substrates

    PubMed Central

    Amano, Mutsuki; Hamaguchi, Tomonari; Shohag, Md. Hasanuzzaman; Kozawa, Kei; Kato, Katsuhiro; Zhang, Xinjian; Yura, Yoshimitsu; Matsuura, Yoshiharu; Kataoka, Chikako; Nishioka, Tomoki

    2015-01-01

    Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases. PMID:26101221

  7. Identification of Protein Kinase Substrates by the Kinase-Interacting Substrate Screening (KISS) Approach.

    PubMed

    Amano, Mutsuki; Nishioka, Tomoki; Yura, Yoshimitsu; Kaibuchi, Kozo

    2016-01-01

    Identifying the substrates of protein kinases to understand their modes of action has been undertaken by various approaches and remains an ongoing challenge. Phosphoproteomic technologies have accelerated the accumulation of data concerning protein phosphorylation and have uncovered vast numbers of phosphorylation sites in vivo. In this unit, a novel in vitro screening approach for protein kinase substrates is presented, based on protein-protein interaction and mass spectrometry-based phosphoproteomic technology. © 2016 by John Wiley & Sons, Inc. PMID:27580705

  8. Revisiting protein kinase-substrate interactions: Toward therapeutic development.

    PubMed

    de Oliveira, Paulo Sérgio L; Ferraz, Felipe Augusto N; Pena, Darlene A; Pramio, Dimitrius T; Morais, Felipe A; Schechtman, Deborah

    2016-01-01

    Despite the efforts of pharmaceutical companies to develop specific kinase modulators, few drugs targeting kinases have been completely successful in the clinic. This is primarily due to the conserved nature of kinases, especially in the catalytic domains. Consequently, many currently available inhibitors lack sufficient selectivity for effective clinical application. Kinases phosphorylate their substrates to modulate their activity. One of the important steps in the catalytic reaction of protein phosphorylation is the correct positioning of the target residue within the catalytic site. This positioning is mediated by several regions in the substrate binding site, which is typically a shallow crevice that has critical subpockets that anchor and orient the substrate. The structural characterization of this protein-protein interaction can aid in the elucidation of the roles of distinct kinases in different cellular processes, the identification of substrates, and the development of specific inhibitors. Because the region of the substrate that is recognized by the kinase can be part of a linear consensus motif or a nonlinear motif, advances in technology beyond simple linear sequence scanning for consensus motifs were needed. Cost-effective bioinformatics tools are already frequently used to predict kinase-substrate interactions for linear consensus motifs, and new tools based on the structural data of these interactions improve the accuracy of these predictions and enable the identification of phosphorylation sites within nonlinear motifs. In this Review, we revisit kinase-substrate interactions and discuss the various approaches that can be used to identify them and analyze their binding structures for targeted drug development. PMID:27016527

  9. Giant protein kinases: domain interactions and structural basis of autoregulation.

    PubMed Central

    Kobe, B; Heierhorst, J; Feil, S C; Parker, M W; Benian, G M; Weiss, K R; Kemp, B E

    1996-01-01

    The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I. Images PMID:9003756

  10. Glycogen Synthase KinaseInteraction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinaseinteraction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  11. West Nile virus methyltransferase domain interacts with protein kinase G

    PubMed Central

    2013-01-01

    Background The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication. PMID:23876037

  12. KLIFS: a structural kinase-ligand interaction database

    PubMed Central

    Kooistra, Albert J.; Kanev, Georgi K.; van Linden, Oscar P.J.; Leurs, Rob; de Esch, Iwan J.P.; de Graaf, Chris

    2016-01-01

    Protein kinases play a crucial role in cell signaling and are important drug targets in several therapeutic areas. The KLIFS database contains detailed structural kinase-ligand interaction information derived from all (>2900) structures of catalytic domains of human and mouse protein kinases deposited in the Protein Data Bank in order to provide insights into the structural determinants of kinase-ligand binding and selectivity. The kinase structures have been processed in a consistent manner by systematically analyzing the structural features and molecular interaction fingerprints (IFPs) of a predefined set of 85 binding site residues with bound ligands. KLIFS has been completely rebuilt and extended (>65% more structures) since its first release as a data set, including: novel automated annotation methods for (i) the assessment of ligand-targeted subpockets and the analysis of (ii) DFG and (iii) αC-helix conformations; improved and automated protocols for (iv) the generation of sequence/structure alignments, (v) the curation of ligand atom and bond typing for accurate IFP analysis and (vi) weekly database updates. KLIFS is now accessible via a website (http://klifs.vu-compmedchem.nl) that provides a comprehensive visual presentation of different types of chemical, biological and structural chemogenomics data, and allows the user to easily access, compare, search and download the data. PMID:26496949

  13. KLIFS: a structural kinase-ligand interaction database.

    PubMed

    Kooistra, Albert J; Kanev, Georgi K; van Linden, Oscar P J; Leurs, Rob; de Esch, Iwan J P; de Graaf, Chris

    2016-01-01

    Protein kinases play a crucial role in cell signaling and are important drug targets in several therapeutic areas. The KLIFS database contains detailed structural kinase-ligand interaction information derived from all (>2900) structures of catalytic domains of human and mouse protein kinases deposited in the Protein Data Bank in order to provide insights into the structural determinants of kinase-ligand binding and selectivity. The kinase structures have been processed in a consistent manner by systematically analyzing the structural features and molecular interaction fingerprints (IFPs) of a predefined set of 85 binding site residues with bound ligands. KLIFS has been completely rebuilt and extended (>65% more structures) since its first release as a data set, including: novel automated annotation methods for (i) the assessment of ligand-targeted subpockets and the analysis of (ii) DFG and (iii) αC-helix conformations; improved and automated protocols for (iv) the generation of sequence/structure alignments, (v) the curation of ligand atom and bond typing for accurate IFP analysis and (vi) weekly database updates. KLIFS is now accessible via a website (http://klifs.vu-compmedchem.nl) that provides a comprehensive visual presentation of different types of chemical, biological and structural chemogenomics data, and allows the user to easily access, compare, search and download the data. PMID:26496949

  14. Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways*

    PubMed Central

    Dickinson, Robin J.; Delavaine, Laurent; Cejudo-Marín, Rocío; Stewart, Graeme; Staples, Christopher J.; Didmon, Mark P.; Trinidad, Antonio Garcia; Alonso, Andrés; Pulido, Rafael; Keyse, Stephen M.

    2011-01-01

    MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. PMID:21908610

  15. Isolation and Characterization of Kinase Interacting Protein 1, a Pollen Protein That Interacts with the Kinase Domain of PRK1, a Receptor-Like Kinase of Petunia1

    PubMed Central

    Skirpan, Andrea L.; McCubbin, Andrew G.; Ishimizu, Takeshi; Wang, Xi; Hu, Yi; Dowd, Peter E.; Ma, Hong; Kao, Teh-hui

    2001-01-01

    Many receptor-like kinases have been identified in plants and have been shown by genetic or transgenic knockouts to play diverse physiological roles; however, to date, the cytosolic interacting proteins of relatively few of these kinases have been identified. We have previously identified a predominantly pollen-expressed receptor-like kinase of petunia (Petunia inflata), named PRK1, and we have shown by the antisense RNA approach that it is required for microspores to progress from the unicellular to bicellular stage. To investigate the PRK1-mediated signal transduction pathway, PRK1-K cDNA, encoding most of the cytoplasmic domain of PRK1, was used as bait in yeast (Saccharomyces cerevisiae) two-hybrid screens of pollen/pollen tube cDNA libraries of petunia. A protein named kinase interacting protein 1 (KIP1) was found to interact very strongly with PRK1-K. This interaction was greatly reduced when lysine-462 of PRK1-K, believed to be essential for kinase activity, was replaced with arginine (the resulting protein is named PRK1-K462R). The amino acid sequence of KIP1 deduced from full-length cDNA contains an EF-hand Ca2+-binding motif and nine predicted coiled-coil regions. The yeast two-hybrid assay and affinity chromatography showed that KIP1 interacts with itself to form a dimer or higher multimer. KIP1 is present in a single copy in the genome, and is expressed predominantly in pollen with a similar temporal pattern to PRK1. In situ hybridization showed that PRK1 and KIP1 transcripts were localized in the cytoplasm of pollen. PRK1-K phosphorylated KIP1-NT (amino acids 1–716), whereas PRK1-K462R only weakly phosphorylated KIP1-NT in vitro. PMID:11500547

  16. Interacting Protein Kinases Involved in the Regulation of Flagellar Length

    PubMed Central

    Erdmann, Maja; Scholz, Anne; Melzer, Inga M.; Schmetz, Christel; Wiese, Martin

    2006-01-01

    A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis. PMID:16467378

  17. Interaction of Ras with phosphoinositide 3-kinase gamma.

    PubMed Central

    Rubio, I; Rodriguez-Viciana, P; Downward, J; Wetzker, R

    1997-01-01

    Phosphoinositide 3-kinase gamma (PI3Kgamma) can be activated in vitro by both alpha and betagamma subunits of heterotrimeric G-proteins and does not interact with p85, the regulatory subunit of PI3Kalpha. Here we demonstrate the binding of Ras to PI3Kgamma in vitro. An N-terminal region of PI3Kgamma was identified as a binding site for Ras. After co-expression with PI3Kgamma in COS-7 cells, Ras induced only a modest increase in PI3K activity compared with the stimulation of PI3Kalpha by Ras in the same cells. PMID:9307042

  18. Protein-tyrosine phosphorylation interaction network in Bacillus subtilis reveals new substrates, kinase activators and kinase cross-talk

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ventroux, Magali; Derouiche, Abderahmane; Bidnenko, Vladimir; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise

    2014-01-01

    Signal transduction in eukaryotes is generally transmitted through phosphorylation cascades that involve a complex interplay of transmembrane receptors, protein kinases, phosphatases and their targets. Our previous work indicated that bacterial protein-tyrosine kinases and phosphatases may exhibit similar properties, since they act on many different substrates. To capture the complexity of this phosphorylation-based network, we performed a comprehensive interactome study focused on the protein-tyrosine kinases and phosphatases in the model bacterium Bacillus subtilis. The resulting network identified many potential new substrates of kinases and phosphatases, some of which were experimentally validated. Our study highlighted the role of tyrosine and serine/threonine kinases and phosphatases in DNA metabolism, transcriptional control and cell division. This interaction network reveals significant crosstalk among different classes of kinases. We found that tyrosine kinases can bind to several modulators, transmembrane or cytosolic, consistent with a branching of signaling pathways. Most particularly, we found that the division site regulator MinD can form a complex with the tyrosine kinase PtkA and modulate its activity in vitro. In vivo, it acts as a scaffold protein which anchors the kinase at the cell pole. This network highlighted a role of tyrosine phosphorylation in the spatial regulation of the Z-ring during cytokinesis. PMID:25374563

  19. Interaction and coalescence of multiple simultaneous and non-simultaneous blast waves

    NASA Astrophysics Data System (ADS)

    Qiu, S.; Eliasson, V.

    2016-05-01

    Interaction of multiple blast waves can be used to direct energy toward a target while simultaneously reducing collateral damage away from the target area. In this paper, simulations of multiple point source explosives were performed and the resulting shock interaction and coalescence behavior were explored. Three to ten munitions were placed concentrically around the target, and conditions at the target area were monitored and compared to those obtained using a single munition. For each simulation, the energy summed over all munitions was kept constant, while the radial distances between target and munitions and the munition initiation times were varied. Each munition was modeled as a point source explosion. The resulting blast wave propagation and shock front coalescence were solved using the inviscid Euler equations of gas dynamics on overlapping grids employing a finite difference scheme. Results show that multiple munitions can be beneficial for creating extreme conditions at the intended target area; over 20 times higher peak pressure is obtained for ten simultaneous munitions compared to a single munition. Moreover, peak pressure at a point away from the target area is reduced by more than a factor of three.

  20. Pneumococcal phosphoglycerate kinase interacts with plasminogen and its tissue activator.

    PubMed

    Fulde, M; Bernardo-García, N; Rohde, M; Nachtigall, N; Frank, R; Preissner, K T; Klett, J; Morreale, A; Chhatwal, G S; Hermoso, J A; Bergmann, S

    2014-03-01

    Streptococcus pneumoniae is not only a commensal of the nasopharyngeal epithelium, but may also cause life-threatening diseases. Immune-electron microscopy studies revealed that the bacterial glycolytic enzyme, phosphoglycerate kinase (PGK), is localised on the pneumococcal surface of both capsulated and non-capsulated strains and colocalises with plasminogen. Since pneumococci may concentrate host plasminogen (PLG) together with its activators on the bacterial cell surface to facilitate the formation of plasmin, the involvement of PGK in this process was studied. Specific binding of human or murine PLG to strain-independent PGK was documented, and surface plasmon resonance analyses indicated a high affinity interaction with the kringle domains 1-4 of PLG. Crystal structure determination of pneumococcal PGK together with peptide array analysis revealed localisation of PLG-binding site in the N-terminal region and provided structural motifs for the interaction with PLG. Based on structural analysis data, a potential interaction of PGK with tissue plasminogen activator (tPA) was proposed and experimentally confirmed by binding studies, plasmin activity assays and thrombus degradation analyses. PMID:24196407

  1. Interaction of LRRK2 with kinase and GTPase signaling cascades

    PubMed Central

    Boon, Joon Y.; Dusonchet, Julien; Trengrove, Chelsea; Wolozin, Benjamin

    2014-01-01

    LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson’s disease (PD). Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however, studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in Caenorhabditis elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the mitogen activated protein kinase (MAPK) pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GTPase-activating proteins and GTPase exchange factors are another strong theme in LRRK2 biology, with LRRK2 binding to rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1, and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy. These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term PD. PMID:25071441

  2. ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

    PubMed Central

    Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N3-ATP) and 8-azidoadenosine 5′-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P1,P5-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

  3. ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.

    PubMed

    Randak, Christoph O; Dong, Qian; Ver Heul, Amanda R; Elcock, Adrian H; Welsh, Michael J

    2013-09-20

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

  4. Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions.

    PubMed Central

    Biondi, Ricardo M; Nebreda, Angel R

    2003-01-01

    Signal transduction pathways use protein kinases for the modification of protein function by phosphorylation. A major question in the field is how protein kinases achieve the specificity required to regulate multiple cellular functions. Here we review recent studies that illuminate the mechanisms used by three families of Ser/Thr protein kinases to achieve substrate specificity. These kinases rely on direct docking interactions with substrates, using sites distinct from the phospho-acceptor sequences. Docking interactions also contribute to the specificity and regulation of protein kinase activities. Mitogen-activated protein kinase (MAPK) family members can associate with and phosphorylate specific substrates by virtue of minor variations in their docking sequences. Interestingly, the same MAPK docking pocket that binds substrates also binds docking sequences of positive and negative MAPK regulators. In the case of glycogen synthase kinase 3 (GSK3), the presence of a phosphate-binding site allows docking of previously phosphorylated (primed) substrates; this docking site is also required for the mechanism of GSK3 inhibition by phosphorylation. In contrast, non-primed substrates interact with a different region of GSK3. Phosphoinositide-dependent protein kinase-1 (PDK1) contains a hydrophobic pocket that interacts with a hydrophobic motif present in all known substrates, enabling their efficient phosphorylation. Binding of the substrate hydrophobic motifs to the pocket in the kinase domain activates PDK1 and other members of the AGC family of protein kinases. Finally, the analysis of protein kinase structures indicates that the sites used for docking substrates can also bind N- and C-terminal extensions to the kinase catalytic core and participate in the regulation of its activity. PMID:12600273

  5. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    PubMed Central

    Lawless, Nathan; Blacklock, Kristin; Berrigan, Elizabeth; Verkhivker, Gennady

    2013-01-01

    A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4) kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock) kinase from the system during client loading (release) stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery. PMID:24287464

  6. Simultaneous Eye Tracking and Blink Detection with Interactive Particle Filters

    NASA Astrophysics Data System (ADS)

    Wu, Junwen; Trivedi, Mohan M.

    2007-12-01

    We present a system that simultaneously tracks eyes and detects eye blinks. Two interactive particle filters are used for this purpose, one for the closed eyes and the other one for the open eyes. Each particle filter is used to track the eye locations as well as the scales of the eye subjects. The set of particles that gives higher confidence is defined as the primary set and the other one is defined as the secondary set. The eye location is estimated by the primary particle filter, and whether the eye status is open or closed is also decided by the label of the primary particle filter. When a new frame comes, the secondary particle filter is reinitialized according to the estimates from the primary particle filter. We use autoregression models for describing the state transition and a classification-based model for measuring the observation. Tensor subspace analysis is used for feature extraction which is followed by a logistic regression model to give the posterior estimation. The performance is carefully evaluated from two aspects: the blink detection rate and the tracking accuracy. The blink detection rate is evaluated using videos from varying scenarios, and the tracking accuracy is given by comparing with the benchmark data obtained using the Vicon motion capturing system. The setup for obtaining benchmark data for tracking accuracy evaluation is presented and experimental results are shown. Extensive experimental evaluations validate the capability of the algorithm.

  7. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

    PubMed Central

    Tse, Amanda; Verkhivker, Gennady M.

    2015-01-01

    Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating

  8. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

    PubMed

    Tse, Amanda; Verkhivker, Gennady M

    2015-01-01

    Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating

  9. Rapamycin enhances eIF4E phosphorylation by activating MAP kinase-interacting kinase 2a (Mnk2a).

    PubMed

    Stead, Rebecca L; Proud, Christopher G

    2013-08-19

    Eukaryotic initiation factor eIF4E and its phosphorylation play key roles in cell transformation and tumorigenesis. eIF4E is phosphorylated by the Mnks (MAP (mitogen-activated protein) kinase-interacting kinases). Rapamycin increases eIF4E phosphorylation in cancer cells, potentially limiting their anti-cancer effects. Here we show that the rapamycin-induced increase in eIF4E phosphorylation reflects increased activity of Mnk2 but not Mnk1. This activation requires a novel phosphorylation site in Mnk2a, Ser437. Our findings have potentially important implications for the use of rapamycin and its analogues in cancer therapy, suggesting that inhibitors of mTOR and Mnk (or Mnk2) may be more efficacious than rapalogs alone. PMID:23831578

  10. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    SciTech Connect

    Knaap, E. van der; Sauter, M.; Kende, H. . DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. . Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  11. Simultaneous Protein Expression and Modification: An Efficient Approach for Production of Unphosphorylated and Biotinylated Receptor Tyrosine Kinases by Triple Infection in the Baculovirus Expression System

    PubMed Central

    Erdmann, Dirk; Zimmermann, Catherine; Fontana, Patrizia; Hau, Jean-Christophe; De Pover, Alain; Chène, Patrick

    2010-01-01

    Protein kinases can adopt multiple protein conformations depending on their activation status. Recently, in drug discovery, a paradigm shift has been initiated, moving from inhibition of fully activated, phosphorylated kinases to targeting the inactive, unphosphorylated forms. For identification and characterization of putative inhibitors, also interacting with the latent kinase conformation outside of the kinase domain, highly purified and homogeneous protein preparations of unphosphorylated kinases are essential. The kinetic parameters of nonphosphorylated kinases cannot be assessed easily by standard kinase enzyme assays as a result of their intrinsic autophosphorylation activity. Kinetic binding rate constants of inhibitor-protein interactions can be measured by biophysical means upon protein immobilization on chips. Protein immobilization can be achieved under mild conditions by binding biotinylated proteins to streptavidin-coated chips, exploiting the strong and highly specific streptavidin–biotin interaction. In the work reported here, the cytoplasmic domains of insulin receptor and insulin-like growth factor-1 receptor fused to a biotin ligase recognition sequence were coexpressed individually with the phosphatase YopH and the biotin-protein ligase BirA upon triple infection in insect cells. Tandem affinity purification yielded pure cytoplasmic kinase domains as judged by gel electrophoresis and HPLC. Liquid chromatography-mass spectrometry analysis showed the absence of any protein phosphorylation. Coexpression of BirA led to quantitative and site-specific biotinylation of the kinases, which had no influence on the catalytic activity of the kinases, as demonstrated by the identical phosphorylation pattern upon autoactivation and by enzymatic assay. This coexpression approach should be applicable to other protein kinases as well and should greatly facilitate the production of protein kinases in their phosphorylated and unphosphorylated state suitable for

  12. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.

    PubMed

    Genheden, Maja; Kenney, Justin W; Johnston, Harvey E; Manousopoulou, Antigoni; Garbis, Spiros D; Proud, Christopher G

    2015-01-21

    Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons. PMID:25609615

  13. Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis.

    PubMed

    Georgescauld, Florian; Moynié, Lucile; Habersetzer, Johann; Cervoni, Laura; Mocan, Iulia; Borza, Tudor; Harris, Pernile; Dautant, Alain; Lascu, Ioan

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg(80)-Asp(93) as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved. PMID:23526954

  14. Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

    PubMed Central

    Georgescauld, Florian; Moynié, Lucile; Habersetzer, Johann; Cervoni, Laura; Mocan, Iulia; Borza, Tudor; Harris, Pernile; Dautant, Alain; Lascu, Ioan

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg80-Asp93 as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved. PMID:23526954

  15. Combined deficiency for MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) delays tumor development

    PubMed Central

    Ueda, Takeshi; Sasaki, Masato; Elia, Andrew J.; Chio, Iok In Christine; Hamada, Koichi; Fukunaga, Rikiro; Mak, Tak W.

    2010-01-01

    MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) are protein-serine/threonine kinases that are activated by ERK or p38 and phosphorylate eIF4E, which is involved in cap-dependent translation initiation. However, Mnk1/2 double knockout (Mnk-DKO) mice show normal cell growth and development despite an absence of eIF4E phosphorylation. Here we show that the tumorigenesis occurring in the Lck-Pten mouse model (referred to here as tPten−/− mice) can be suppressed by the loss of Mnk1/2. Phosphorylation of eIF4E was greatly enhanced in lymphomas of parental tPten−/− mice compared with lymphoid tissues of wild-type mice, but was totally absent in lymphomas of tPten−/−; Mnk-DKO mice. Notably, stable knockdown of Mnk1 in the human glioma cell line U87MG resulted in dramatically decreased tumor formation when these cells were injected into athymic nude mice. Our data demonstrate an oncogenic role for Mnk1/2 in tumor development, and highlight these molecules as potential anticancer drug targets that could be inactivated with minimal side effects. PMID:20679220

  16. Do Src Kinase and Caveolin Interact Directly with Na,K-ATPase?

    PubMed

    Yosef, Eliyahu; Katz, Adriana; Peleg, Yoav; Mehlman, Tevie; Karlish, Steven J D

    2016-05-27

    Much evidence points to a role of Na,K-ATPase in ouabain-dependent signal transduction. Based on experiments with different cell lines and native tissue membranes, a current hypothesis postulates direct interactions between the Na,K-ATPase and Src kinase (non-receptor tyrosine kinase). Na,K-ATPase is proposed to bind Src kinase and inhibit its activity, whereas ouabain, the specific Na,K-ATPase inhibitor, binds and stabilizes the E2 conformation, thus exposing the Src kinase domain and its active site Tyr-418 for activation. Ouabain-dependent signaling is thought to be mediated within caveolae by a complex consisting of Na,K-ATPase, caveolin, and Src kinase. In the current work, we have looked for direct interactions utilizing purified recombinant Na,K-ATPase (human α1β1FXYD1 or porcine α1D369Nβ1FXYD1) and purified human Src kinase and human caveolin 1 or interactions between these proteins in native membrane vesicles isolated from rabbit kidney. By several independent criteria and techniques, no stable interactions were detected between Na,K-ATPase and purified Src kinase. Na,K-ATPase was found to be a substrate for Src kinase phosphorylation at Tyr-144. Clear evidence for a direct interaction between purified human Na,K-ATPase and human caveolin was obtained, albeit with a low molar stoichiometry (1:15-30 caveolin 1/Na,K-ATPase). In native renal membranes, a specific caveolin 14-5 oligomer (95 kDa) was found to be in direct interaction with Na,K-ATPase. We inferred that a small fraction of the renal Na,K-ATPase molecules is in a ∼1:1 complex with a caveolin 14-5 oligomer. Thus, overall, whereas a direct caveolin 1/Na,K-ATPase interaction is confirmed, the lack of direct Src kinase/Na,K-ATPase binding requires reassessment of the mechanism of ouabain-dependent signaling. PMID:27022017

  17. Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry

    PubMed Central

    Park, Chorong; Min, Saehong; Park, Eun-Mee; Lim, Yun-Sook; Kang, Sangmin; Suzuki, Tetsuro; Shin, Eui-Cheol

    2015-01-01

    ABSTRACT The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both in vitro pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry. IMPORTANCE Pim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCV-infected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell

  18. Phonetic Variation and Interactional Contingencies in Simultaneous Responses

    ERIC Educational Resources Information Center

    Walker, Gareth

    2016-01-01

    An auspicious but unexplored environment for studying phonetic variation in naturalistic interaction is where two or more participants say the same thing at the same time. Working with a core dataset built from the multimodal Augmented Multi-party Interaction corpus, the principles of conversation analysis were followed to analyze the sequential…

  19. Dataset of integrin-linked kinase protein: Protein interactions in cardiomyocytes identified by mass spectrometry.

    PubMed

    Traister, Alexandra; Lu, Mingliang; Coles, John G; Maynes, Jason T

    2016-06-01

    Using hearts from mice overexpressing integrin linked kinase (ILK) behind the cardiac specific promoter αMHC, we have performed immunoprecipitation and mass spectrometry to identify novel ILK protein:protein interactions that regulate cardiomyocyte activity and calcium flux. Integrin linked kinase complexes were captured from mouse heart lysates using a commercial antibody, with subsequent liquid chromatography tandem mass spectral analysis. Interacting partners were identified using the MASCOT server, and important interactions verified using reverse immunoprecipitation and mass spectrometry. All ILK interacting proteins were identified in a non-biased manner, and are stored in the ProteomeXchange Consortium via the PRIDE partner repository (reference ID PRIDE: PXD001053). The functional role of identified ILK interactions in cardiomyocyte function and arrhythmia were subsequently confirmed in human iPSC-cardiomyocytes. PMID:27408918

  20. Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

    PubMed

    Santafé, M M; Garcia, N; Lanuza, M A; Tomàs, M; Tomàs, J

    2009-02-15

    We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release. PMID:18816790

  1. Hyperscanning: simultaneous fMRI during linked social interactions.

    PubMed

    Montague, P Read; Berns, Gregory S; Cohen, Jonathan D; McClure, Samuel M; Pagnoni, Giuseppe; Dhamala, Mukesh; Wiest, Michael C; Karpov, Igor; King, Richard D; Apple, Nathan; Fisher, Ronald E

    2002-08-01

    "Plain question and plain answer make the shortest road out of most perplexities." Mark Twain-Life on the Mississippi. A new methodology for the measurement of the neural substrates of human social interaction is described. This technology, termed "Hyperscan," embodies both the hardware and the software necessary to link magnetic resonance scanners through the internet. Hyperscanning allows for the performance of human behavioral experiments in which participants can interact with each other while functional MRI is acquired in synchrony with the behavioral interactions. Data are presented from a simple game of deception between pairs of subjects. Because people may interact both asymmetrically and asynchronously, both the design and the analysis must accommodate this added complexity. Several potential approaches are described. PMID:12202103

  2. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex

    PubMed Central

    Frey, Stefan; Reschka, Eva J.; Pöggeler, Stefanie

    2015-01-01

    The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain. PMID:26418262

  3. Compensation for channel interaction in a simultaneous cochlear implant coding strategy.

    PubMed

    Bader, Paul; Kals, Mathias; Schatzer, Reinhold; Griessner, Andreas; Zierhofer, Clemens

    2013-06-01

    This study evaluated a concept to reduce detrimental effects of spatial channel interaction in case of simultaneous stimulation with cochlear implants. The hypothesis was that effects of simultaneous channel interaction can be compensated by an algorithm such that no difference in hearing performance between simultaneous pulsatile stimulation and a strictly sequential reference strategy can be found. The simultaneous strategies used in this study stimulated two or three electrodes simultaneously in a monopolar configuration and used a specific compensation algorithm to reduce detrimental effects of simultaneous channel interaction. Overall stimulation rate was kept constant throughout conditions. Three of the configurations applied extended pulse phase durations. The German Oldenburg sentence and a German vowel test were used to measure speech recognition in 12 cochlear implant users. The results support the initial hypothesis. No significant differences in performance were found. A small spatial distance between simultaneous electrodes yielded slightly better results than a large distance. Extending the pulse phase durations had no significant effect on hearing performance. However, it significantly reduced stimulation amplitudes. Thus strategies implementing channel interaction compensated simultaneous stimulation with extended pulse phase durations might be a viable option for reducing power consumption and increasing battery life in cochlear implants. PMID:23742364

  4. Leucine-rich repeat kinase 2 functionally interacts with microtubules and kinase-dependently modulates cell migration.

    PubMed

    Caesar, Mareike; Zach, Susanne; Carlson, Coby B; Brockmann, Kathrin; Gasser, Thomas; Gillardon, Frank

    2013-06-01

    Recent studies indicate that the Parkinson's disease-linked leucine-rich repeat kinase 2 (LRRK2) modulates cytoskeletal functions by regulating actin and tubulin dynamics, thereby affecting neurite outgrowth. By interactome analysis we demonstrate that the binding of LRRK2 to tubulins is significantly enhanced by pharmacological LRRK2 inhibition in cells. Co-incubation of LRRK2 with microtubules increased the LRRK2 GTPase activity in a cell-free assay. Destabilization of microtubules causes a rapid decrease in cellular LRRK2(S935) phosphorylation indicating a decreased LRRK2 kinase activity. Moreover, both human LRRK2(G2019S) fibroblasts and mouse LRRK2(R1441G) fibroblasts exhibit alterations in cell migration in culture. Treatment of mouse fibroblasts with the selective LRRK2 inhibitor LRRK2-IN1 reduces cell motility. These findings suggest that LRRK2 and microtubules mutually interact both in non-neuronal cells and in neurons, which might contribute to our understanding of its pathogenic effects in Parkinson's disease. PMID:23318930

  5. Interaction of pigeon-liver nicotinamide-adenine dinucleotide kinase with cibacron blue F3GA.

    PubMed Central

    Apps, D K; Gleed, C D

    1976-01-01

    The interaction of pigeon liver NAD kinase with Cibacron Blue F3GA was investigated. By using steady-state rate measurements, spectrophotometric titration and chromatography of the enzyme on immobilized dye, it was shown that binding occurs at two nucleotide sites with different affinities, and also at a site distinct from the substrate-binding region. PMID:187176

  6. Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain

    SciTech Connect

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G.

    2012-03-16

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its 20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

  7. Control of Paip1-eukayrotic translation initiation factor 3 interaction by amino acids through S6 kinase.

    PubMed

    Martineau, Yvan; Wang, Xiaoshan; Alain, Tommy; Petroulakis, Emmanuel; Shahbazian, David; Fabre, Bertrand; Bousquet-Dubouch, Marie-Pierre; Monsarrat, Bernard; Pyronnet, Stéphane; Sonenberg, Nahum

    2014-03-01

    The simultaneous interaction of poly(A)-binding protein (PABP) with eukaryotic translation initiation factor 4G (eIF4G) and the mRNA 3' poly(A) tail promotes translation initiation. We previously showed that the interaction of PABP-interacting protein 1 (Paip1) with PABP and eukaryotic translation initiation factor 3 (eIF3; via the eIF3g subunit) further stimulates translation. Here, we demonstrate that the interaction of eIF3 with Paip1 is regulated by amino acids through the mTORC1 signaling pathway. The Paip1-eIF3 interaction is impaired by the mTORC1 inhibitors, rapamycin and PP242. We show that ribosomal protein S6 kinases 1 and 2 (S6K1/2) promote the interaction of eIF3 with Paip1. The enhancement of Paip1-eIF3 interaction by amino acids is abrogated by an S6K inhibitor or shRNA against S6K1/2. S6K1 interacts with eIF3f and, in vitro, phosphorylates eIF3. Finally, we show that S6K inhibition leads to a reduction in translation by Paip1. We propose that S6K1/2 phosphorylate eIF3 to stimulate Paip1-eIF3 interaction and consequent translation initiation. Taken together, these data demonstrate that eIF3 is a new translation target of the mTOR/S6K pathway. PMID:24396066

  8. Interaction between light harvesting chlorophyll-a/b protein (LHCII) kinase and cytochrome b6/f complex. In vitro control of kinase activity.

    PubMed

    Gal, A; Hauska, G; Herrmann, R; Ohad, I

    1990-11-15

    We have previously reported that the cytochrome b6/f complex may be involved in the redox activation of light harvesting chlorophyll-a/b protein complex of photosystem II (LHCII) kinase in higher plants (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210). The aim of this work was to establish whether a relation between the cytochrome b6/f and LHCII kinase activation can be demonstrated in vitro. Preparations enriched in cytochrome b6/f obtained from spinach thylakoids by detergent extraction and precipitation with ammonium sulfate followed by different procedures of purification, contained various amounts of LHCII kinase activity. Analysis of the cytochrome b6/f content and kinase activity of fractions obtained by histone-Sepharose and immunoaffinity columns, immunoprecipitation and sucrose density centrifugation, indicate functional association of kinase and cytochrome b6/f. Phosphorylation of LHCII by fractions containing both cytochrome b6/f and kinase was enhanced by addition of plastoquinol-1. LHCII phosphorylation and kinase activation could be obtained in fractions prepared by use of beta-D-octyl glucoside but not when 3-[(cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate was used as the solubilizing detergent. Kinase activity could be inhibited by halogenated quinone analogues (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2,3-diiodo-5-t-butyl-p-benzoquinone) known to inhibit cytochrome b6/f activity. However, kinase activity was inhibited by these analogues in all preparations including those which could not phosphorylate LHCII. We thus propose that the redox activation of LHCII phosphorylation is mediated by kinase interaction with cytochrome b6/f while the deactivation may be related to a distinct quinone binding site of the enzyme molecule. PMID:2246258

  9. Exact solutions to a spatially extended model of kinase-receptor interaction

    NASA Astrophysics Data System (ADS)

    Szopa, Piotr; Lipniacki, Tomasz; Kazmierczak, Bogdan

    2011-10-01

    B and Mast cells are activated by the aggregation of the immune receptors. Motivated by this phenomena we consider a simple spatially extended model of mutual interaction of kinases and membrane receptors. It is assumed that kinase activates membrane receptors and in turn the kinase molecules bound to the active receptors are activated by transphosphorylation. Such a type of interaction implies positive feedback and may lead to bistability. In this study we apply the Steklov eigenproblem theory to analyze the linearized model and find exact solutions in the case of non-uniformly distributed membrane receptors. This approach allows us to determine the critical value of receptor dephosphorylation rate at which cell activation (by arbitrary small perturbation of the inactive state) is possible. We found that cell sensitivity grows with decreasing kinase diffusion and increasing anisotropy of the receptor distribution. Moreover, these two effects are cooperating. We showed that the cell activity can be abruptly triggered by the formation of the receptor aggregate. Since the considered activation mechanism is not based on receptor crosslinking by polyvalent antigens, the proposed model can also explain B cell activation due to receptor aggregation following binding of monovalent antigens presented on the antigen presenting cell.

  10. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    PubMed Central

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2014-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit. PMID:24286368

  11. Interactions among three species of cereal aphids simultaneously infesting wheat

    PubMed Central

    Qureshi, Jawwad A.; Michaud, J. P.

    2005-01-01

    Interactions among greenbug, Schizaphis graminum (Rondani), Russian wheat aphid, Diuraphis noxia (Mordvilko), and bird cherry-oat aphid Rhopalosiphum padi (L.) were examined on wheat plants (Triticum aestivum L., cultivar TAM 107). Nymphs were released on the plants as conspecific and heterospecific pairs of either first or fourth instars and evaluated for survival, developmental time, fecundity, intra-plant movement, and affinity to plant tissues. Survival from first instar to onset of reproduction averaged 90–100% across all pair combinations. Diuraphis noxia developed faster as conspecifics than in any heterospecific combination, and faster as conspecifics feeding on the same plant tissue than on different tissues. Fecundity of S. graminum was higher for conspecifics that developed on the same plant tissue than for those feeding separately. There was evidence of amensalism (one species was harmed while the other was unaffected) in that D. noxia experienced delayed development feeding in tandem with S. graminum, and reduced fecundity with both S. graminum and R. padi. Furthermore, S. graminum nymphs had reduced survival when their mothers matured on a same plant with R. padi. Both D. noxia and R. padi changed position on the plant more often when developing with S. graminum. Survival of second generation S. graminum nymphs was reduced when this species developed and reproduced in tandem with R. padi. Preferred feeding locations were S. graminum - primary leaf, D. noxia - tertiary leaf and R. padi - stem and these were not altered in any heterospecific combinations. Heterospecific aphids had no impact on fecundity or progeny survival in any species combination when fourth instars matured and reproduced on plants not previously exposed to aphid feeding, supporting the inference that systemic, aphid-induced changes in plant physiology mediated the effects observed when first instars developed and reproduced on the same plants. PMID:16341245

  12. A novel calmodulin-β-PIX interaction and its implication in receptor tyrosine kinase regulation.

    PubMed

    Singh, Vinay K; Munro, Kim; Jia, Zongchao

    2012-09-01

    Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates numerous cellular processes, primarily in response to calcium flux. We have identified and characterized a novel interaction between CaM and β-p21-activated kinase interacting exchange factor (β-PIX), a putative guanine exchange factor implicated in cell signaling, using affinity pull-down assays, co-immunoprecipitation, co-localization and circular dichroism studies. Fluorescence-based titration and isothermal titration calorimetry experiments revealed a Ca(2+)-dependent binding mechanism (K(D)≤10μM). Further, we show that CaM participates in a multi-protein complex involving β-PIX and E3 ubiquitin ligase c-Cbl (casitas B-cell lymphoma), which may play a critical role in receptor tyrosine kinase regulation and downstream signaling. PMID:22588125

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

    PubMed

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

    2014-12-01

    The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase. PMID:25040100

  14. Physical and functional interactions between ZIP kinase and UbcH5

    SciTech Connect

    Ohbayashi, Norihiko; Okada, Katsuya; Kawakami, Shiho; Togi, Sumihito; Sato, Noriko; Ikeda, Osamu; Kamitani, Shinya; Muromoto, Ryuta; Sekine, Yuichi; Kawai, Taro; Akira, Shizuo; Matsuda, Tadashi

    2008-08-08

    Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. In our previous study, we showed that leukemia inhibitory factor stimulated threonine-265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription 3. Here, we identified UbcH5c as a novel ZIPK-binding partner by yeast two-hybrid screening. Importantly, we found that UbcH5c induced ubiquitination of ZIPK. Small-interfering RNA-mediated reduction of endogenous UbcH5 expression decreased ZIPK ubiquitination. Furthermore, coexpression of UbcH5c with ZIPK influenced promyelocytic leukemia protein nuclear body (PML-NB) formation. These results suggest that UbcH5 regulates ZIPK accumulation in PML-NBs by interacting with ZIPK and stimulating its ubiquitination.

  15. Making the Auroras glow: regulation of Aurora A and B kinase function by interacting proteins

    PubMed Central

    Carmena, Mar; Ruchaud, Sandrine; Earnshaw, William C

    2009-01-01

    The conserved Aurora family of protein kinases have emerged as crucial regulators of mitosis and cytokinesis. Despite their high degree of homology, Aurora A and B have very distinctive localisations and functions: Aurora A associates with the spindle poles to regulate entry into mitosis, centrosome maturation and spindle assembly; Aurora B is a member of the Chromosomal Passenger Complex (CPC) that transfers from the inner centromere in early mitosis to the spindle midzone, equatorial cortex and midbody in late mitosis and cytokinesis. Aurora B functions include regulation of chromosome–microtubule interactions, cohesion, spindle stability and cytokinesis. This review will focus on how interacting proteins make this functional diversity possible by targeting the kinases to different subcellular locations and regulating their activity. PMID:19836940

  16. Making the Auroras glow: regulation of Aurora A and B kinase function by interacting proteins.

    PubMed

    Carmena, Mar; Ruchaud, Sandrine; Earnshaw, William C

    2009-12-01

    The conserved Aurora family of protein kinases have emerged as crucial regulators of mitosis and cytokinesis. Despite their high degree of homology, Aurora A and B have very distinctive localisations and functions: Aurora A associates with the spindle poles to regulate entry into mitosis, centrosome maturation and spindle assembly; Aurora B is a member of the Chromosomal Passenger Complex (CPC) that transfers from the inner centromere in early mitosis to the spindle midzone, equatorial cortex and midbody in late mitosis and cytokinesis. Aurora B functions include regulation of chromosome-microtubule interactions, cohesion, spindle stability and cytokinesis. This review will focus on how interacting proteins make this functional diversity possible by targeting the kinases to different subcellular locations and regulating their activity. PMID:19836940

  17. Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases.

    PubMed

    Zeke, András; Bastys, Tomas; Alexa, Anita; Garai, Ágnes; Mészáros, Bálint; Kirsch, Klára; Dosztányi, Zsuzsanna; Kalinina, Olga V; Reményi, Attila

    2015-11-01

    Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles. PMID:26538579

  18. In Vitro Interactions between Target of Rapamycin Kinase Inhibitor and Antifungal Agents against Aspergillus Species.

    PubMed

    Gao, Lujuan; Ding, Xiaozhen; Liu, Zhun; Wu, Qingzhi; Zeng, Tongxiang; Sun, Yi

    2016-06-01

    In vitro interactions of INK128, a target of rapamycin (TOR) kinase inhibitor, and antifungals, including itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin, against Aspergillus spp. were assessed with the broth microdilution checkerboard technique. Our results suggested synergistic effects between INK128 and all azoles tested, against multiple Aspergillus fumigatus and Aspergillus flavus isolates. However, no synergistic effects were observed when INK128 was combined with amphotericin B or caspofungin. No antagonism was observed for any combination. PMID:26976874

  19. Regulatory Interactions between a Bacterial Tyrosine Kinase and Its Cognate Phosphatase*

    PubMed Central

    Temel, Deniz B.; Dutta, Kaushik; Alphonse, Sébastien; Nourikyan, Julien; Grangeasse, Christophe; Ghose, Ranajeet

    2013-01-01

    The cyclic process of autophosphorylation of the C-terminal tyrosine cluster (YC) of a bacterial tyrosine kinase and its subsequent dephosphorylation following interactions with a counteracting tyrosine phosphatase regulates diverse physiological processes, including the biosynthesis and export of polysaccharides responsible for the formation of biofilms or virulence-determining capsules. We provide here the first detailed insight into this hitherto uncharacterized regulatory interaction at residue-specific resolution using Escherichia coli Wzc, a canonical bacterial tyrosine kinase, and its opposing tyrosine phosphatase, Wzb. The phosphatase Wzb utilizes a surface distal to the catalytic elements of the kinase, Wzc, to dock onto its catalytic domain (WzcCD). WzcCD binds in a largely YC-independent fashion near the Wzb catalytic site, inducing allosteric changes therein. YC dephosphorylation is proximity-mediated and reliant on the elevated concentration of phosphorylated YC near the Wzb active site resulting from WzcCD docking. Wzb principally recognizes the phosphate of its phosphotyrosine substrate and further stabilizes the tyrosine moiety through ring stacking interactions with a conserved active site tyrosine. PMID:23543749

  20. Synaptotagmin-1 C2B domain interacts simultaneously with SNAREs and membranes to promote membrane fusion

    PubMed Central

    Wang, Shen; Li, Yun; Ma, Cong

    2016-01-01

    Synaptotagmin-1 (Syt1) acts as a Ca2+ sensor for neurotransmitter release through its C2 domains. It has been proposed that Syt1 promotes SNARE-dependent fusion mainly through its C2B domain, but the underlying mechanism is poorly understood. In this study, we show that the C2B domain interacts simultaneously with acidic membranes and SNARE complexes via the top Ca2+-binding loops, the side polybasic patch, and the bottom face in response to Ca2+. Disruption of the simultaneous interactions completely abrogates the triggering activity of the C2B domain in liposome fusion. We hypothesize that the simultaneous interactions endow the C2B domain with an ability to deform local membranes, and this membrane-deformation activity might underlie the functional significance of the Syt1 C2B domain in vivo. DOI: http://dx.doi.org/10.7554/eLife.14211.001 PMID:27083046

  1. Synaptotagmin-1 C2B domain interacts simultaneously with SNAREs and membranes to promote membrane fusion.

    PubMed

    Wang, Shen; Li, Yun; Ma, Cong

    2016-01-01

    Synaptotagmin-1 (Syt1) acts as a Ca(2+) sensor for neurotransmitter release through its C2 domains. It has been proposed that Syt1 promotes SNARE-dependent fusion mainly through its C2B domain, but the underlying mechanism is poorly understood. In this study, we show that the C2B domain interacts simultaneously with acidic membranes and SNARE complexes via the top Ca(2+)-binding loops, the side polybasic patch, and the bottom face in response to Ca(2+). Disruption of the simultaneous interactions completely abrogates the triggering activity of the C2B domain in liposome fusion. We hypothesize that the simultaneous interactions endow the C2B domain with an ability to deform local membranes, and this membrane-deformation activity might underlie the functional significance of the Syt1 C2B domain in vivo. PMID:27083046

  2. Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta-gonad-mapharsen hematopoiesis

    SciTech Connect

    Ohtsu, Naoki; Nobuhisa, Ikuo; Mochita, Miyuki; Taga, Tetsuya . E-mail: taga@kaiju.medic.kumamoto-u.ac.jp

    2007-01-01

    Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45{sup +} hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45{sup low}c-Kit{sup +} cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.

  3. Speech perception with interaction-compensated simultaneous stimulation and long pulse durations in cochlear implant users.

    PubMed

    Schatzer, Reinhold; Koroleva, Inna; Griessner, Andreas; Levin, Sergey; Kusovkov, Vladislav; Yanov, Yuri; Zierhofer, Clemens

    2015-04-01

    Early multi-channel designs in the history of cochlear implant development were based on a vocoder-type processing of frequency channels and presented bands of compressed analog stimulus waveforms simultaneously on multiple tonotopically arranged electrodes. The realization that the direct summation of electrical fields as a result of simultaneous electrode stimulation exacerbates interactions among the stimulation channels and limits cochlear implant outcome led to the breakthrough in the development of cochlear implants, the continuous interleaved (CIS) sampling coding strategy. By interleaving stimulation pulses across electrodes, CIS activates only a single electrode at each point in time, preventing a direct summation of electrical fields and hence the primary component of channel interactions. In this paper we show that a previously presented approach of simultaneous stimulation with channel interaction compensation (CIC) may also ameliorate the deleterious effects of simultaneous channel interaction on speech perception. In an acute study conducted in eleven experienced MED-EL implant users, configurations involving simultaneous stimulation with CIC and doubled pulse phase durations have been investigated. As pairs of electrodes were activated simultaneously and pulse durations were doubled, carrier rates remained the same. Comparison conditions involved both CIS and fine structure (FS) strategies, either with strictly sequential or paired-simultaneous stimulation. Results showed no statistical difference in the perception of sentences in noise and monosyllables for sequential and paired-simultaneous stimulation with doubled phase durations. This suggests that CIC can largely compensate for the effects of simultaneous channel interaction, for both CIS and FS coding strategies. A simultaneous stimulation paradigm has a number of potential advantages over a traditional sequential interleaved design. The flexibility gained when dropping the requirement of

  4. Homeodomain-interacting protein kinase 2 is the ionizing radiation-activated p53 serine 46 kinase and is regulated by ATM.

    PubMed

    Dauth, Ilka; Krüger, Jana; Hofmann, Thomas G

    2007-03-01

    Phosphorylation of p53 at Ser(46) is important to activate the apoptotic program. The protein kinase that phosphorylates p53 Ser(46) in response to DNA double-strand breaks is currently unknown. The identification of this kinase is of particular interest because it may contribute to the outcome of cancer therapy. Here, we report that ionizing radiation (IR) provokes homeodomain-interacting protein kinase 2 (HIPK2) accumulation, activation, and complex formation with p53. IR-induced HIPK2 up-regulation strictly correlates with p53 Ser(46) phosphorylation. Down-regulation of HIPK2 by RNA interference specifically inhibits IR-induced phosphorylation of p53 at Ser(46). Moreover, we show that HIPK2 activation after IR is regulated by the DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM). Cells from ataxia telangiectasia patients show defects in HIPK2 accumulation. Concordantly, IR-induced HIPK2 accumulation is blocked by pharmacologic inhibition of ATM. Furthermore, ATM down-regulation by RNA interference inhibited IR-induced HIPK2 accumulation, whereas checkpoint kinase 2 deficiency showed no effect. Taken together, our findings indicate that HIPK2 is the IR-activated p53 Ser(46) kinase and is regulated by ATM. PMID:17332358

  5. Oestrogen receptors interact with the α-catalytic subunit of AMP-activated protein kinase

    PubMed Central

    Lipovka, Yulia; Chen, Hao; Vagner, Josef; Price, Theodore J.; Tsao, Tsu-Shuen; Konhilas, John P.

    2015-01-01

    Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signalling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity and cancer. AMPK is targeted by 17β-oestradiol (E2), the main circulating oestrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an oestrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit-binding domain. Finally, both ERs interacted with the upstream liver kinase B 1 (LKB1) kinase complex, which is required for E2-dependent activation of AMPK. We conclude that E2 activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα. PMID:26374855

  6. Focal Adhesion Kinase Is Involved in Rabies Virus Infection through Its Interaction with Viral Phosphoprotein P

    PubMed Central

    Fouquet, Baptiste; Nikolic, Jovan; Larrous, Florence; Bourhy, Hervé; Wirblich, Christoph

    2014-01-01

    ABSTRACT The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition, RABV P has been identified as an interferon antagonist. Here, a yeast two-hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the 106-to-131 domain, corresponding to the dimerization domain of P and the C-terminal domain of FAK containing the proline-rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by coimmunoprecipitation and colocalization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Ala for Arg in position 109 in P (P.R109A), which did not interact with FAK, is affected at a posttranscriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RABV with FAK that positively regulates infection. IMPORTANCE Rabies virus exhibits a small genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional, such as the phosphoprotein P. We and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the focal adhesion kinase (FAK). Our data shed light on the implication of FAK in RABV infection and provide evidence that P-FAK interaction has a proviral function. PMID:25410852

  7. Interaction of brain areas of visual and vestibular simultaneous activity with fMRI.

    PubMed

    Della-Justina, Hellen M; Gamba, Humberto R; Lukasova, Katerina; Nucci-da-Silva, Mariana P; Winkler, Anderson M; Amaro, Edson

    2015-01-01

    Static body equilibrium is an essential requisite for human daily life. It is known that visual and vestibular systems must work together to support equilibrium. However, the relationship between these two systems is not fully understood. In this work, we present the results of a study which identify the interaction of brain areas that are involved with concurrent visual and vestibular inputs. The visual and the vestibular systems were individually and simultaneously stimulated, using flickering checkerboard (without movement stimulus) and galvanic current, during experiments of functional magnetic resonance imaging. Twenty-four right-handed and non-symptomatic subjects participated in this study. Single visual stimulation shows positive blood-oxygen-level-dependent (BOLD) responses (PBR) in the primary and associative visual cortices. Single vestibular stimulation shows PBR in the parieto-insular vestibular cortex, inferior parietal lobe, superior temporal gyrus, precentral gyrus and lobules V and VI of the cerebellar hemisphere. Simultaneous stimulation shows PBR in the middle and inferior frontal gyri and in the precentral gyrus. Vestibular- and somatosensory-related areas show negative BOLD responses (NBR) during simultaneous stimulation. NBR areas were also observed in the calcarine gyrus, lingual gyrus, cuneus and precuneus during simultaneous and single visual stimulations. For static visual and galvanic vestibular simultaneous stimulation, the reciprocal inhibitory visual-vestibular interaction pattern is observed in our results. The experimental results revealed interactions in frontal areas during concurrent visual-vestibular stimuli, which are affected by intermodal association areas in occipital, parietal, and temporal lobes. PMID:25300959

  8. Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids.

    PubMed Central

    Dynan, W S; Yoo, S

    1998-01-01

    The Ku protein-DNA-dependent protein kinase system is one of the major pathways by which cells of higher eukaryotes respond to double-strand DNA breaks. The components of the system are evolutionarily conserved and homologs are known from a number of organisms. The Ku protein component binds directly to DNA ends and may help align them for ligation. Binding of Ku protein to DNA also nucleates formation of an active enzyme complex containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The interaction between Ku protein, DNA-PKcs and nucleic acids has been extensively investigated. This review summarizes the results of these biochemical investigations and relates them to recent molecular genetic studies that reveal highly characteristic repair and recombination defects in mutant cells lacking Ku protein or DNA-PKcs. PMID:9512523

  9. Metabolite regulation of the interaction between Arabidopsis thaliana PII and N-acetyl-l-glutamate kinase.

    PubMed

    Feria Bourrellier, Ana Belén; Ferrario-Méry, Sylvie; Vidal, Jean; Hodges, Michael

    2009-10-01

    The metabolic control of the interaction between ArabidopsisN-acetyl-l-glutamate kinase (NAGK) and the PII protein has been studied. Both gel exclusion and affinity chromatography analyses of recombinant, affinity-purified PII (trimeric complex) and NAGK (hexameric complex) showed that NAGK strongly interacted with PII only in the presence of Mg-ATP, and that this process was reversed by 2-oxoglutarate (2-OG). Furthermore, metabolites such as arginine, glutamate, citrate, and oxalacetate also exerted a negative effect on the PII-NAGK complex formation in the presence of Mg-ATP. Using chloroplast protein extracts and PII affinity chromatography, NAGK interacted with PII only in the presence of ATP-Mg(2+), and this process was antagonized by 2-OG. These results reveal a complex metabolic control of the PII interaction with NAGK in the chloroplast stroma of higher plants. PMID:19631611

  10. Protein-protein interactions in plant mitogen-activated protein kinase cascades.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2016-02-01

    Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense. However, the molecular mechanisms underlying MAPK cascades are still elusive, due largely to our poor understanding of how they relay the signals. Extensive effort has been devoted to characterization of MAPK-substrate interactions to illustrate phosphorylation-based signaling. The diverse MAPK substrates identified also shed light on how spatiotemporal-specific protein-protein interactions function in distinct MAPK cascade-mediated biological processes. This review surveys various technologies used for characterizing MAPK-substrate interactions and presents case studies of MPK4 and MPK6, highlighting the multiple functions of MAPKs. Mass spectrometry-based approaches in identifying MAPK-interacting proteins are emphasized due to their increasing utility and effectiveness. The potential for using MAPKs and their substrates in enhancing plant stress tolerance is also discussed. PMID:26646897

  11. Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with Proapoptotic Kinase Mst1 to Promote Cardiomyocyte Apoptosis

    PubMed Central

    You, Bei; Huang, Shengdong; Qin, Qing; Yi, Bing; Yuan, Yang; Xu, Zhiyun; Sun, Jianxin

    2013-01-01

    Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease. PMID:23527007

  12. Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain.

    PubMed

    Civiero, Laura; Cirnaru, Maria Daniela; Beilina, Alexandra; Rodella, Umberto; Russo, Isabella; Belluzzi, Elisa; Lobbestael, Evy; Reyniers, Lauran; Hondhamuni, Geshanthi; Lewis, Patrick A; Van den Haute, Chris; Baekelandt, Veerle; Bandopadhyay, Rina; Bubacco, Luigi; Piccoli, Giovanni; Cookson, Mark R; Taymans, Jean-Marc; Greggio, Elisa

    2015-12-01

    Leucine-rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21-activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21-activated kinases are serine-threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post-mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock-out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2-mediated pathophysiology. We propose p21-activated kinase 6 (PAK6) as a novel interactor of leucine-rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2-linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6. PMID:26375402

  13. Quasi-simultaneous interaction method for solving 2D boundary layer flows over plates and airfoils

    NASA Astrophysics Data System (ADS)

    Bijleveld, H. A.; Veldman, A. E. P.

    2012-11-01

    This paper studies unsteady 2D boundary layer flows over dented plates and a NACA 0012 airfoil. An inviscid flow is assumed to exist outside the boundary layer and is solved iteratively with the boundary layer flow together with the interaction method until a matching solution is achieved. Hereto a quasi-simultaneous interaction method is applied, in which the integral boundary layer equations are solved together with an interaction-law equation. The interaction-law equation is an approximation of the external flow and based on thin-airfoil theory. It is an algebraic relation between the velocity and displacement thickness. The interaction-law equation ensures that the eigenvalues of the system of equations do not have a sign change and that no singularities occur. Three numerical schemes are used to solve the boundary layer flow with the interaction method. These are: a standard scheme, a splitting method and a characteristics solver. All schemes use a finite difference discretization. The three schemes yield comparable results for the simulations carried out. The standard scheme is deviating most from the splitting and characteristics solvers. The results show that the eigenvalues remain positive, even in separation. As expected, the addition of the interaction-law equation prevents a sign change of the eigenvalues. The quasi-simultaneous interaction scheme is applicable to the three numerical schemes tested.

  14. Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin.

    PubMed

    Sun, F; Hug, M J; Bradbury, N A; Frizzell, R A

    2000-05-12

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR. PMID:10799517

  15. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    SciTech Connect

    Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

    2012-10-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  16. IκB kinase epsilon expression in adipocytes is upregulated by interaction with macrophages.

    PubMed

    Sanada, Yohei; Kumoto, Takahiro; Suehiro, Haruna; Yamamoto, Takafumi; Nishimura, Fusanori; Kato, Norihisa; Yanaka, Noriyuki

    2014-01-01

    Macrophage infiltration in the adipose tissue, and the interaction with adipocytes, is well documented to be involved in fat inflammation and obesity-associated complications. In this study, we isolated IκB kinase ε (IKKε) as a key adipocyte factor that is potentially affected by interaction with macrophages in adipose tissue in vivo. We showed that IKKε mRNA expression levels in white adipose tissue were increased in both genetic and diet-induced obese mouse. Furthermore, IKKε mRNA expression was decreased by the administration of vitamin B6, an anti-inflammatory vitamin, and that IKKε expression levels in adipose tissue were closely correlated with the numbers of infiltrating macrophages. In a co-culture system, we showed that IKKε expression in adipocytes was upregulated by interaction with activated macrophages. This study provides novel insight into IKKε, which is involved in adipose tissue inflammation during the development of obesity. PMID:25130737

  17. Molecular Interaction Study of N1-p-fluorobenzyl-cymserine with TNF-α, p38 Kinase and JNK Kinase

    PubMed Central

    Batool, Sidra; Nawaz, Muhammad Sulaman; Greig, Nigel H.; Rehan, Mohd; Kamal, Mohammad A.

    2016-01-01

    Alzheimer’s disease (AD) is an age-related neurodegenerative disease distinguished by progressive memory loss and cognitive decline. It is accompanied by classical neuropathological changes, including cerebral deposits of amyloid-beta peptide (Aβ) containing senile plaques, neurofibrillary tangles (NFTs) of phosphorylated tau (p-tau), and clusters of activated glial cells. Postmortem studies strongly support a critical role for neuroinflammation in the pathogenesis of AD, with activated microglia and reactive astrocytes surrounding senile plaques and NFTs. These are accompanied by an elevated expression of inflammatory mediators that further drives Ab and p-tau generation. Although epidemiological and experimental studies suggested that long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may lessen AD risk by mitigating inflammatory responses, primary NSAID treatment trials of AD have not proved successful. Elevated systemic butyrylcholinesterase (BuChE) levels have been considered a marker of low-grade systemic inflammation, and BuChE levels are reported elevated in AD brain. Recent research indicates that selective brain inhibition of BuChE elevates acetylcholine (ACh) and augments cognition in rodents free of the characteristic undesirable actions of acetylcholinesterase-inhibitors (AChE-Is). Hence, centrally active BuChE-selective-inhibitors, cymserine analogs, have been developed to test the hypothesis that BuChE-Is would be efficacious and better tolerated than AChE-Is in AD. The focus of the current study was to undertake an in-silico evaluation of an agent to assess its potential to halt the self-propagating interaction between inflammation, Ab and p-tau generation. Molecular docking studies were performed between the novel BuChE-I, N1-p-fluorobenzyl-cymserine (FBC) and inflammatory targets to evaluate the potential of FBC as an inhibitor of p38, JNK kinases and TNF-α with respect to putative binding free energy and IC50 values. Our in

  18. Drosophila tribbles antagonizes insulin signaling-mediated growth and metabolism via interactions with Akt kinase.

    PubMed

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  19. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis.

    PubMed

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering. PMID:27446127

  20. Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

    PubMed Central

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L.

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  1. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis

    PubMed Central

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U.; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering. PMID:27446127

  2. Protein-protein interactions between histidine kinases and response regulators of Mycobacterium tuberculosis H37Rv.

    PubMed

    Lee, Ha-Na; Jung, Kwang-Eun; Ko, In-Jeong; Baik, Hyung Suk; Oh, Jeong-Il

    2012-04-01

    Using yeast two-hybrid assay, we investigated protein-protein interactions between all orthologous histidine kinase (HK)/response regulator (RR) pairs of M. tuberculosis H37Rv and identified potential protein-protein interactions between a noncognate HK/RR pair, DosT/NarL. The protein interaction between DosT and NarL was verified by phosphotransfer reaction from DosT to NarL. Furthermore, we found that the DosT and DosS HKs, which share considerable sequence similarities to each other and form a two-component system with the DosR RR, have different cross-interaction capabilities with NarL: DosT interacted with NarL, while DosS did not. The dimerization domains of DosT and DosS were shown to be sufficient to confer specificity for DosR, and the different cross-interaction abilities of DosS and DosT with NarL were demonstrated to be attributable to variations in the amino acid sequences of the α2-helices of their dimerization domains. PMID:22538656

  3. The juxtamembrane regions of human receptor tyrosine kinases exhibit conserved interaction sites with anionic lipids

    PubMed Central

    Hedger, George; Sansom, Mark S. P.; Koldsø, Heidi

    2015-01-01

    Receptor tyrosine kinases (RTKs) play a critical role in diverse cellular processes and their activity is regulated by lipids in the surrounding membrane, including PIP2 (phosphatidylinositol-4,5-bisphosphate) in the inner leaflet, and GM3 (monosialodihexosylganglioside) in the outer leaflet. However, the precise details of the interactions at the molecular level remain to be fully characterised. Using a multiscale molecular dynamics simulation approach, we comprehensively characterise anionic lipid interactions with all 58 known human RTKs. Our results demonstrate that the juxtamembrane (JM) regions of RTKs are critical for inducing clustering of anionic lipids, including PIP2, both in simple asymmetric bilayers, and in more complex mixed membranes. Clustering is predominantly driven by interactions between a conserved cluster of basic residues within the first five positions of the JM region, and negatively charged lipid headgroups. This highlights a conserved interaction pattern shared across the human RTK family. In particular predominantly the N-terminal residues of the JM region are involved in the interactions with PIP2, whilst residues within the distal JM region exhibit comparatively less lipid specificity. Our results suggest that JM–lipid interactions play a key role in RTK structure and function, and more generally in the nanoscale organisation of receptor-containing cell membranes. PMID:25779975

  4. Therapeutic potential of mitotic interaction between the nucleoporin Tpr and aurora kinase A

    PubMed Central

    Kobayashi, Akiko; Hashizume, Chieko; Dowaki, Takayuki; Wong, Richard W

    2015-01-01

    Spindle poles are defined by centrosomes; therefore, an abnormal number or defective structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. Previously, we showed that Tpr (translocated promoter region), a component of the nuclear pore complex (NPC), interacts with Mad1 and dynein to promote proper chromosome segregation during mitosis. Tpr also associates with p53 to induce autophagy. Here, we report that Tpr depletion induces mitotic catastrophe and enhances the rate of tetraploidy and polyploidy. Mechanistically, Tpr interacts, via its central domain, with Aurora A but not Aurora B kinase. In Tpr-depleted cells, the expression levels, centrosomal localization and phosphorylation of Aurora A were all reduced. Surprisingly, an Aurora A inhibitor, Alisertib (MLN8237), also disrupted centrosomal localization of Tpr and induced mitotic catastrophe and cell death in a time- and dose-dependent manner. Strikingly, over-expression of Aurora A disrupted Tpr centrosomal localization only in cells with supernumerary centrosomes but not in bipolar cells. Our results highlight the mutual regulation between Tpr and Aurora A and further confirm the importance of nucleoporin function in spindle pole organization, bipolar spindle assembly, and mitosis; functions that are beyond the conventional nucleocytoplasmic transport and NPC structural roles of nucleoporins. Furthermore, the central coiled-coil domain of Tpr binds to and sequesters extra Aurora A to safeguard bipolarity. This Tpr domain merits further investigation for its ability to inhibit Aurora kinase and as a potential therapeutic agent in cancer treatment. PMID:25789545

  5. Protein-Protein Interaction for the De Novo Design of Cyclin-Dependent Kinase Peptide Inhibitors.

    PubMed

    Arumugasamy, Karthiga; Tripathi, Sunil Kumar; Singh, Poonam; Singh, Sanjeev Kumar

    2016-01-01

    The homology of the inhibitor binding site regions on the surface of cyclin-dependent kinases (CDKs) makes actual CDK inhibitors unable to bind specifically to their molecular targets. Most of them are ATP competitive inhibitors with low specificity that also affect the phosphorylation mechanisms of other nontarget kinases giving rise to harmful side effects. So, the search of specific and potent inhibitors able to bind to the desired CDK target is still a pending issue. Structure based drug design minimized the erroneous binding and increased the affinity of the inhibitor interaction. In the case of CDKs their activation and regulation mechanisms mainly depend on protein-protein interactions (PPIs). The design of drugs targeting these PPIs makes feasible and promising towards the discovery of new and specific CDK inhibitors. Development of peptide inhibitors for a target protein is an emerging approach in computer aided drug designing. This chapter describes in detail methodology for use of the VitAL-Viterbi algorithm for de novo peptide design of CDK2 inhibitors. PMID:26231708

  6. Impact of kinase activating and inactivating patient mutations on binary PKA interactions

    PubMed Central

    Röck, Ruth; Mayrhofer, Johanna E.; Bachmann, Verena; Stefan, Eduard

    2015-01-01

    The second messenger molecule cAMP links extracellular signals to intracellular responses. The main cellular cAMP effector is the compartmentalized protein kinase A (PKA). Upon receptor initiated cAMP-mobilization, PKA regulatory subunits (R) bind cAMP thereby triggering dissociation and activation of bound PKA catalytic subunits (PKAc). Mutations in PKAc or RIa subunits manipulate PKA dynamics and activities which contribute to specific disease patterns. Mutations activating cAMP/PKA signaling contribute to carcinogenesis or hormone excess, while inactivating mutations cause hormone deficiency or resistance. Here we extended the application spectrum of a Protein-fragment Complementation Assay based on the Renilla Luciferase to determine binary protein:protein interactions (PPIs) of the PKA network. We compared time- and dose-dependent influences of cAMP-elevation on mutually exclusive PPIs of PKAc with the phosphotransferase inhibiting RIIb and RIa subunits and the protein kinase inhibitor peptide (PKI). We analyzed PKA dynamics following integration of patient mutations into PKAc and RIa. We observed that oncogenic modifications of PKAc(L206R) and RIa(Δ184-236) as well as rare disease mutations in RIa(R368X) affect complex formation of PKA and its responsiveness to cAMP elevation. With the cell-based PKA PPI reporter platform we precisely quantified the mechanistic details how inhibitory PKA interactions and defined patient mutations contribute to PKA functions. PMID:26347651

  7. Interactions of calmodulin with death-associated protein kinase peptides: experimental and modeling studies.

    PubMed

    Kuczera, Krzysztof; Kursula, Petri

    2012-01-01

    We have studied the interactions between calmodulin (CaM) and three target peptides from the death-associated protein kinase (DAPK) protein family using both experimental and modeling methods, aimed at determining the details of the underlying biological regulation mechanisms. Experimentally, calorimetric binding free energies were determined for the complexes of CaM with peptides representing the DAPK2 wild-type and S308D mutant, as well as DAPK1. The observed affinity of CaM was very similar for all three studied peptides. The DAPK2 and DAPK1 peptides differ significantly in sequence and total charge, while the DAPK2 S308D mutant is designed to model the effects of DAPK2 Ser308 phosphorylation. The crystal structure of the CaM-DAPK2 S308D mutant peptide is also reported. The structures of CaM-DAPK peptide complexes present a mode of CaM-kinase interaction, in which bulky hydrophobic residues at positions 10 and 14 are both bound to the same hydrophobic cleft. To explain the microscopic effects underlying these interactions, we performed free energy calculations based on the approximate MM-PBSA approach. For these highly charged systems, standard MM-PBSA calculations did not yield satisfactory results. We proposed a rational modification of the approach which led to reasonable predictions of binding free energies. All three complexes are strongly stabilized by two effects: electrostatic interactions and buried surface area. The strong favorable interactions are to a large part compensated by unfavorable entropic terms, in which vibrational entropy is the largest contributor. The electrostatic component of the binding free energy followed the trend of the overall peptide charge, with strongest interactions for DAPK1 and weakest for the DAPK2 mutant. The electrostatics was dominated by interactions of the positively charged residues of the peptide with the negatively charged residues of CaM. The nonpolar binding free energy was comparable for all three peptides, the

  8. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata

    PubMed Central

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism. PMID:26911653

  9. POLLEN TUBE LOCALIZATION IMPLIES A ROLE IN POLLEN-PISTIL INTERACTIONS FOR THE TOMATO RECEPTOR-LIKE PROTEIN KINASES LEPRK1 AND LEPRK2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We screened for pollen-specific kinase genes, which are potential signal transduction components of pollen-pistil interactions, and isolated two structurally related receptor-like kinases RLKs from tomato, LePRK1 and LePRK2. These kinases are similar to a pollen-expressed RLK from petunia, but they ...

  10. Kinase Substrate Sensor (KISS), a mammalian in situ protein interaction sensor.

    PubMed

    Lievens, Sam; Gerlo, Sarah; Lemmens, Irma; De Clercq, Dries J H; Risseeuw, Martijn D P; Vanderroost, Nele; De Smet, Anne-Sophie; Ruyssinck, Elien; Chevet, Eric; Van Calenbergh, Serge; Tavernier, Jan

    2014-12-01

    Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges. PMID:25154561

  11. SUMOylation regulates polo-like kinase 1-interacting checkpoint helicase (PICH) during mitosis.

    PubMed

    Sridharan, Vinidhra; Park, Hyewon; Ryu, Hyunju; Azuma, Yoshiaki

    2015-02-01

    Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres. PMID:25564610

  12. Targeting the interaction of Aurora kinases and SIRT1 mediated by Wnt signaling pathway in colorectal cancer: A critical review.

    PubMed

    Subramaniyan, Boopathi; Jagadeesan, Kaviya; Ramakrishnan, Sabitha; Mathan, Ganeshan

    2016-08-01

    The Aurora kinases belong to the family of serine/threonine kinase, a central regulator of mitosis and their expression increased during G2/M phase. It is classified into Aurora A, B and C, each has distinct roles in cellular processes, which includes regulation of spindle assembly, function of centrosomes, cytoskeleton and cytokinesis. During cancer growth, their rapid increase makes most attractive marker for cancer treatment at present. However Aurora A kinase is known to be a marker for cancer therapy, the most important serine/threonine kinase of Aurora B kinase involvement in cancer is still inadequate. Subsequently, the recent findings revealed that the class III histone deacetylase of SIRT1 is a key regulator to activate Aurora kinases from S phase damaged DNA through Wnt signaling pathway. Even if both Aurora A kinase and SIRT1 serve as a marker for cancer therapy, the present review reveals it is interaction in Wnt signaling pathway that solely for colorectal cancer. PMID:27470380

  13. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    PubMed

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis. PMID:14551255

  14. The SONB(NUP98) nucleoporin interacts with the NIMA kinase in Aspergillus nidulans.

    PubMed Central

    De Souza, Colin P C; Horn, Kevin P; Masker, Kathryn; Osmani, Stephen A

    2003-01-01

    The Aspergillus nidulans NIMA kinase is essential for mitotic entry. At restrictive temperature, temperature-sensitive nimA alleles arrest in G2, before accumulation of NIMA in the nucleus. We performed a screen for extragenic suppressors of the nimA1 allele and isolated two cold-sensitive son (suppressor of nimA1) mutants. The sonA1 mutant encoded a nucleoporin that is a homolog of yeast Gle2/Rae1. We have now cloned SONB, a second nucleoporin genetically interacting with NIMA. sonB is essential and encodes a homolog of the human NUP98/NUP96 precursor. Similar to NUP98/NUP96, SONB(NUP98/NUP96) is autoproteolytically cleaved to generate SONB(NUP98) and SONB(NUP96). SONB(NUP98) localizes to the nuclear pore complex and contains a GLEBS domain (Gle2 binding sequence) that binds SONA(GLE2). A point mutation within the GLEBS domain of SONB1(NUP98) suppresses the temperature sensitivity of the nimA1 allele and compromises the physical interaction between SONA(GLE2) and SONB1(NUP98). The sonB1 mutation also causes sensitivity to hydroxyurea. We isolated the histone H2A-H2B gene pair as a copy-number suppressor of sonB1 cold sensitivity and hydroxyurea sensitivity. The data suggest that the nucleoporins SONA(GLE2) and SONB(NUP98) and the NIMA kinase interact and regulate nuclear accumulation of mitotic regulators to help promote mitosis. PMID:14668365

  15. Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7

    PubMed Central

    Chia, Ruth; Haddock, Sara; Beilina, Alexandra; Rudenko, Iakov N; Mamais, Adamantios; Kaganovich, Alice; Li, Yan; Kumaran, Ravindran; Nalls, Michael A; Cookson, Mark R

    2014-01-01

    LRRK2, a gene relevant to Parkinson's disease, encodes a scaffolding protein with both GTPase and kinase activities. LRRK2 protein is itself phosphorylated and therefore subject to regulation by cell signaling but the kinase(s) responsible for this event have not been definitively identified. Here, using an unbiased siRNA kinome screen, we identify and validate casein kinase 1α (CK1α) as being responsible for LRRK2 phosphorylation, including in the adult mouse striatum. We further show that LRRK2 recruitment to TGN46-positive Golgi-derived vesicles is modulated by constitutive LRRK2 phosphorylation by CK1α. These effects are mediated by differential protein interactions of LRRK2 with a guanine nucleotide exchange factor, ARHGEF7. These pathways are therefore likely involved in the physiological maintenance of the Golgi in cells, which may play a role in the pathogenesis of Parkinson's disease. PMID:25500533

  16. TOUSLED Kinase Activity Oscillates during the Cell Cycle and Interacts with Chromatin Regulators1

    PubMed Central

    Ehsan, Hashimul; Reichheld, Jean-Philippe; Durfee, Tim; Roe, Judith L.

    2004-01-01

    The TOUSLED (TSL)-like nuclear protein kinase family is highly conserved in plants and animals. tsl loss of function mutations cause pleiotropic defects in both leaf and flower development, and growth and initiation of floral organ primordia is abnormal, suggesting that basic cellular processes are affected. TSL is more highly expressed in exponentially growing Arabidopsis culture cells than in stationary, nondividing cells. While its expression remains constant throughout the cell cycle in dividing cells, TSL kinase activity is higher in enriched late G2/M-phase and G1-phase populations of Arabidopsis suspension culture cells compared to those in S-phase. tsl mutants also display an aberrant pattern and increased expression levels of the mitotic cyclin gene CycB1;1, suggesting that TSL represses CycB1;1 expression at certain times during development or that cells are delayed in mitosis. TSL interacts with and phosphorylates one of two Arabidopsis homologs of the nucleosome assembly/silencing protein Asf1 and histone H3, as in humans, and a novel plant SANT/myb-domain protein, TKI1, suggesting that TSL plays a role in chromatin metabolism. PMID:15047893

  17. Characterization of interactions and pharmacophore development for DFG-out inhibitors to RET tyrosine kinase.

    PubMed

    Gao, Chunxia; Grøtli, Morten; Eriksson, Leif A

    2015-07-01

    RET (rearranged during transfection) tyrosine kinase is a promising target for several human cancers. Abt-348, Birb-796, Motesanib and Sorafenib are DFG-out multi-kinase inhibitors that have been reported to inhibit RET activity with good IC50 values. Although the DFG-out conformation has attracted great interest in the design of type II inhibitors, the structural requirements for binding to the RET DFG-out conformation remains unclear. Herein, the DFG-out conformation of RET was determined by homology modelling, the four inhibitors were docked, and the binding modes investigated by molecular dynamics simulation. Binding free energies were calculated using the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method. The trends in predicted binding free affinities correlated well with experimental data and were used to explain the activity difference of the studied inhibitors. Per-residue energy decomposition analyses provided further information on specific interaction properties. Finally, we also conducted a detailed e-pharmacophore modelling of the different RET-inhibitor complexes, explaining the common and specific pharmacophore features of the different complexes. The results reported herein will be useful in future rational design of novel DFG-out RET inhibitors. PMID:26044359

  18. Physical and functional interaction of the TPL2 kinase with nucleophosmin.

    PubMed

    Kanellis, D C; Bursac, S; Tsichlis, P N; Volarevic, S; Eliopoulos, A G

    2015-05-01

    Tumor Progression Locus 2 (TPL2) is widely recognized as a cytoplasmic mitogen-activated protein 3 kinase with a prominent role in the regulation of inflammatory and oncogenic signal transduction. Herein we report that TPL2 may also operate in the nucleus as a physical and functional partner of nucleophosmin (NPM/B23), a major nucleolar phosphoprotein with diverse cellular activities linked to malignancy. We demonstrate that TPL2 mediates the phosphorylation of a fraction of NPM at threonine 199, an event required for its proteasomal degradation and maintenance of steady-state NPM levels. Upon exposure to ultraviolet C, Tpl2 is required for the translocation of de-phosphorylated NPM from the nucleolus to the nucleoplasm. NPM is an endogenous inhibitor of HDM2:p53 interaction and knockdown of TPL2 was found to result in reduced binding of NPM to HDM2, with concomitant defects in p53 accumulation following genotoxic or ribosomal stress. These findings expand our understanding of the function of TPL2 as a negative regulator of carcinogenesis by defining a nuclear role for this kinase in the topological sequestration of NPM, linking p53 signaling to the generation of threonine 199-phosphorylated NPM. PMID:24998852

  19. Short-Form Ron Promotes Spontaneous Breast Cancer Metastasis through Interaction with Phosphoinositide 3-Kinase

    PubMed Central

    Liu, Xuemei; Zhao, Ling; DeRose, Yoko S.; Lin, Yi-Chun; Bieniasz, Magdalena; Eyob, Henok; Buys, Saundra S.; Neumayer, Leigh

    2011-01-01

    Receptor tyrosine kinases (RTKs) have been the subject of intense investigation due to their widespread deregulation in cancer and the prospect of developing targeted therapeutics against these proteins. The Ron RTK has been implicated in tumor aggressiveness and is a developing target for therapy, but its function in tumor progression and metastasis is not fully understood. We examined Ron activity in human breast cancers and found striking predominance of an activated Ron isoform known as short-form Ron (sfRon), whose function in breast tumors has not been explored. We found that sfRon plays a significant role in aggressiveness of breast cancer in vitro and in vivo. sfRon expression was sufficient to convert slow-growing, nonmetastatic tumors into rapidly growing tumors that spontaneously metastasized to liver and bones. Mechanistic studies revealed that sfRon promotes epithelial-mesenchymal transition, invasion, tumor growth, and metastasis through interaction with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K activity, or introduction of a single mutation in the p85 docking site on sfRon, completely eliminated the ability of sfRon to promote tumor growth, invasion, and metastasis. These findings reveal sfRon as an important new player in breast cancer and validate Ron and PI3K as therapeutic targets in this disease. PMID:22207901

  20. The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage.

    PubMed

    Reuven, Nina; Adler, Julia; Porat, Ziv; Polonio-Vallon, Tilman; Hofmann, Thomas G; Shaul, Yosef

    2015-07-01

    The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate. PMID:25944899

  1. AMP-activated Protein Kinase Up-regulates Mitogen-activated Protein (MAP) Kinase-interacting Serine/Threonine Kinase 1a-dependent Phosphorylation of Eukaryotic Translation Initiation Factor 4E.

    PubMed

    Zhu, Xiaoqing; Dahlmans, Vivian; Thali, Ramon; Preisinger, Christian; Viollet, Benoit; Voncken, J Willem; Neumann, Dietbert

    2016-08-12

    AMP-activated protein kinase (AMPK) is a molecular energy sensor that acts to sustain cellular energy balance. Although AMPK is implicated in the regulation of a multitude of ATP-dependent cellular processes, exactly how these processes are controlled by AMPK as well as the identity of AMPK targets and pathways continues to evolve. Here we identify MAP kinase-interacting serine/threonine protein kinase 1a (MNK1a) as a novel AMPK target. Specifically, we show AMPK-dependent Ser(353) phosphorylation of the human MNK1a isoform in cell-free and cellular systems. We show that AMPK and MNK1a physically interact and that in vivo MNK1a-Ser(353) phosphorylation requires T-loop phosphorylation, in good agreement with a recently proposed structural regulatory model of MNK1a. Our data suggest a physiological role for MNK1a-Ser(353) phosphorylation in regulation of the MNK1a kinase, which correlates with increased eIF4E phosphorylation in vitro and in vivo. PMID:27413184

  2. Untangling interactions: do temperature and habitat fragmentation gradients simultaneously impact biotic relationships?

    PubMed Central

    Lakeman-Fraser, Poppy; Ewers, Robert M.

    2014-01-01

    Gaining insight into the impact of anthropogenic change on ecosystems requires investigation into interdependencies between multiple drivers of ecological change and multiple biotic responses. Global environmental change drivers can act simultaneously to impact the abundance and diversity of biota, but few studies have also measured the impact across trophic levels. We firstly investigated whether climate (using temperature differences across a latitudinal gradient as a surrogate) interacts with habitat fragmentation (measured according to fragment area and distance to habitat edges) to impact a New Zealand tri-trophic food chain (plant, herbivore and natural enemy). Secondly, we examined how these interactions might differentially impact both the density and biotic processes of species at each of the three trophic levels. We found evidence to suggest that these drivers act non-additively across trophic levels. The nature of these interactions however varied: location synergistically interacted with fragmentation measures to exacerbate the detrimental effects on consumer density; and antagonistically interacted to ameliorate the impact on plant density and on the interactions between trophic levels (herbivory and parasitoid attack rate). Our findings indicate that the ecological consequences of multiple global change drivers are strongly interactive and vary according to the trophic level studied and whether density or ecological processes are investigated. PMID:24898374

  3. Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

    SciTech Connect

    Murphy, James M.; Korzhnev, Dmitry M.; Ceccarelli, Derek F.; Briant, Douglas J.; Zarrine-Afsar, Arash; Sicheri, Frank; Kay, Lewis E.; Pawson, Tony

    2012-10-23

    The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the N- and C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.

  4. Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin

    PubMed Central

    Maddika, S; Wiechec, E; Ande, SR; Poon, IK; Fischer, U; Wesselborg, S; Jans, DA; Schulze-Osthoff, K; Los, M

    2010-01-01

    Apoptin, a small protein from the chicken anemia virus, has attracted attention because of its specificity in killing tumor cells. Localization of apoptin in the nucleus of tumor cells has been shown to be vital for proapoptotic activity, however, targeted expression of apoptin in the nucleus of normal cells does not harm the cells, indicating that nuclear localization of apoptin is insufficient for its cytotoxicity. Here, we demonstrate for the first time that apoptin interacts with the SH3 domain of p85, the regulatory subunit of phosphoinositide 3-kinase (PI3-K), through its proline-rich region. Apoptin derivatives devoid of this proline-rich region do not interact with p85, are unable to activate PI3-K, and show impaired apoptosis induction. Moreover, apoptin mutants containing the proline-rich domain are sufficient to elevate PI3-K activity and to induce apoptosis in cancer cells. Downregulation of p85 leads to nuclear exclusion of apoptin and impairs cell death induction, indicating that interaction with the p85 PI3-K subunit essentially contributes to the cytotoxic activity of apoptin. PMID:18059340

  5. Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction.

    PubMed

    Krenn, Veronica; Wehenkel, Annemarie; Li, Xiaozheng; Santaguida, Stefano; Musacchio, Andrea

    2012-02-20

    The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR-KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1. PMID:22331848

  6. Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex1[OPEN

    PubMed Central

    Shapiguzov, Alexey; Chai, Xin; Fucile, Geoffrey; Longoni, Paolo; Zhang, Lixin

    2016-01-01

    Photosynthetic organisms have the ability to adapt to changes in light quality by readjusting the cross sections of the light-harvesting systems of photosystem II (PSII) and photosystem I (PSI). This process, called state transitions, maintains the redox poise of the photosynthetic electron transfer chain and ensures a high photosynthetic yield when light is limiting. It is mediated by the Stt7/STN7 protein kinase, which is activated through the cytochrome b6f complex upon reduction of the plastoquinone pool. Its probable major substrate, the light-harvesting complex of PSII, once phosphorylated, dissociates from PSII and docks to PSI, thereby restoring the balance of absorbed light excitation energy between the two photosystems. Although the kinase is known to be inactivated under high-light intensities, the molecular mechanisms governing its regulation remain unknown. In this study we monitored the redox state of a conserved and essential Cys pair of the Stt7/STN7 kinase and show that it forms a disulfide bridge. We could not detect any change in the redox state of these Cys during state transitions and high-light treatment. It is only after prolonged anaerobiosis that this disulfide bridge is reduced. It is likely to be mainly intramolecular, although kinase activation may involve a transient covalently linked kinase dimer with two intermolecular disulfide bonds. Using the yeast two-hybrid system, we have mapped one interaction site of the kinase on the Rieske protein of the cytochrome b6f complex. PMID:26941194

  7. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

    PubMed

    D'Souza, Serena A; Rajendran, Luckshika; Bagg, Rachel; Barbier, Louis; van Pel, Derek M; Moshiri, Houtan; Roy, Peter J

    2016-04-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue. PMID:27123983

  8. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans

    PubMed Central

    D’Souza, Serena A.; Rajendran, Luckshika; Bagg, Rachel; van Pel, Derek M.; Moshiri, Houtan; Roy, Peter J.

    2016-01-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue. PMID:27123983

  9. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling

    PubMed Central

    Wang, Jun; Rouse, Clay; Jasper, Jeff S.; Pendergast, Ann Marie

    2016-01-01

    Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. Here, we report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast activating factors interleukin 6 (IL6) and matrix metalloproteinase-1 (MMP1). Furthermore, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for targeting both tumor and the bone microenvironment with ABL-specific inhibitors. PMID:26838548

  10. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling.

    PubMed

    Wang, Jun; Rouse, Clay; Jasper, Jeff S; Pendergast, Ann Marie

    2016-02-01

    Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. We report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor cells and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast-activating factors interleukin-6 (IL-6) and matrix metalloproteinase 1 (MMP1). Furthermore, in breast cancer cells, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for using ABL-specific inhibitors to limit breast cancer metastasis to bone. PMID:26838548

  11. Simultaneous suppression of the MAP kinase and NF-κB pathways provides a robust therapeutic potential for thyroid cancer.

    PubMed

    Tsumagari, Koji; Abd Elmageed, Zakaria Y; Sholl, Andrew B; Friedlander, Paul; Abdraboh, Mohamed; Xing, Mingzhao; Boulares, A Hamid; Kandil, Emad

    2015-11-01

    The MAP kinase and NF-κB signaling pathways play an important role in thyroid cancer tumorigenesis. We aimed to examine the therapeutic potential of dually targeting the two pathways using AZD6244 and Bortezomib in combination. We evaluated their effects on cell proliferation, cell-cycle progression, apoptosis, cell migration assay, and the activation of the MAPK pathway in vitro and the in vivo using tumor size and immunohistochemical changes of Ki67 and ppRB. We found inhibition of cell growth rate by 10%, 20%, and 56% (p <0.05), migration to 55%, 61%, and 29% (p <0.05), and induction of apoptosis to 10%, 15%, and 38% (p <0.05) with AZD6244, Bortezomib, or combination, respectively. Induction of cell cycle arrest occurred only with drug combination. Dual drug treatment in the xenograft model caused a 94% reduction in tumor size (p <0.05) versus 15% with AZD6244 and 34% with Bortezomib (p < 0.05) and also reduced proliferative marker Ki67, and increased pRb dephosphorylation. Our results demonstrate a robust therapeutic potential of combining AZD6244 and Bortezomib as an effective strategy to overcome drug resistance encountered in monotherapy in the treatment of thyroid cancer, strongly supporting clinical trials to further test this strategy. PMID:26208433

  12. Human-computer interaction in freeform object design and simultaneous manufacturing

    NASA Astrophysics Data System (ADS)

    Zhang, Wei; Lin, Heng; Ma, Liang; Chen, Delin

    2004-03-01

    Freeform object design and simultaneous manufacturing is a novel virtual design and manufacturing method that aims to enable creative and individualized product geometry design and rapid manufacturing of the designed model. The geometry is defined through the process of "virtual sculpting" during which the designer can touch and visualize the designed object in a virtual environment. Natural human-computer interaction is a key issue for this method. This paper first briefly reviewed the principle of the method, including the system configuration, data flow, and fundamental algorithm. Then an input/output device was developed to achieve natural human-computer interaction. Structure of the device and algorithms of calculating the input coordinates and output force were presented. Finally a feedback model was proposed and discussed to apply force feedback during virtual sculpting design.

  13. Simultaneous silence organizes structured higher-order interactions in neural populations

    PubMed Central

    Shimazaki, Hideaki; Sadeghi, Kolia; Ishikawa, Tomoe; Ikegaya, Yuji; Toyoizumi, Taro

    2015-01-01

    Activity patterns of neural population are constrained by underlying biological mechanisms. These patterns are characterized not only by individual activity rates and pairwise correlations but also by statistical dependencies among groups of neurons larger than two, known as higher-order interactions (HOIs). While HOIs are ubiquitous in neural activity, primary characteristics of HOIs remain unknown. Here, we report that simultaneous silence (SS) of neurons concisely summarizes neural HOIs. Spontaneously active neurons in cultured hippocampal slices express SS that is more frequent than predicted by their individual activity rates and pairwise correlations. The SS explains structured HOIs seen in the data, namely, alternating signs at successive interaction orders. Inhibitory neurons are necessary to maintain significant SS. The structured HOIs predicted by SS were observed in a simple neural population model characterized by spiking nonlinearity and correlated input. These results suggest that SS is a ubiquitous feature of HOIs that constrain neural activity patterns and can influence information processing. PMID:25919985

  14. Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages

    PubMed Central

    Wex, Katharina; Schmid, Ursula; Just, Sissy; Wang, Xu; Wurm, Rebecca; Naumann, Michael; Schlüter, Dirk; Nishanth, Gopala

    2016-01-01

    Upon infection with intracellular bacteria, nucleotide oligomerization domain protein 2 recognizes bacterial muramyl dipeptide and binds, subsequently, to receptor-interacting serine/threonine kinase 2 (RIPK2), which activates immune responses via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinase (ERK) pathways. Activation of RIPK2 depends on its K63 ubiquitination by E3 ligases, whereas the deubiquitinating enzyme A20 counter regulates RIPK2 activity by cleaving K63-polyubiquitin chains from RIPK2. Here, we newly identify the deubiquitinating enzyme CYLD as a new inhibitor of RIPK2. We show that CYLD binds to and removes K63-polyubiquitin chains from RIPK2 in Listeria monocytogenes (Lm) infected murine bone marrow-derived macrophages. CYLD-mediated K63 deubiquitination of RIPK2 resulted in an impaired activation of both NF-κB and ERK1/2 pathways, reduced production of proinflammatory cytokines interleukin-6 (IL-6), IL-12, anti-listerial reactive oxygen species (ROS) and nitric oxide (NO), and, finally, impaired pathogen control. In turn, RIPK2 inhibition by siRNA prevented activation of NF-κB and ERK1/2 and completely abolished the protective effect of CYLD deficiency with respect to the production of IL-6, NO, ROS, and pathogen control. Noteworthy, CYLD also inhibited autophagy of Listeria in a RIPK2-ERK1/2-dependent manner. The protective function of CYLD deficiency was dependent on interferon gamma (IFN-γ) prestimulation of infected macrophages. Interestingly, the reduced NF-κB activation in CYLD-expressing macrophages limited the protective effect of IFN-γ by reducing NF-κB-dependent signal transducers and activators of transcription-1 (STAT1) activation. Taken together, our study identifies CYLD as an important inhibitor of RIPK2-dependent antibacterial immune responses in macrophages. PMID:26834734

  15. Phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2.

    PubMed

    Hikasa, Hiroki; Sokol, Sergei Y

    2011-04-01

    Wnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component β-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized β-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with β-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos. PMID:21285352

  16. Simultaneous observations of aerosol–cloud–albedo interactions with three stacked unmanned aerial vehicles

    PubMed Central

    Roberts, G. C.; Ramana, M. V.; Corrigan, C.; Kim, D.; Ramanathan, V.

    2008-01-01

    Aerosol impacts on climate change are still poorly understood, in part, because the few observations and methods for detecting their effects are not well established. For the first time, the enhancement in cloud albedo is directly measured on a cloud-by-cloud basis and linked to increasing aerosol concentrations by using multiple autonomous unmanned aerial vehicles to simultaneously observe the cloud microphysics, vertical aerosol distribution, and associated solar radiative fluxes. In the presence of long-range transport of dust and anthropogenic pollution, the trade cumuli have higher droplet concentrations and are on average brighter. Our observations suggest a higher sensitivity of radiative forcing by trade cumuli to increases in cloud droplet concentrations than previously reported owing to a constrained droplet radius such that increases in droplet concentrations also increase cloud liquid water content. This aerosol-cloud forcing efficiency is as much as −60 W m−2 per 100% percent cloud fraction for a doubling of droplet concentrations and associated increase of liquid water content. Finally, we develop a strategy for detecting aerosol–cloud interactions based on a nondimensional scaling analysis that relates the contribution of single clouds to albedo measurements and illustrates the significance of characterizing cloud morphology in resolving radiometric measurements. This study demonstrates that aerosol–cloud–albedo interactions can be directly observed by simultaneous observations below, in, and above the clouds. PMID:18499803

  17. Simultaneous estimation of cortical activity during social interactions by using EEG hyperscannings.

    PubMed

    Astolfi, L; Cincotti, F; Mattia, D; De Vico Fallani, F; Salinari, S; Vecchiato, G; Toppi, J; Wilke, C; Doud, A; Yuan, H; He, B; Babiloni, F

    2010-01-01

    In this paper we show how the possibility of recording simultaneously the cerebral neuroelectric activity in different subjects (EEG hyperscanning) during the execution of different tasks could return useful information about the "internal" cerebral state of the subjects. We present the results obtained by EEG hyperscannings during ecological task (such as the execution of a card game) as well as that obtained in a series of couples of subjects during the performance of the Prisoner's Dilemma Game. The simultaneous recordings of couples of interacting subjects allows to observe and to model directly the neural signature of human interactions in order to understand the cerebral processes generating and generated by social cooperation or competition. Results obtained in a study of different groups recorded during the card game revealed a larger activity in prefrontal and anterior cingulated cortex in different frequency bands for the player that leads the game when compared to other players. Results collected in a population of 10 subjects during the performance of the Prisoner's Dilemma suggested that the most consistently activated structure is the orbitofrontal region (roughly described by the Brodmann area 10) during the condition of competition in both the tasks. It could be speculated whether the pattern of cortical connectivity between different cortical areas in different subjects could be employed as a tool for assessing the outcome of the task in advance. PMID:21096219

  18. Simultaneous observations of aerosol-cloud-albedo interactions with three stacked unmanned aerial vehicles.

    PubMed

    Roberts, G C; Ramana, M V; Corrigan, C; Kim, D; Ramanathan, V

    2008-05-27

    Aerosol impacts on climate change are still poorly understood, in part, because the few observations and methods for detecting their effects are not well established. For the first time, the enhancement in cloud albedo is directly measured on a cloud-by-cloud basis and linked to increasing aerosol concentrations by using multiple autonomous unmanned aerial vehicles to simultaneously observe the cloud microphysics, vertical aerosol distribution, and associated solar radiative fluxes. In the presence of long-range transport of dust and anthropogenic pollution, the trade cumuli have higher droplet concentrations and are on average brighter. Our observations suggest a higher sensitivity of radiative forcing by trade cumuli to increases in cloud droplet concentrations than previously reported owing to a constrained droplet radius such that increases in droplet concentrations also increase cloud liquid water content. This aerosol-cloud forcing efficiency is as much as -60 W m(-2) per 100% percent cloud fraction for a doubling of droplet concentrations and associated increase of liquid water content. Finally, we develop a strategy for detecting aerosol-cloud interactions based on a nondimensional scaling analysis that relates the contribution of single clouds to albedo measurements and illustrates the significance of characterizing cloud morphology in resolving radiometric measurements. This study demonstrates that aerosol-cloud-albedo interactions can be directly observed by simultaneous observations below, in, and above the clouds. PMID:18499803

  19. Multiple faces of protein interacting with C kinase 1 (PICK1): Structure, function, and diseases.

    PubMed

    Li, Yun-Hong; Zhang, Nan; Wang, Ya-Nan; Shen, Ying; Wang, Yin

    2016-09-01

    Protein interacting with C-kinase 1 (PICK1) has received considerable attention because it is the only protein that contains both PSD-95/DlgA/ZO-1 (PDZ) domain and Bin-Amphiphysin-Rvs (BAR) domain. Through PDZ and BAR domains, PICK1 binds to a large number of membrane proteins and lipid molecules, and is thereby of multiple functions. PICK1 is widely expressed in various tissues, particularly abundant in the brain and testis. In the central nervous system (CNS), PICK1 interacts with numerous neurotransmitters receptors, transporters, ion channels, and enzymes, and controls their trafficking. The best characterized function of PICK1 is that it regulates trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunit GluA2 during long-term depression and long-term potentiation. Recent evidence shows that PICK1 participates in various diseases including neurobiological disorders, such as chronic pain, epilepsy, oxidative stress, stroke, Parkinson's disease, amyotrophic lateral sclerosis, schizophrenia, and non-neurological disorders, such as globozoospermia, breast cancer, and heart failure. In this review, we will summarize recent advances focusing on the structure and regulation of PICK1 and its functions in protein trafficking, neurological and non-neurological diseases. PMID:26970394

  20. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat

    PubMed Central

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na+ and superfluous accumulation of Na+ in transgenic wheat lines. TaCIPK25 expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, transmembrane Na+/H+ exchange was impaired in the root cells of transgenic wheat. These results suggested that TaCIPK25 negatively regulated salt response in wheat. Additionally, yeast-one-hybrid, β-glucuronidase activity and DNA-protein-interaction-enzyme-linked-immunosorbent assays showed that the transcription factor TaWRKY9 bound W-box in the TaCIPK25 promoter region. Quantitative real-time polymerase chain reaction assays showed concomitantly inverted expression patterns of TaCIPK25 and TaWRKY9 in wheat roots under salt treatment, ABA application and inhibition of endogenous ABA condition. Overall, based on our results, in a salt stress condition, the negative salt response in wheat involved TaCIPK25 with the expression regulated by TaWRKY9. PMID:27358166

  1. Inositol hexakisphosphate kinase-1 interacts with perilipin1 to modulate lipolysis.

    PubMed

    Ghoshal, Sarbani; Tyagi, Richa; Zhu, Qingzhang; Chakraborty, Anutosh

    2016-09-01

    Lipolysis leads to the breakdown of stored triglycerides (TAG) to release free fatty acids (FFA) and glycerol which is utilized by energy expenditure pathways to generate energy. Therefore, a decrease in lipolysis augments fat accumulation in adipocytes which promotes weight gain. Conversely, if lipolysis is not complemented by energy expenditure, it leads to FFA induced insulin resistance and type-2 diabetes. Thus, lipolysis is under stringent physiological regulation, although the precise mechanism of the regulation is not known. Deletion of inositol hexakisphosphate kinase-1 (IP6K1), the major inositol pyrophosphate biosynthetic enzyme, protects mice from high fat diet (HFD) induced obesity and insulin resistance. IP6K1-KO mice are lean due to enhanced energy expenditure. Therefore, IP6K1 is a target in obesity and type-2 diabetes. However, the mechanism/s by which IP6K1 regulates adipose tissue lipid metabolism is yet to be understood. Here, we demonstrate that IP6K1-KO mice display enhanced basal lipolysis. IP6K1 modulates lipolysis via its interaction with the lipolytic regulator protein perilipin1 (PLIN1). Furthermore, phosphorylation of IP6K1 at a PKC/PKA motif modulates its interaction with PLIN1 and lipolysis. Thus, IP6K1 is a novel regulator of PLIN1 mediated lipolysis. PMID:27373682

  2. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2.

    PubMed

    Kovács, Krisztián A; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J; Cardinaux, Jean-René

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. PMID:26247811

  3. Interaction of N-acetylglutamate kinase with a PII-like protein in rice.

    PubMed

    Sugiyama, Kenjiro; Hayakawa, Toshihiko; Kudo, Toru; Ito, Takashi; Yamaya, Tomoyuki

    2004-12-01

    PII protein in bacteria is a sensor for 2-oxoglutarate and a transmitter for glutamine signaling. We identified an OsGlnB gene that encoded a bacterial PII-like protein in rice. Yeast two-hybrid analysis showed that an OsGlnB gene product interacted with N-acetylglutamate kinase 1 (OsNAGK1) and PII-like protein (OsGlnB) itself in rice. In cyanobacteria, NAGK is a key enzyme in arginine biosynthesis. Transient expression of OsGlnB cDNA or OsNAGK1 cDNA fused with sGFP in rice leaf blades strongly suggested that the PII-like protein as well as OsNAGK1 protein is located in chloroplasts. Both OsGlnB and OsNAGK1 genes were expressed in roots, leaf blades, leaf sheaths and spikelets of rice, and these two genes were coordinately expressed in leaf blades during the life span. Thus, PII-like protein in rice plants is potentially able to interact with OsNAGK1 protein in vivo. This finding will provide a clue to the precise physiological function of PII-like protein in rice. PMID:15653795

  4. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat.

    PubMed

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na(+) and superfluous accumulation of Na(+) in transgenic wheat lines. TaCIPK25 expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, transmembrane Na(+)/H(+) exchange was impaired in the root cells of transgenic wheat. These results suggested that TaCIPK25 negatively regulated salt response in wheat. Additionally, yeast-one-hybrid, β-glucuronidase activity and DNA-protein-interaction-enzyme-linked-immunosorbent assays showed that the transcription factor TaWRKY9 bound W-box in the TaCIPK25 promoter region. Quantitative real-time polymerase chain reaction assays showed concomitantly inverted expression patterns of TaCIPK25 and TaWRKY9 in wheat roots under salt treatment, ABA application and inhibition of endogenous ABA condition. Overall, based on our results, in a salt stress condition, the negative salt response in wheat involved TaCIPK25 with the expression regulated by TaWRKY9. PMID:27358166

  5. Cortical activity and functional hyperconnectivity by simultaneous EEG recordings from interacting couples of professional pilots.

    PubMed

    Astolfi, L; Toppi, J; Borghini, G; Vecchiato, G; He, E J; Roy, A; Cincotti, F; Salinari, S; Mattia, D; He, B; Babiloni, F

    2012-01-01

    Controlling an aircraft during a flight is a compelling condition, which requires a strict and well coded interaction between the crew. The interaction level between the Captain and the First Officer changes during the flight, ranging from a maximum (during takeoff and landing, as well as in case of a failure of the instrumentation or other emergency situations) to a minimum during quiet mid-flight. In this study, our aim is to investigate the neural correlates of different kinds and levels of interaction between couples of professional crew members by means of the innovative technique called brain hyperscanning, i.e. the simultaneous recording of the hemodynamic or neuroelectrical activity of different human subjects involved in interaction tasks. This approach allows the observation and modeling of the neural signature specifically dependent on the interaction between subjects, and, even more interestingly, of the functional links existing between the brain activities of the subjects interacting together. In this EEG hyperscanning study, different phases of a flight were reproduced in a professional flight simulator, which allowed, on one side, to reproduce the ecological setting of a real flight, and, on the other, to keep under control the different levels of interaction induced in the crew by means of systematic and simulated failures of the aircraft instrumentation. Results of the procedure of linear inverse estimation, together with functional hyperconnectivity estimated by means of Partial Directed Coherence, showed a dense network of connections between the activity in the two brains in the takeoff and landing phases, when the cooperation between the crew is maximal, while conversely no significant links were shown during the phases in which the activity of the two pilots was independent. PMID:23366990

  6. Receptor tyrosine kinase inhibition causes simultaneous bone loss and excess bone formation within growing bone in rats

    SciTech Connect

    Nurmio, Mirja; Joki, Henna; Kallio, Jenny; Maeaettae, Jorma A.; Vaeaenaenen, H. Kalervo; Toppari, Jorma; Jahnukainen, Kirsi; Laitala-Leinonen, Tiina

    2011-08-01

    During postnatal skeletal growth, adaptation to mechanical loading leads to cellular activities at the growth plate. It has recently become evident that bone forming and bone resorbing cells are affected by the receptor tyrosine kinase (RTK) inhibitor imatinib mesylate (STI571, Gleevec (registered)) . Imatinib targets PDGF, ABL-related gene, c-Abl, c-Kit and c-Fms receptors, many of which have multiple functions in the bone microenvironment. We therefore studied the effects of imatinib in growing bone. Young rats were exposed to imatinib (150 mg/kg on postnatal days 5-7, or 100 mg/kg on postnatal days 5-13), and the effects of RTK inhibition on bone physiology were studied after 8 and 70 days (3-day treatment), or after 14 days (9-day treatment). X-ray imaging, computer tomography, histomorphometry, RNA analysis and immunohistochemistry were used to evaluate bone modeling and remodeling in vivo. Imatinib treatment eliminated osteoclasts from the metaphyseal osteochondral junction at 8 and 14 days. This led to a resorption arrest at the growth plate, but also increased bone apposition by osteoblasts, thus resulting in local osteopetrosis at the osteochondral junction. The impaired bone remodelation observed on day 8 remained significant until adulthood. Within the same bone, increased osteoclast activity, leading to bone loss, was observed at distal bone trabeculae on days 8 and 14. Peripheral quantitative computer tomography (pQCT) and micro-CT analysis confirmed that, at the osteochondral junction, imatinib shifted the balance from bone resorption towards bone formation, thereby altering bone modeling. At distal trabecular bone, in turn, the balance was turned towards bone resorption, leading to bone loss. - Research Highlights: > 3-Day imatinib treatment. > Causes growth plate anomalies in young rats. > Causes biomechanical changes and significant bone loss at distal trabecular bone. > Results in loss of osteoclasts at osteochondral junction.

  7. A role for Hsp90 in cell cycle control: Wee1 tyrosine kinase activity requires interaction with Hsp90.

    PubMed Central

    Aligue, R; Akhavan-Niak, H; Russell, P

    1994-01-01

    Wee1 protein kinase regulates the length of G2 phase by carrying out the inhibitory tyrosyl phosphorylation of Cdc2-cyclin B kinase. Mutations were isolated that suppressed the G2 cell cycle arrest caused by overproduction of Wee1. One class of swo (suppressor of wee1 overproduction) mutation, exemplified by swo1-26, also caused a temperature sensitive lethal phenotype in a wee1+ background. The swo1+ gene encodes a member of the Hsp90 family of stress proteins. Swo1 is essential for viability at all temperatures. Swo1 coimmunoprecipitates with Wee1, showing that the two proteins interact. The swo1-26 mutant undergoes premature mitosis when grown at a semi-permissive temperature. These data strongly indicate that formation of active Wee1 tyrosine kinase requires interaction with Swo1, perhaps in a manner analogous to the previously demonstrated interaction between Hsp90 and v-src tyrosine kinase. These observations demonstrate a unexpected role for Hsp90 in cell cycle control. Images PMID:7813446

  8. Specific effects of c-Jun NH2-terminal kinase-interacting protein 1 in neuronal axons

    PubMed Central

    Tang, Shu; Wen, Qiang; Zhang, Xiao-jian; Kan, Quan-cheng

    2016-01-01

    c-Jun NH2-terminal kinase (JNK)-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B (TrkB) anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of TrkB anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed TrkB complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of TrkB gradually increased in axon terminals. However, the distribution of TrkB reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of TrkB after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of TrkB in dendrites. These findings confirm that JNK-interacting protein 1 can interact with TrkB in neuronal cells, and can regulate the transport of TrkB in axons, but not in dendrites. PMID:26981098

  9. Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

    PubMed

    Akaike, Y; Kuwano, Y; Nishida, K; Kurokawa, K; Kajita, K; Kano, S; Masuda, K; Rokutan, K

    2015-06-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent

  10. Induction of Viral, 7-Methyl-Guanosine Cap-Independent Translation and Oncolysis by Mitogen-Activated Protein Kinase-Interacting Kinase-Mediated Effects on the Serine/Arginine-Rich Protein Kinase

    PubMed Central

    Brown, Michael C.; Bryant, Jeffrey D.; Dobrikova, Elena Y.; Shveygert, Mayya; Bradrick, Shelton S.; Chandramohan, Vidyalakshmi; Bigner, Darell D.

    2014-01-01

    ABSTRACT Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m7G) “cap”-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. IMPORTANCE We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. PMID:25187541

  11. Identification of an interaction between EI and a histidine kinase-response regulator hybrid protein in Gluconobacter oxydans.

    PubMed

    Li, Shan; Ma, Yushu; Wei, Dongzhi

    2016-02-01

    Gluconobacter oxydans may contain an incomplete phosphoenolpyruvate: carbohydrate phosphotransferase system consisting of three components - EI, HPr and EIIA, while the function of individual members of the system remains unknown. In this research, a specific interaction between EI and a histidine kinase-response regulator hybrid protein was screened by yeast two-hybrid assay, and the interaction was further identified with GST pull-down assay and bimolecular fluorescence complementation assay in vitro and in vivo, respectively. As the histidine kinase-response regulator hybrid protein serves as a member of two-component system in G. oxydans, its interaction with EI implied that PTS may play certain roles in bacteria under stress. PMID:26792729

  12. p120 Catenin-Associated Fer and Fyn Tyrosine Kinases Regulate β-Catenin Tyr-142 Phosphorylation and β-Catenin-α-Catenin Interaction

    PubMed Central

    Piedra, Jose; Miravet, Susana; Castaño, Julio; Pálmer, Héctor G.; Heisterkamp, Nora; García de Herreros, Antonio; Duñach, Mireia

    2003-01-01

    β-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and α-catenin. We show here that interaction of β-catenin with α-catenin is regulated by the phosphorylation of β-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases. Transfection of these kinases to epithelial cells disrupted the association between both catenins. We have also examined whether these kinases are involved in the regulation of this interaction by K-ras. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little α-catenin-β-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of β-catenin-α-catenin interaction. PMID:12640114

  13. Temporal Multiscale Approach for Nanocarrier Motion with Simultaneous Adhesion and Hydrodynamic Interactions in Targeted Drug Delivery

    PubMed Central

    Radhakrishnan, R.; Uma, B.; Liu, J.; Ayyaswamy, P. S.; Eckmann, D. M.

    2012-01-01

    We present a fluctuating hydrodynamics approach and a hybrid approach combining fluctuating hydrodynamics with generalized Langevin dynamics to resolve the motion of a nanocarrier when subject to both hydrodynamic interactions and adhesive interactions. Specifically, using these approaches, we compute equilibrium probability distributions at constant temperature as well as velocity autocorrelation functions of the nanocarrier subject to thermal motion in a quiescent Newtonian fluid medium, when tethered by a harmonic spring force mimicking a tether due to a single receptor-ligand bond. We demonstrate that the thermal equipartition of translation, rotation, and spring degrees of freedom are preserved by our formalism while simultaneously resolving the nature of the hydrodynamic correlations. Additionally, we evaluate the potential of mean force (or free energy density) along a specified reaction coordinate to faciltate extensive conformational sampling of the nanocarrier motion. We show that our results are in excellent agreement with analytical results and Monte Carlo simulations, thereby validating our methodologies. The frameworks we have presented provide a comprehensive platform for temporal multiscale modeling of hydrodynamic and microscopic interactions mediating nanocarrier motion and adhesion in vascular targeted drug delivery. PMID:23853388

  14. Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans

    PubMed Central

    Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

    2016-01-01

    Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development. PMID:26791749

  15. Signaling between two interacting sensor kinases promotes biofilms and colonization by a bacterial symbiont

    PubMed Central

    Norsworthy, Allison N.; Visick, Karen L.

    2015-01-01

    Summary Cells acclimate to fluctuating environments by utilizing sensory circuits. One common sensory pathway used by bacteria is two-component signaling (TCS), composed of an environmental sensor (the sensor kinase, SK) and a cognate, intracellular effector (the response regulator, RR). The squid symbiont Vibrio fischeri uses an elaborate TCS phosphorelay containing a hybrid SK, RscS, and two RRs, SypE and SypG, to control biofilm formation and host colonization. Here, we found that another hybrid SK, SypF, was essential for biofilms by functioning downstream of RscS to directly control SypE and SypG. Surprisingly, although wild-type SypF functioned as a SK in vitro, this activity was dispensable for colonization. In fact, only a single non-enzymatic domain within SypF, the HPt domain, was critical in vivo. Remarkably, this domain within SypF interacted with RscS to permit a bypass of RscS’s own HPt domain and SypF’s enzymatic function. This represents the first in vivo example of a functional SK that exploits the enzymatic activity of another SK, an adaptation that demonstrates the elegant plasticity in the arrangement of TCS regulators. PMID:25586643

  16. Molecular interactions of polo-like kinase 1 in human cancers.

    PubMed

    Weng Ng, Wayne Tiong; Shin, Joo-Shik; Roberts, Tara Laurine; Wang, Bin; Lee, Cheok Soon

    2016-07-01

    Polo-like kinase 1 (PLK1) is an essential protein in communicating cell-cycle progression and DNA damage. Overexpression of PLK1 has been validated as a marker for poor prognosis in many cancers. PLK1 knockdown decreases the survival of cancer cells. PLK1 is therefore an attractive target for anticancer treatments. Several inhibitors have been developed, and some have been clinically tested to show additive effects with conventional therapies. Upstream regulation of PLK1 involves multiple interactions of proteins such as FoxM1, E2F and p21. Other cancer-related proteins such as pRB and p53 also indirectly influence PLK1 expression. With the high mutation rates of these genes seen in cancers, they may be associated with PLK1 deregulation. This raises the question of whether PLK1 overexpression is a cause or a consequence of oncogenesis. In addition, hypomethylation of the CpG island of the PLK1 promoter region contributes to its upregulation. PLK1 expression can be affected by many factors; thus, it is possible that PLK1 deregulation in each individual patient tumours could be due to different underlying mechanisms. PMID:26941182

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

    PubMed Central

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

    2016-01-01

    Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA. PMID:27057269

  18. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    PubMed

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells. PMID:23387972

  19. Effects of Interactive versus Simultaneous Display of Multimedia Glosses on L2 Reading Comprehension and Incidental Vocabulary Learning

    ERIC Educational Resources Information Center

    Türk, Emine; Erçetin, Gülcan

    2014-01-01

    This study examines the effects of interactive versus simultaneous display of visual and verbal multimedia information on incidental vocabulary learning and reading comprehension of learners of English with lower proficiency levels. In the interactive display condition, learners were allowed to select the type of multimedia information whereas the…

  20. Quantifying foodweb interactions with simultaneous linear equations: Stable isotope models of the Truckee River, USA

    USGS Publications Warehouse

    Saito, L.; Redd, C.; Chandra, S.; Atwell, L.; Fritsen, C.H.; Rosen, Michael R.

    2007-01-01

    Aquatic foodweb models for 2 seasons (relatively high- [March] and low-flow [August] conditions) were constructed for 4 reaches on the Truckee River using ??13C and ??15N data from periphyton, macroinvertebrate, and fish samples collected in 2003 and 2004. The models were constructed with isotope values that included measured periphyton signatures and calculated mean isotope values for detritus and seston as basal food sources of each food web. The pseudo-optimization function in Excel's Solver module was used to minimize the sum of squared error between predicted and observed stable-isotope values while simultaneously solving for diet proportions for all foodweb consumers and estimating ??13C and ??15N trophic enrichment factors. This approach used an underdetermined set of simultaneous linear equations and was tested by running the pseudo-optimization procedure for 500 randomly selected sets of initial conditions. Estimated diet proportions had average standard deviations (SDs) of 0.03 to 0.04??? and SDs of trophic enrichment factors ranged from <0.005 to 0.05??? based on the results of the 500 runs, indicating that the modeling approach was very robust. However, sensitivity analysis of calculated detritus and seston ??13C and ??15N values indicated that the robustness of the approach is dependent on having accurate measures of all observed foodweb-component ??13c and ??15N values. Model results from the 500 runs using the mean isotope values for detritus and seston indicated that upstream food webs were the simplest, with fewer feeding groups and trophic interactions (e.g., 21 interactions for 10 feeding groups), whereas food webs for the reach downstream of the Reno-Sparks metropolitan area were the most complex (e.g., 58 interactions for 16 feeding groups). Nonnative crayfish were important omnivores in each reach and drew energy from multiple sources, but appeared to be energetic dead ends because they generally were not consumed. Predatory macroinvertebrate

  1. A Complex Network of Interactions between Mitotic Kinases, Phosphatases and ESCRT Proteins Regulates Septation and Membrane Trafficking in S. pombe

    PubMed Central

    Bhutta, Musab S.; Roy, Brinta; Gould, Gwyn W.; McInerny, Christopher J.

    2014-01-01

    Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast. PMID:25356547

  2. Interaction of the C-terminal acidic domain of the insulin receptor with histone modulates the receptor kinase activity.

    PubMed

    Baron, V; Kaliman, P; Alengrin, F; Van Obberghen, E

    1995-04-01

    In this study, we investigated the role of the insulin receptor domain 1270-1280, an acid-rich sequence located in the receptor C-terminus. Antipeptide IgG raised against this sequence were obtained and used to analyze their effect on receptor function. Antipeptide IgG inhibited receptor autophosphorylation at Tyr1146, Tyr1150 and Tyr1151. These sites are known to be key modulators of the receptor activity. Autophosphorylation at other sites may also have been inhibited. The antipeptide antibody decreased the receptor kinase activity measured with poly(Glu80Tyr20) and a synthetic peptide corresponding to the proreceptor sequence 1142-1158. We provide evidence that the effect of the antibody on substrate phosphorylation may result from the control of the phosphorylation level of the receptor. Concerning the action of the antipeptide IgG on the receptor kinase activity, histone did not behave similarly to poly(Glu80Tyr20). The antibody recognizing sequence 1270-1280 competed with histone for an overlapping binding site. Histone also modulated insulin receptor autophosphorylation, supporting the idea that interference with domain 1270-1280 alters the receptor kinase. Our data suggest that the acidic region including residues 1270-1280 of the insulin receptor C-terminus is involved in the following events: (a) receptor binding with histone, an exogenous substrate of the receptor kinase, and (b) the regulation of receptor autophosphorylation and kinase activity. Based on these observations, we would like to propose that this insulin receptor domain could interact with cellular proteins modulating the receptor kinase. PMID:7744039

  3. IRBIT Interacts with the Catalytic Core of Phosphatidylinositol Phosphate Kinase Type Iα and IIα through Conserved Catalytic Aspartate Residues

    PubMed Central

    Ando, Hideaki; Hirose, Matsumi; Gainche, Laura; Kawaai, Katsuhiro; Bonneau, Benjamin; Ijuin, Takeshi; Itoh, Toshiki; Takenawa, Tadaomi; Mikoshiba, Katsuhiko

    2015-01-01

    Phosphatidylinositol phosphate kinases (PIPKs) are lipid kinases that generate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a critical lipid signaling molecule that regulates diverse cellular functions, including the activities of membrane channels and transporters. IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate) is a multifunctional protein that regulates diverse target proteins. Here, we report that IRBIT forms signaling complexes with members of the PIPK family. IRBIT bound to all PIPK isoforms in heterologous expression systems and specifically interacted with PIPK type Iα (PIPKIα) and type IIα (PIPKIIα) in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are involved in the interaction with IRBIT. Furthermore, phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfered with the interaction, suggesting that IRBIT interacts with catalytic cores of PIPKs. Mutations of phosphorylation sites in the serine-rich region of IRBIT affected the selectivity of its interaction with PIPKIα and PIPKIIα. The structural flexibility of the serine-rich region, located in the intrinsically disordered protein region, is assumed to underlie the mechanism of this interaction. Furthermore, in vitro binding experiments and immunocytochemistry suggest that IRBIT and PIPKIα interact with the Na+/HCO3− cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIα and NBCe1-B, whose activity is regulated by PI(4,5)P2. PMID:26509711

  4. Simultaneous Multi-angle Radar Observations of Langmuir Turbulence Excited by RF Ionospheric Interactions at HAARP

    NASA Astrophysics Data System (ADS)

    Sheerin, J. P.; Watanabe, N.; Rayyan, N.; Spry, D.; Adham, N.; Watkins, B. J.; Bristow, W. A.; Spaleta, J.; Bernhardt, P. A.

    2012-12-01

    The high power HAARP HF transmitter is employed to generate and study strong Langmuir turbulence (SLT) in the interaction region of overdense ionospheric plasma. Diagnostics included the Modular UHF Ionospheric Radar (MUIR) sited at HAARP, the SuperDARN-Kodiak HF radar, and HF receivers to record stimulated electromagnetic emissions (SEE). Dependence of diagnostic signals on HAARP HF parameters, including pulselength, duty-cycle, aspect angle, and frequency were recorded. Short pulse, low duty cycle experiments demonstrate control of artificial field-aligned irregularities (AFAI) and isolation of ponderomotive effects. Among the effects observed and studied are: SLT spectra including cascade, collapse, and co-existence spectra and an outshifted plasma line under certain ionospheric conditions. High time resolution studies of the temporal evolution of the plasma line reveal the appearance of an overshoot effect on ponderomotive timescales. Bursty turbulence is observed in the collapse and cascade lines. For the first time, simultaneous multi-angle radar measurements of plasma line spectra are recorded demonstrating marked dependence on aspect angle with the strongest interaction region observed displaced southward of the HF zenith pointing angle. Numerous measurements of the outshifted plasma line are observed. Experimental results are compared to previous high latitude experiments and predictions from recent modeling efforts.

  5. Simultaneous Multi-angle Radar Observations of Langmuir Turbulence Excited by RF Ionospheric Interactions at HAARP

    NASA Astrophysics Data System (ADS)

    Sheerin, J. P.; Rayyan, N.; Watanabe, N.; Watkins, B. J.; Bristow, W. A.; Bernhardt, P. A.

    2013-10-01

    The high power HAARP HF transmitter is employed to generate and study strong Langmuir turbulence (SLT) in the interaction region of overdense ionospheric plasma. Diagnostics included the Modular UHF Ionospheric Radar (MUIR) sited at HAARP, the SuperDARN-Kodiak HF radar, and HF receivers to record stimulated electromagnetic emissions (SEE). Dependence of diagnostic signals on HAARP HF parameters, including pulselength, duty-cycle, aspect angle, and frequency were recorded. Short pulse, low duty cycle experiments demonstrate control of artificial field-aligned irregularities (AFAI) and isolation of ponderomotive effects. Among the effects observed and studied are: SLT spectra including cascade, collapse, and co-existence spectra and an outshifted plasma line under certain ionospheric conditions. High time resolution studies of the temporal evolution of the plasma line reveal the appearance of an overshoot effect on ponderomotive timescales. Bursty turbulence is observed in the collapse and cascade lines. For the first time, simultaneous multi-angle radar measurements of plasma line spectra are recorded demonstrating marked dependence on aspect angle with the strongest interaction region observed displaced southward of the HF zenith pointing angle. Numerous measurements of the outshifted plasma line are observed. Experimental results are compared to previous high latitude experiments and predictions from recent modeling efforts.

  6. Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice.

    PubMed

    Liu, Lingtong; Zheng, Canhui; Kuang, Baijan; Wei, Liqin; Yan, Longfeng; Wang, Tai

    2016-07-01

    During sexual reproduction of flowering plants, the pollen tube grows fast and over a long distance within the pistil to deliver two sperms for double fertilization. Growing plant cells need to communicate constantly with external stimuli as well as monitor changes in surface tension of the cell wall and plasma membrane to coordinate these signals and internal growth machinery; however, the underlying mechanisms remain largely unknown. Here we show that the rice member of plant-specific receptor-like kinase CrRLK1Ls subfamily, Ruptured Pollen tube (RUPO), is specifically expressed in rice pollen. RUPO localizes to the apical plasma membrane and vesicle of pollen tubes and is required for male gamete transmission. K+ levels were greater in pollen of homozygous CRISPR-knockout lines than wild-type plants, and pollen tubes burst shortly after germination. We reveal the interaction of RUPO with high-affinity potassium transporters. Phosphorylation of RUPO established and dephosphorylation abolished the interaction. These results have revealed the receptor-like kinase as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction, and demonstrated a novel receptor-like kinase signaling pathway that mediates K+ homeostasis required for pollen tube growth and integrity. PMID:27447945

  7. Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice

    PubMed Central

    Liu, Lingtong; Zheng, Canhui; Kuang, Baijan; Wei, Liqin; Yan, Longfeng; Wang, Tai

    2016-01-01

    During sexual reproduction of flowering plants, the pollen tube grows fast and over a long distance within the pistil to deliver two sperms for double fertilization. Growing plant cells need to communicate constantly with external stimuli as well as monitor changes in surface tension of the cell wall and plasma membrane to coordinate these signals and internal growth machinery; however, the underlying mechanisms remain largely unknown. Here we show that the rice member of plant-specific receptor-like kinase CrRLK1Ls subfamily, Ruptured Pollen tube (RUPO), is specifically expressed in rice pollen. RUPO localizes to the apical plasma membrane and vesicle of pollen tubes and is required for male gamete transmission. K+ levels were greater in pollen of homozygous CRISPR-knockout lines than wild-type plants, and pollen tubes burst shortly after germination. We reveal the interaction of RUPO with high-affinity potassium transporters. Phosphorylation of RUPO established and dephosphorylation abolished the interaction. These results have revealed the receptor-like kinase as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction, and demonstrated a novel receptor-like kinase signaling pathway that mediates K+ homeostasis required for pollen tube growth and integrity. PMID:27447945

  8. Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis1

    PubMed Central

    Pandey, Girdhar K.; Kanwar, Poonam; Singh, Amarjeet; Steinhorst, Leonie; Pandey, Amita; Yadav, Akhlilesh K.; Tokas, Indu; Sanyal, Sibaji K.; Kim, Beom-Gi; Lee, Sung-Chul; Cheong, Yong-Hwa; Kudla, Jörg; Luan, Sheng

    2015-01-01

    The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes. PMID:26198257

  9. Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis.

    PubMed

    Pandey, Girdhar K; Kanwar, Poonam; Singh, Amarjeet; Steinhorst, Leonie; Pandey, Amita; Yadav, Akhlilesh K; Tokas, Indu; Sanyal, Sibaji K; Kim, Beom-Gi; Lee, Sung-Chul; Cheong, Yong-Hwa; Kudla, Jörg; Luan, Sheng

    2015-09-01

    The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes. PMID:26198257

  10. The MAP kinase-interacting kinases regulate cell migration, vimentin expression and eIF4E/CYFIP1 binding.

    PubMed

    Beggs, James E; Tian, Shuye; Jones, Greg G; Xie, Jianling; Iadevaia, Valentina; Jenei, Veronika; Thomas, Gareth; Proud, Christopher G

    2015-04-01

    The MAP kinase-interacting kinases (Mnk1 and Mnk2) are activated by ERK and are best known for phosphorylating the translation initiation factor eIF4E. Genetic knockout of the Mnks impaired the migration of embryonic fibroblasts both in two-dimensional wound-healing experiments and in three-dimensional migration assays. Furthermore, a novel and selective Mnk inhibitor, Mnk-I1, which potently blocks eIF4E phosphorylation, blocked the migration of fibroblasts and cancer cells, without exerting 'off-target' effects on other signalling pathways such as Erk. Mnk-I1 or genetic knockout of the Mnks decreased the expression of vimentin, a marker of mesenchymal cells, without affecting vimentin mRNA levels. Vimentin protein levels were much lower in Mnk1/2-knockout cells than in controls, although mRNA levels were similar. Our data suggest that the Mnks regulate the translation of the vimentin mRNA and the stability of the vimentin protein. Inhibition or genetic knockout of the Mnks increased the binding of eIF4E to the cytoplasmic FMRP-interacting protein 1 (CYFIP1), which binds the fragile-X mental retardation protein, FMRP, a translational repressor. Since FMRP binds mRNAs for proteins involved in metastasis, the Mnk-dependent release of CYFIP1 from eIF4E is expected to release the repression of translation of FMRP-bound mRNAs, potentially providing a molecular mechanism for the control of cell migration by the Mnks. As Mnk1/2 are not essential for viability, inhibition of the Mnks may be a useful approach to tackling cancer metastasis, a key process contributing to mortality in cancer patients. PMID:25588502

  11. Nuclear lymphocyte-specific protein tyrosine kinase and its interaction with CR6-interacting factor 1 promote the survival of human leukemic T cells

    PubMed Central

    VAHEDI, SHAHROOZ; CHUEH, FU-YU; DUTTA, SUJOY; CHANDRAN, BALA; YU, CHAO-LAN

    2015-01-01

    Overexpression and hyperactivation of lymphocyte-specific protein tyrosine kinase (Lck) have been associated with leukemia development. We previously showed that, other than its known function as a cytoplasmic signal transducer, Lck also acts as a nuclear transcription factor in mouse leukemic cells. In the present study, we demonstrated the presence of nuclear Lck in human leukemic T cells and in primary cells. We further established a positive correlation between Lck nuclear localization and its kinase activity. Proteomic analysis identified CR6-interacting factor 1 (CRIF1) as one of the Lck-interacting proteins. CRIF1 and Lck association in the nucleus was confirmed both by immunofluorescence microscopy and co-immunoprecipitation in human leukemic T cells. Close-range interaction between Lck and CRIF1 was validated by in situ proximity ligation assay (PLA). Consistent with the role of nuclear CRIF1 as a tumor suppressor, CRIF1 silencing promotes leukemic T cell survival in the absence of growth factors. This protective effect can be recapitulated by endogenous Lck or reconstituted Lck in leukemic T cells. All together, our results support a novel function of nuclear Lck in promoting human leukemic T cell survival through interaction with a tumor suppressor. It has important implications in defining a paradigm shift of non-canonical protein tyrosine kinase signaling. PMID:25997448

  12. Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

    PubMed Central

    Laudet, Béatrice; Barette, Caroline; Dulery, Vincent; Renaudet, Olivier; Dumy, Pascal; Metz, Alexandra; Prudent, Renaud; Deshiere, Alexandre; Dideberg, Otto; Filhol, Odile; Cochet, Claude

    2007-01-01

    X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2β which contacts at the centre of the CK2α/CK2β interface dominates affinity. The results indicate that a double mutation in CK2β of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2α, is the most disruptive to CK2α binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188–Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2β-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference. PMID:17714077

  13. Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction.

    PubMed

    Laudet, Béatrice; Barette, Caroline; Dulery, Vincent; Renaudet, Olivier; Dumy, Pascal; Metz, Alexandra; Prudent, Renaud; Deshiere, Alexandre; Dideberg, Otto; Filhol, Odile; Cochet, Claude

    2007-12-15

    X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2beta which contacts at the centre of the CK2alpha/CK2beta interface dominates affinity. The results indicate that a double mutation in CK2beta of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2alpha, is the most disruptive to CK2alpha binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188-Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2beta-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference. PMID:17714077

  14. Methyl-CpG-binding protein 2 is phosphorylated by homeodomain-interacting protein kinase 2 and contributes to apoptosis.

    PubMed

    Bracaglia, Giorgia; Conca, Barbara; Bergo, Anna; Rusconi, Laura; Zhou, Zhaolan; Greenberg, Michael E; Landsberger, Nicoletta; Soddu, Silvia; Kilstrup-Nielsen, Charlotte

    2009-12-01

    Mutations in the methyl-CpG-binding protein 2 (MeCP2) are associated with Rett syndrome and other neurological disorders. MeCP2 represses transcription mainly by recruiting various co-repressor complexes. Recently, MeCP2 phosphorylation at Ser 80, Ser 229 and Ser 421 was shown to occur in the brain and modulate MeCP2 silencing activities. However, the kinases directly responsible for this are largely unknown. Here, we identify the homeodomain-interacting protein kinase 2 (HIPK2) as a kinase that binds MeCP2 and phosphorylates it at Ser 80 in vitro and in vivo. HIPK2 modulates cell proliferation and apoptosis, and the neurological defects of Hipk2-null mice indicate its role in proper brain functions. We show that MeCP2 cooperates with HIPK2 in induction of apoptosis and that Ser 80 phosphorylation is required together with the DNA binding of MeCP2. These data are, to our knowledge, the first that describe a kinase associating with MeCP2, causing its specific phosphorylation in vivo and, furthermore, they reinforce the role of MeCP2 in regulating cell growth. PMID:19820693

  15. Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage.

    PubMed

    de la Vega, Laureano; Hornung, Juliane; Kremmer, Elisabeth; Milanovic, Maja; Schmitz, M Lienhard

    2013-06-01

    Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis. PMID:23620283

  16. Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage

    PubMed Central

    de la Vega, Laureano; Hornung, Juliane; Kremmer, Elisabeth; Milanovic, Maja; Schmitz, M. Lienhard

    2013-01-01

    Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis. PMID:23620283

  17. Methyl-CpG-binding protein 2 is phosphorylated by homeodomain-interacting protein kinase 2 and contributes to apoptosis

    PubMed Central

    Bracaglia, Giorgia; Conca, Barbara; Bergo, Anna; Rusconi, Laura; Zhou, Zhaolan; Greenberg, Michael E; Landsberger, Nicoletta; Soddu, Silvia; Kilstrup-Nielsen, Charlotte

    2009-01-01

    Mutations in the methyl-CpG-binding protein 2 (MeCP2) are associated with Rett syndrome and other neurological disorders. MeCP2 represses transcription mainly by recruiting various co-repressor complexes. Recently, MeCP2 phosphorylation at Ser 80, Ser 229 and Ser 421 was shown to occur in the brain and modulate MeCP2 silencing activities. However, the kinases directly responsible for this are largely unknown. Here, we identify the homeodomain-interacting protein kinase 2 (HIPK2) as a kinase that binds MeCP2 and phosphorylates it at Ser 80 in vitro and in vivo. HIPK2 modulates cell proliferation and apoptosis, and the neurological defects of Hipk2-null mice indicate its role in proper brain functions. We show that MeCP2 cooperates with HIPK2 in induction of apoptosis and that Ser 80 phosphorylation is required together with the DNA binding of MeCP2. These data are, to our knowledge, the first that describe a kinase associating with MeCP2, causing its specific phosphorylation in vivo and, furthermore, they reinforce the role of MeCP2 in regulating cell growth. PMID:19820693

  18. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

    PubMed Central

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  19. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

    PubMed

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  20. Yeast two-hybrid interactions between Arabidopsis lyrata S Receptor Kinase and the ARC1 E3 ligase.

    PubMed

    Indriolo, Emily; Goring, Daphne R

    2016-06-01

    Here we describe protein-protein interactions between signaling components in the conserved self-incompatibility pathway from Brassica spp. and Arabidopsis lyrata. Previously, we had demonstrated that ARC1 is necessary in A. lyrata for the rejection of self-pollen by the self-incompatibility pathway. The results described here demonstrate that A. lyrata ARC1 interacts with A. lyrata S Receptor Kinase (SRK1) in the yeast 2-hybrid system. A. lyrata ARC1 also interacted with B. napus SRK910 illustrating that interactions in this pathway are conserved across species. Finally, we discuss how the more widely occurring interactions between SRK and ARC1-related family members may be modulated in vivo by expression and subcellular localization patterns resulting in a particular response. PMID:27175603

  1. Interaction of yeast repressor-activator protein Ume6p with glycogen synthase kinase 3 homolog Rim11p.

    PubMed Central

    Malathi, K; Xiao, Y; Mitchell, A P

    1997-01-01

    Meiosis and expression of early meiotic genes in the budding yeast Saccharomyces cerevisiae depend upon Rim11p, Ume6p, and Ime1p. Rim11p (also called Mds1p and ScGSK3) is a protein kinase related to glycogen synthase kinase 3 (GSK3); Ume6p is an architectural transcription factor; and Imelp is a Ume6p-binding protein that provides a transcriptional activation domain. Rim11p is required for Ime1p-Ume6p interaction, and prior studies have shown that Rim11p binds to and phosphorylates Ime1p. We show here that Rim11p binds to and phosphorylates Ume6p, as well. Amino acid substitutions in Ume6p that alter a consensus GSK3 site reduce or abolish Rim11p-Ume6p interaction and Rim11p-dependent phosphorylation, and they cause defects in interaction between Ume6p and Ime1p and in meiotic gene expression. Therefore, interaction between Rim11p and Ume6p, resulting in phosphorylation of Ume6p, is required for Ime1p-Ume6p complex formation. Rim11p, like metazoan GSK3beta, phosphorylates both interacting subunits of a target protein complex. PMID:9372955

  2. Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

    PubMed

    Geering, Barbara; Zokouri, Zina; Hürlemann, Samuel; Gerrits, Bertran; Ausländer, David; Britschgi, Adrian; Tschan, Mario P; Simon, Hans-Uwe; Fussenegger, Martin

    2016-01-01

    Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions. PMID:26483415

  3. Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

    PubMed Central

    Lageix, Sebastien; Rothenburg, Stefan; Dever, Thomas E.; Hinnebusch, Alan G.

    2014-01-01

    The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn− substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd− substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd− substitutions enhance YKD-KD interactions in vitro, whereas Gcn− substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd− substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD. PMID:24811037

  4. Enhanced interaction between pseudokinase and kinase domains in Gcn2 stimulates eIF2α phosphorylation in starved cells.

    PubMed

    Lageix, Sebastien; Rothenburg, Stefan; Dever, Thomas E; Hinnebusch, Alan G

    2014-05-01

    The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn- substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd- substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd- substitutions enhance YKD-KD interactions in vitro, whereas Gcn- substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd- substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD. PMID:24811037

  5. A frequent kinase domain mutation that changes the interaction between PI3K[alpha] and the membrane

    SciTech Connect

    Mandelker, Diana; Gabelli, Sandra B.; Schmidt-Kittler, Oleg; Zhu, Jiuxiang; Cheong, Ian; Huang, Chuan-Hsiang; Kinzler, Kenneth W.; Vogelstein, Bert; Amzel, L. Mario

    2009-12-01

    Mutations in oncogenes often promote tumorigenesis by changing the conformation of the encoded proteins, thereby altering enzymatic activity. The PIK3CA oncogene, which encodes p110{alpha}, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3K{alpha}), is one of the two most frequently mutated oncogenes in human cancers. We report the structure of the most common mutant of p110{alpha} in complex with two interacting domains of its regulatory partner (p85{alpha}), both free and bound to an inhibitor (wortmannin). The N-terminal SH2 (nSH2) domain of p85{alpha} is shown to form a scaffold for the entire enzyme complex, strategically positioned to communicate extrinsic signals from phosphopeptides to three distinct regions of p110{alpha}. Moreover, we found that Arg-1047 points toward the cell membrane, perpendicular to the orientation of His-1047 in the WT enzyme. Surprisingly, two loops of the kinase domain that contact the cell membrane shift conformation in the oncogenic mutant. Biochemical assays revealed that the enzymatic activity of the p110{alpha} His1047Arg mutant is differentially regulated by lipid membrane composition. These structural and biochemical data suggest a previously undescribed mechanism for mutational activation of a kinase that involves perturbation of its interaction with the cellular membrane.

  6. Simultaneous analysis of multiple neurotransmitters by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry.

    PubMed

    Tufi, Sara; Lamoree, Marja; de Boer, Jacob; Leonards, Pim

    2015-05-22

    Neurotransmitters are endogenous metabolites that allow the signal transmission across neuronal synapses. Their biological role is crucial for many physiological functions and their levels can be changed by several diseases. Because of their high polarity, hydrophilic interaction liquid chromatography (HILIC) is a promising tool for neurotransmitter analysis. Due to the large number of HILIC stationary phases available, an evaluation of the column performances and retention behaviors has been performed on five different commercial HILIC packing materials (silica, amino, amide and two zwitterionic stationary phases). Several parameters like the linear correlation between retention and the distribution coefficient (logD), the separation factor k and the column resolution Rs have been investigated and the column performances have been visualized with a heat map and hierarchical clustering analysis. An optimized and validated HILIC-MS/MS method based on the ZIC-cHILIC column is proposed for the simultaneous detection and quantification of twenty compounds consisting of neurotransmitters, precursors and metabolites: 3-methoxytyramine (3-MT), 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxy-L-tripthophan, acetylcholine, choline, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, epinephrine, γ-aminobutyric acid (GABA), glutamate, glutamine, histamine, histidine, L-tryptophan, L-tyrosine, norepinephrine, normetanephrine, phenylalanine, serotonin and tyramine. The method was applied to neuronal metabolite profiling of the central nervous system of the freshwater snail Lymnaea stagnalis. This method is suitable to explore neuronal metabolism and its alteration in different biological matrices. PMID:25869798

  7. The effects of knockdown of rho-associated kinase 1 and zipper-interacting protein kinase on gene expression and function in cultured human arterial smooth muscle cells.

    PubMed

    Deng, Jing-Ti; Wang, Xiu-Ling; Chen, Yong-Xiang; O'Brien, Edward R; Gui, Yu; Walsh, Michael P

    2015-01-01

    Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and

  8. The Effects of Knockdown of Rho-Associated Kinase 1 and Zipper-Interacting Protein Kinase on Gene Expression and Function in Cultured Human Arterial Smooth Muscle Cells

    PubMed Central

    Deng, Jing-Ti; Wang, Xiu-Ling; Chen, Yong-Xiang; O’Brien, Edward R.; Gui, Yu; Walsh, Michael P.

    2015-01-01

    Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and

  9. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    PubMed

    Borthwick, Lee A; Kerbiriou, Mathieu; Taylor, Christopher J; Cozza, Giorgio; Lascu, Ioan; Postel, Edith H; Cassidy, Diane; Trouvé, Pascal; Mehta, Anil; Robson, Louise; Muimo, Richmond

    2016-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia. PMID:26950439

  10. Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

    PubMed

    Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

    2016-02-12

    The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. PMID:26668315

  11. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions.

    PubMed Central

    Reszka, A A; Bulinski, J C; Krebs, E G; Fischer, E H

    1997-01-01

    The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that act as key components in a signaling cascade linking growth factor receptors to the cytoskeleton and the nucleus. ERK2 mutants have been used to alter cytoskeletal regulation in Chinese hamster ovary cells without affecting cell growth or feedback signaling. Mutation of the unique loop L6 (residues 91-95), which is in a portion of the molecule that is cryptic upon the binding of ERK2 to the microtubules (MTs), generated significant morphological alterations. Most notable phenotypes were observed after expression of a combined mutant incorporating changes to both L6 and the TEY phosphorylation lip, including a 70% increase in cell spreading. Actin stress fibers in these cells, which normally formed a single broad parallel array, were arranged in three or more orientations or in fan-like arrays. MTs, which ordinarily extend longitudinally from the centrosome, spread radially, covering a larger surface area. Single, but not the double, mutations of the Thr and Tyr residues of the TEY phosphorylation lip caused a ca. 25% increase in cell spreading, accompanied by a threefold increase in chemotactic cell migration. Mutation of Lys-52 triggered a 48% increase in cell spreading but no alteration to chemotaxis. These findings suggest that wild-type ERK2 inhibits the organization of the cytoskeleton, the spreading of the cell, and chemotactic migration. This involves control of the orientation of actin and MTs and the positioning of focal adhesions via regulatory interactions that may occur on the MTs. Images PMID:9243503

  12. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A

    PubMed Central

    Borthwick, Lee A.; Kerbiriou, Mathieu; Taylor, Christopher J.; Cozza, Giorgio; Lascu, Ioan; Postel, Edith H.; Cassidy, Diane; Trouvé, Pascal; Mehta, Anil; Robson, Louise; Muimo, Richmond

    2016-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36–54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351–727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia. PMID:26950439

  13. Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing

    PubMed Central

    Yang, Weng-Lang; Wang, Zhimin; Nicastro, Jeffrey; Coppa, Gene F.; Wang, Ping

    2015-01-01

    Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression

  14. Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing.

    PubMed

    Godwin, Andrew; Sharma, Archna; Yang, Weng-Lang; Wang, Zhimin; Nicastro, Jeffrey; Coppa, Gene F; Wang, Ping

    2015-01-01

    Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression

  15. Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase.

    PubMed Central

    Yoakim, M; Hou, W; Liu, Y; Carpenter, C L; Kapeller, R; Schaffhausen, B S

    1992-01-01

    The binding of phosphatidylinositol-3-kinase to the polyomavirus middle T antigen is facilitated by tyrosine phosphorylation of middle T on residue 315. The pp85 subunit of phosphatidylinositol-3-kinase contains two SH2 domains, one in the middle of the molecule and one at the C terminus. When assayed by blotting with phosphorylated middle T, the more N-terminal SH2 domain is responsible for binding to middle T. When assayed in solution with glutathione S transferase fusions, both SH2s are capable of binding phosphorylated middle T. While both SH2 fusions can compete with intact pp85 for binding to middle T, the C-terminal SH2 is the more efficient of the two. Interaction between pp85 or its SH2 domains and middle T can be blocked by a synthetic peptide comprising the tyrosine phosphorylation sequence around middle T residue 315. Despite the fact that middle T can interact with both SH2s, these domains are not equivalent. Only the C-terminal SH2-middle T interaction was blocked by anti-SH2 antibody; the two SH2 fusions also interact with different cellular proteins. Images PMID:1380095

  16. Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization.

    PubMed

    Valero, María Llanos; Sabariegos, Rosario; Cimas, Francisco J; Perales, Celia; Domingo, Esteban; Sánchez-Prieto, Ricardo; Mas, Antonio

    2016-06-01

    Hepatitis C virus (HCV) interacts with cellular components and modulates their activities for its own benefit. These interactions have been postulated as a target for antiviral treatment, and some candidate molecules are currently in clinical trials. The multifunctional cellular kinase Akt/protein kinase B (PKB) must be activated to increase the efficacy of HCV entry but is rapidly inactivated as the viral replication cycle progresses. Viral components have been postulated to be responsible for Akt/PKB inactivation, but the underlying mechanism remained elusive. In this study, we show that HCV polymerase NS5B interacts with Akt/PKB. In the presence of transiently expressed NS5B or in replicon- or virus-infected cells, NS5B changes the cellular localization of Akt/PKB from the cytoplasm to the perinuclear region. Sequestration of Akt/PKB by NS5B could explain its exclusion from its participation in early Akt/PKB inactivation. The NS5B-Akt/PKB interaction represents a new regulatory step in the HCV infection cycle, opening possibilities for new therapeutic options. PMID:27021315

  17. Mitogen-activated protein kinase p38b interaction with delta class glutathione transferases from the fruit fly, Drosophila melanogaster.

    PubMed

    Wongtrakul, Jeerang; Sukittikul, Suchada; Saisawang, Chonticha; Ketterman, Albert J

    2012-01-01

    Glutathione transferases (GSTs) are a family of multifunctional enzymes involved in xenobiotic biotransformation, drug metabolism, and protection against oxidative damage. The p38b mitogen-activated protein kinase is involved in cellular stress response. This study screened interactions between Drosophila melanogaster Meigen (Diptera: Drosophilidae) Delta class glutathione transferases (DmGSTs) and the D. melanogaster p38b MAPK. Therefore, 12 DmGSTs and p38b kinase were obtained as recombinant proteins. The study showed that DmGSTD8 and DmGSTD11b significantly increased p38b activity toward ATF2 and jun, which are transcription factor substrates. DmGSTD3 and DmGSTD5 moderately increased p38b activity for jun. In addition, GST activity in the presence of p38b was also measured. It was found that p38b affected substrate specificity toward CDNB (1-chloro-2,4-dinitrobenzene) and DCNB (1,2-dichloro-4-nitrobenzene) of several GST isoforms, i.e., DmGSTD2, DmGSTD5, DmGSTD8, and DmGSTD11b. The interaction of a GST and p38b can affect the substrate specificity of either enzyme, which suggests induced conformational changes affecting catalysis. Similar interactions do not occur for all the Delta enzymes and p38b, which suggests that these interactions could be specific. PMID:23438069

  18. Protein Kinase C ζ Interacts with a Novel Binding Region of Gαq to Act as a Functional Effector.

    PubMed

    Sánchez-Fernández, Guzmán; Cabezudo, Sofía; Caballero, Álvaro; García-Hoz, Carlota; Tall, Gregory G; Klett, Javier; Michnick, Stephen W; Mayor, Federico; Ribas, Catalina

    2016-04-29

    Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. We have recently reported that GPCR activation promotes a direct interaction between Gαq and protein kinase C ζ (PKCζ), leading to the stimulation of the ERK5 pathway independent of the canonical effector PLCβ. We report herein that the activation-dependent Gαq/PKCζ complex involves the basic PB1-type II domain of PKCζ and a novel interaction module in Gαq different from the classical effector-binding site. Point mutations in this Gαq region completely abrogate ERK5 phosphorylation, indicating that Gαq/PKCζ association is required for the activation of the pathway. Indeed, PKCζ was demonstrated to directly bind ERK5 thus acting as a scaffold between Gαq and ERK5 upon GPCR activation. The inhibition of these protein complexes by G protein-coupled receptor kinase 2, a known Gαq modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal that Gαq/PKCζ complexes link Gαq to apoptotic cell death pathways. Our data suggest that the interaction between this novel region in Gαq and the effector PKCζ is a key event in Gαq signaling. PMID:26887939

  19. Protein kinase CK2 interacts with Chk2 and phosphorylates Mre11 on serine 649

    SciTech Connect

    Kim, Seong-Tae . E-mail: stkim@med.skku.ac.kr

    2005-05-27

    The Mre11-Rad50-Nbs1 protein complex has been known to be involved in a variety of DNA metabolic events that involve DNA double-strand breaks (DSBs). The phosphorylation of Mre11 is increased in response to ionizing radiation, which suggests that phosphorylation of Mre11 may be an important regulatory mechanism of this complex. Mre11-phosphorylating kinase activities were observed in Chk2 immunoprecipitates and HeLa nuclear extracts. Through the tandem affinity tagging system and conventional chromatography, this kinase was purified and identified as protein kinase CK2. CK2 phosphorylates Mre11 in vitro. In vitro kinase assay with a series of truncated Mre11 proteins as substrates for CK2 and site-directed mutagenesis showed that serine 649 of Mre11 is mainly phosphorylated by CK2 in vitro. In vivo labeling and phosphopeptide mapping analysis revealed that this phosphorylation occurs in vivo. These data implicate CK2 as a potential upstream regulator of Mre11 function.

  20. Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity*

    PubMed Central

    Hughes, Siobhan; Jenkins, Victoria; Dar, Mohd Jamal; Engelman, Alan; Cherepanov, Peter

    2010-01-01

    Lens epithelium-derived growth factor (LEDGF) is an important co-factor of human immunodeficiency virus DNA integration; however, its cellular functions are poorly characterized. We now report identification of the Cdc7-activator of S-phase kinase (ASK) heterodimer as a novel interactor of LEDGF. Both kinase subunits co-immunoprecipitated with endogenous LEDGF from human cell extracts. Truncation analyses identified the integrase-binding domain of LEDGF as essential and minimally sufficient for the interaction with Cdc7-ASK. Reciprocally, the interaction required autophosphorylation of the kinase and the presence of 50 C-terminal residues of ASK. The kinase phosphorylated LEDGF in vitro, with Ser-206 being the major target, and LEDGF phosphorylated at this residue could be detected during S phase of the cell cycle. LEDGF potently stimulated the enzymatic activity of Cdc7-ASK, increasing phosphorylation of MCM2 in vitro by more than 10-fold. This enzymatic stimulation as well as phosphorylation of LEDGF depended on the protein-protein interaction. Intriguingly, removing the C-terminal region of ASK, involved in the interaction with LEDGF, resulted in a hyperactive kinase. Our results indicate that the interaction with LEDGF relieves autoinhibition of Cdc7-ASK kinase, imposed by the C terminus of ASK. PMID:19864417

  1. Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase.

    PubMed Central

    Pleiman, C M; Clark, M R; Gauen, L K; Winitz, S; Coggeshall, K M; Johnson, G L; Shaw, A S; Cambier, J C

    1993-01-01

    Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly. Images PMID:8395016

  2. Interaction of cCMP with the cGK, cAK and MAPK Kinases in Murine Tissues

    PubMed Central

    Wolfertstetter, Stefanie; Reinders, Jörg; Schwede, Frank; Ruth, Peter; Schinner, Elisabeth; Schlossmann, Jens

    2015-01-01

    cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-32P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung. PMID:25978317

  3. Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways.

    PubMed

    Guan, Hongtao; Shuaib, Aban; Leon, David Davila De; Angyal, Adrienn; Salazar, Maria; Velasco, Guillermo; Holcombe, Mike; Dower, Steven K; Kiss-Toth, Endre

    2016-01-01

    Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways. PMID:27600771

  4. Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways

    PubMed Central

    Guan, Hongtao; Shuaib, Aban; Leon, David Davila De; Angyal, Adrienn; Salazar, Maria; Velasco, Guillermo; Holcombe, Mike; Dower, Steven K.; Kiss-Toth, Endre

    2016-01-01

    Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways. PMID:27600771

  5. Dehydroglyasperin D Inhibits the Proliferation of HT-29 Human Colorectal Cancer Cells Through Direct Interaction With Phosphatidylinositol 3-kinase

    PubMed Central

    Jung, Sung Keun; Jeong, Chul-Ho

    2016-01-01

    Background: Despite recent advances in therapy, colorectal cancer still has a grim prognosis. Although licorice has been used in East Asian traditional medicine, the molecular properties of its constituents including dehydroglyasperin D (DHGA-D) remain unknown. We sought to evaluate the inhibitory effect of DHGA-D on colorectal cancer cell proliferation and identify the primary signaling molecule targeted by DHGA-D. Methods: We evaluated anchorage-dependent and -independent cell growth in HT-29 human colorectal adenocarcinoma cells. The target protein of DHGA-D was identified by Western blot analysis with a specific antibody, and direct interaction between DHGA-D and the target protein was confirmed by kinase and pull-down assays. Cell cycle analysis by flow cytometry and further Western blot analysis was performed to identify the signaling pathway involved. Results: DHGA-D significantly suppressed anchorage-dependent and -independent HT-29 colorectal cancer cell proliferation. DHGA-D directly suppressed phosphatidylinositol 3-kinase (PI3K) activity and subsequent Akt phosphorylation and bound to the p110 subunit of PI3K. DHGA-D also significantly induced G1 cell cycle arrest, together with the suppression of glycogen synthase kinase 3β and retinoblastoma phosphorylation and cyclin D1 expression. Conclusions: DHGA-D has potent anticancer activity and targets PI3K in human colorectal adenocarcinoma HT-29 cells. To our knowledge, this is the first report to detail the molecular basis of DHGA-D in suppressing colorectal cancer cell growth. PMID:27051646

  6. Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1.

    PubMed

    Benzeno, Sharon; Narla, Goutham; Allina, Jorge; Cheng, George Z; Reeves, Helen L; Banck, Michaela S; Odin, Joseph A; Diehl, J Alan; Germain, Doris; Friedman, Scott L

    2004-06-01

    Kruppel-like factor 6 (KLF6) is a tumor suppressor gene inactivated in prostate and colon cancers, as well as in astrocytic gliomas. Here, we establish that KLF6 mediates growth inhibition through an interaction with cyclin D1, leading to reduced phosphorylation of the retinoblastoma protein (Rb) at Ser(795). Furthermore, introduction of KLF6 disrupts cyclin D1-cyclin-dependent kinase (cdk) 4 complexes and forces the redistribution of p21(Cip/Kip) onto cdk2, which promotes G(1) cell cycle arrest. Our data suggest that KLF6 converges with the Rb pathway to inhibit cyclin D1/cdk4 activity, resulting in growth suppression. PMID:15172998

  7. Selective Phosphorylation Inhibitor of Delta Protein Kinase C-Pyruvate Dehydrogenase Kinase Protein-Protein Interactions: Application for Myocardial Injury in Vivo.

    PubMed

    Qvit, Nir; Disatnik, Marie-Hélène; Sho, Eiketsu; Mochly-Rosen, Daria

    2016-06-22

    Protein kinases regulate numerous cellular processes, including cell growth, metabolism, and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set out to develop an inhibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an in vitro Kd of ∼50 nM and reducing cardiac injury IC50 of ∼5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 μM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors. PMID:27218445

  8. Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

    PubMed

    Yamada, T; Takeuchi, S; Fujita, N; Nakamura, A; Wang, W; Li, Q; Oda, M; Mitsudomi, T; Yatabe, Y; Sekido, Y; Yoshida, J; Higashiyama, M; Noguchi, M; Uehara, H; Nishioka, Y; Sone, S; Yano, S

    2013-09-12

    Despite initial dramatic response, epidermal growth factor receptor (EGFR) mutant lung cancer patients always acquire resistance to EGFR-tyrosine kinase inhibitors (TKIs). Gatekeeper T790M mutation in EGFR is the most prevalent genetic alteration underlying acquired resistance to EGFR-TKI, and EGFR mutant lung cancer cells are reported to be addictive to EGFR/Akt signaling even after acquired T790M mutation. Here, we focused on Akt kinase-interacting protein1 (Aki1), a scaffold protein of PI3K (phosphoinositide 3-kinase)/PDK1 (3-phosphoinositide-dependent protein kinase)/Akt that determines receptor signal selectivity for non-mutated EGFR, and assessed its role in EGFR mutant lung cancer with or without gatekeeper T790M mutation. Cell line-based assays showed that Aki1 constitutively associates with mutant EGFR in lung cancer cells with (H1975) or without (PC-9 and HCC827) T790M gatekeeper mutation. Silencing of Aki1 induced apoptosis of EGFR mutant lung cancer cells. Treatment with Aki1 siRNA dramatically inhibited growth of H1975 cells in a xenograft model. Moreover, silencing of Aki1 further potentiated growth inhibitory effect of new generation EGFR-TKIs against H1975 cells in vitro. Aki1 was frequently expressed in tumor cells of EGFR mutant lung cancer patients (53/56 cases), including those with acquired resistance to EGFR-TKI treatment (7/7 cases). Our data suggest that Aki1 may be a critical mediator of survival signaling from mutant EGFR to Akt, and may therefore be an ideal target for EGFR mutant lung cancer patients, especially those with acquired EGFR-TKI resistance due to EGFR T790M gatekeeper mutation. PMID:23045273

  9. Protein kinase A modulates transforming growth factor-β signaling through a direct interaction with Smad4 protein.

    PubMed

    Yang, Huibin; Li, Gangyong; Wu, Jing-Jiang; Wang, Lidong; Uhler, Michael; Simeone, Diane M

    2013-03-22

    Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo. PMID:23362281

  10. Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration.

    PubMed

    Zurnic, Irena; Hütter, Sylvia; Rzeha, Ute; Stanke, Nicole; Reh, Juliane; Müllers, Erik; Hamann, Martin V; Kern, Tobias; Gerresheim, Gesche K; Lindel, Fabian; Serrao, Erik; Lesbats, Paul; Engelman, Alan N; Cherepanov, Peter; Lindemann, Dirk

    2016-08-01

    Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells. PMID:27579920

  11. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    NASA Technical Reports Server (NTRS)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  12. Interaction and cooperation of the CCAAT-box enhancer-binding protein β (C/EBPβ) with the homeodomain-interacting protein kinase 2 (Hipk2).

    PubMed

    Steinmann, Simone; Coulibaly, Anna; Ohnheiser, Johanna; Jakobs, Anke; Klempnauer, Karl-Heinz

    2013-08-01

    CCAAT box/enhancer-binding protein β (C/EBPβ) is a bZip transcription factor that plays crucial roles in important cellular processes such as differentiation and proliferation of specific cell types. Previously, we showed that C/EBPβ cooperates with the coactivator p300 through a novel mechanism that involves the C/EBPβ-induced phosphorylation of multiple sites in the carboxyl-terminal domain of p300 by protein kinase Hipk2. We have now examined the interaction and cooperation of C/EBPβ, p300, and Hipk2 in more detail. We show that Hipk2 and C/EBPβ are direct physical binding partners whose interaction is mediated by sequences located in the amino-terminal and central domains of Hipk2 and the amino-terminal part of C/EBPβ. In addition to phosphorylating p300 recruited to C/EBPβ, Hipk2 also phosphorylates C/EBPβ at sites that have previously been shown to plays key roles in the regulation of C/EBPβ activity. Silencing of Hipk2 expression disrupts adipocyte differentiation of 3T3-L1 cells, a physiological C/EBPβ-dependent differentiation process indicating that the cooperation of C/EBPβ and Hipk2 is functionally relevant. Finally, we demonstrate that C/EBPα, a related C/EBP family member whose amino-terminal sequences differ significantly from that of C/EBPβ, is unable to interact and cooperate with Hipk2. Instead, our data suggest that C/EBPα cooperates with the protein kinase Jnk to induce phosphorylation of p300. Overall, our data identify Hipk2 as a novel regulator of C/EBPβ and implicate different protein kinases in the cooperation of p300 with C/EBPβ and C/EBPα. PMID:23782693

  13. Rapeseed calcineurin B-like protein CBL4, interacting with CBL-interacting protein kinase CIPK24, modulates salt tolerance in plants.

    PubMed

    Liu, Wu-Zhen; Deng, Min; Li, Liang; Yang, Bo; Li, Hongwei; Deng, Hanqing; Jiang, Yuan-Qing

    2015-11-20

    Calcium is a ubiquitous intracellular secondary messenger in eukaryotes. Upon stress challenge, cytosolic Ca(2+) fluctuation could be sensed and bound by calcineurin B-like proteins (CBLs), which further regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs) to relay the signal and induce cellular responses. Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in rapeseed. In the present study, we characterized CBL4 gene from rapeseed. We found that CBL4 is localized at the plasma membrane and it interacted with CIPK24 in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Unlike the orthologs in Arabidopsis, rapeseed CIPK24 did not interact with CBL10. Furthermore, expression of rapeseed CBL4 rescued the salt-sensitive phenotype of sos3-1 mutant and overexpression of rapeseed CBL4 in Arabidopsis showed enhanced tolerance of salt stress than wild-type. Overall, the results clarified the function of CBL4 in rapeseed. PMID:26462466

  14. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    PubMed

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein. PMID:27231351

  15. Specific chlamydial inclusion membrane proteins associate with active Src family kinases in microdomains that interact with the host microtubule network

    PubMed Central

    Mital, Jeffrey; Miller, Natalie J.; Fischer, Elizabeth R.; Hackstadt, Ted

    2010-01-01

    Summary Chlamydiae are gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impact. Chlamydia trachomatis replicates within a vacuole termed an inclusion, which is extensively modified by the insertion of a number of bacterial effector proteins known as inclusion membrane proteins (Incs). Once modified, the inclusion is trafficked in a dynein-dependent manner to the microtubule organizing center (MTOC), where it associates with host centrosomes. Here we describe a novel structure on the inclusion membrane comprised of both host and bacterial proteins. Members of the Src family of kinases are recruited to the chlamydial inclusion in an active form. These kinases display a distinct, localized punctate microdomain-like staining pattern on the inclusion membrane that colocalizes with four chlamydial inclusion membrane proteins (Incs) and is enriched in cholesterol. Biochemical studies show that at least two of these Incs stably interact with one another. Furthermore, host centrosomes associate with these microdomain proteins in C. trachomatis-infected cells and in uninfected cells exogenously expressing one of the chlamydial effectors. Together, the data suggest that a specific structure on the C. trachomatis inclusion membrane may be responsible for the known interactions of chlamydiae with the microtubule network and resultant effects on centrosome stability. PMID:20331642

  16. Metabolism-related pharmacokinetic drug−drug interactions with tyrosine kinase inhibitors: current understanding, challenges and recommendations

    PubMed Central

    Teo, Yi Ling; Ho, Han Kiat; Chan, Alexandre

    2015-01-01

    Drug−drug interactions (DDIs) occur when a patient's response to the drug is modified by administration or co-exposure to another drug. The main cytochrome P450 (CYP) enzyme, CYP3A4, is implicated in the metabolism of almost all of the tyrosine kinase inhibitors (TKIs). Therefore, there is a substantial potential for interaction between TKIs and other drugs that modulate the activity of this metabolic pathway. Cancer patients are susceptible to DDIs as they receive many medications, either for supportive care or for treatment of toxicity. Differences in DDI outcomes are generally negligible because of the wide therapeutic window of common drugs. However for anticancer agents, serious clinical consequences may occur from small changes in drug metabolism and pharmacokinetics. Therefore, the objective of this review is to highlight the current understanding of DDIs among TKIs, with a focus on metabolism, as well as to identify challenges in the prediction of DDIs and provide recommendations. PMID:25125025

  17. Simultaneous Mapping of Interactions between Scientific and Technological Knowledge Bases: The Case of Space Communications.

    ERIC Educational Resources Information Center

    Hassan, E.

    2003-01-01

    Examines the knowledge structure of the field of space communications using bibliometric mapping techniques based on textual analysis. Presents a new approach with the aim of visualizing simultaneously the configuration of the scientific and technological knowledge bases at a worldwide level, and discusses results that show different…

  18. Selective glucocorticoid control of Rho kinase isoforms regulate cell-cell interactions

    PubMed Central

    Rubenstein, Nicola M.; Callahan, Joseph A.; Lo, Daniel H.; Firestone, Gary L.

    2007-01-01

    The two Rho kinase isoforms ROCK1 and ROCK2 are downstream effectors of the small GTPase RhoA, although relatively little is known about potential isoform specific functions or the selective control of their cellular activities. Using Con8 rat mammary epithelial cells, we show that the synthetic glucocorticoid dexamethasone strongly stimulates the level of ROCK2 protein, which accounts for the increase in total cellular ROCK2 activity, whereas, steroid treatment down-regulated ROCK1 specific kinase activity without altering ROCK1 protein levels. In Con8 cells, the glucocorticoid induced formation of tight junctions requires the steroid-mediated down-regulation RhoA and function of the RhoA antagonist Rnd3. Treatment with the ROCK inhibitor Y-27632 ablated both the glucocorticoid-induced and Rnd3-mediated stimulation in tight junction sealing. Taken together, our results demonstrate that the expression and activity of ROCK1 and ROCK2 can be uncoupled in a signal-dependent manner, and further implicate a new function for ROCK2 in the steroid control of tight junction dynamics. PMID:17240358

  19. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    PubMed Central

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  20. Interactions between Eph kinases and ephrins provide a mechanism to support platelet aggregation once cell-to-cell contact has occurred

    PubMed Central

    Prevost, Nicolas; Woulfe, Donna; Tanaka, Takako; Brass, Lawrence F.

    2002-01-01

    Eph kinases are receptor tyrosine kinases whose ligands, the ephrins, are also expressed on the surface of cells. Interactions between Eph kinases and ephrins on adjacent cells play a central role in neuronal patterning and vasculogenesis. Here we examine the expression of ephrins and Eph kinases on human blood platelets and explore their role in the formation of the hemostatic plug. The results show that human platelets express EphA4 and EphB1, and the ligand, ephrinB1. Forced clustering of EphA4 or ephrinB1 led to cytoskeletal reorganization, adhesion to fibrinogen, and α-granule secretion. Clustering of ephrinB1 also caused activation of the Ras family member, Rap1B. In platelets that had been activated by ADP and allowed to aggregate, EphA4 formed complexes with two tyrosine kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Blockade of Eph/ephrin interactions prevented the formation of these complexes and caused platelet aggregation at low ADP concentrations to become more readily reversible. We propose that when sustained contacts between platelets have occurred in response to agonists such as collagen, ADP, and thrombin, the binding of ephrins to Eph kinases on adjacent platelets provides a mechanism to perpetuate signaling and promote stable platelet aggregation. PMID:12084815

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

    PubMed Central

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

    2014-01-01

    Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading and that this association can be regulated by factors in the tumor microenvironment. PMID:24219282

  2. Phosphorylation of the amino-terminus of the AGC kinase Gad8 prevents its interaction with TORC2.

    PubMed

    Du, Wei; Forte, Gabriella M; Smith, Duncan; Petersen, Janni

    2016-03-01

    Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKC(Pck2) expression prevented Gad8-TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress. PMID:26935949

  3. Negative regulation of beta4 integrin transcription by homeodomain-interacting protein kinase 2 and p53 impairs tumor progression.

    PubMed

    Bon, Giulia; Di Carlo, Selene E; Folgiero, Valentina; Avetrani, Paolo; Lazzari, Chiara; D'Orazi, Gabriella; Brizzi, Maria Felice; Sacchi, Ada; Soddu, Silvia; Blandino, Giovanni; Mottolese, Marcella; Falcioni, Rita

    2009-07-15

    Increased expression of alpha(6)beta(4) integrin in several epithelial cancers promotes tumor progression; however, the mechanism underlying its transcriptional regulation remains unclear. Here, we show that depletion of homeodomain-interacting protein kinase 2 (HIPK2) activates beta(4) transcription that results in a strong increase of beta(4)-dependent mitogen-activated protein kinase and Akt phosphorylation, anchorage-independent growth, and invasion. In contrast, stabilization of HIPK2 represses beta(4) expression in wild-type p53 (wtp53)-expressing cells but not in p53-null cells or cells expressing mutant p53, indicating that HIPK2 requires a wtp53 to inhibit beta(4) transcription. Consistent with our in vitro findings, a strong correlation between beta(4) overexpression and HIPK2 inactivation by cytoplasmic relocalization was observed in wtp53-expressing human breast carcinomas. Under loss of function of HIPK2 or p53, the p53 family members TAp63 and TAp73 strongly activate beta(4) transcription. These data, by revealing that beta(4) expression is transcriptionally repressed in tumors by HIPK2 and p53 to impair beta(4)-dependent tumor progression, suggest that loss of p53 function favors the formation of coactivator complex with the TA members of the p53 family to allow beta(4) transcription. PMID:19567674

  4. Homeodomain-interacting protein kinase 2 (HIPK2) targets beta-catenin for phosphorylation and proteasomal degradation.

    PubMed

    Kim, Eun-A; Kim, Ji Eon; Sung, Ki Sa; Choi, Dong Wook; Lee, Byeong Jae; Choi, Cheol Yong

    2010-04-16

    The regulation of intracellular beta-catenin levels is central in the Wnt/beta-catenin signaling cascade and the activation of the Wnt target genes. Here, we show that homeodomain-interacting protein kinase 2 (HIPK2) acts as a negative regulator of the Wnt/beta-catenin pathway. Knock-down of endogenous HIPK2 increases the stability of beta-catenin and results in the accumulation of beta-catenin in the nucleus, consequently enhancing the expression of Wnt target genes and cell proliferation both in vivo and in cultured cells. HIPK2 inhibits TCF/LEF-mediated target gene activation via degradation of beta-catenin. HIPK2 phosphorylates beta-catenin at its Ser33 and Ser37 residues without the aid of a priming kinase. Substitutions of Ser33 and Ser37 for alanines abolished the degradation of beta-catenin associated with HIPK2. In ex vivo mouse model, HIPK2 knock-down resulted in accumulation of beta-catenin, thereby potentiated beta-catenin-mediated cell proliferation and tumor formation. Furthermore, the axis duplication induced by the ectopic expression of beta-catenin was blocked by co-injection of HIPK2 mRNAs into Xenopus embryos. Taken together, HIPK2 appears to function as a novel negative regulator of beta-catenin through its phosphorylation and proteasomal degradation. PMID:20307497

  5. Homeodomain-interacting protein kinase 2 (HIPK2) targets {beta}-catenin for phosphorylation and proteasomal degradation

    SciTech Connect

    Kim, Eun-A; Kim, Ji Eon; Sung, Ki Sa; Choi, Dong Wook; Lee, Byeong Jae; Choi, Cheol Yong

    2010-04-16

    The regulation of intracellular {beta}-catenin levels is central in the Wnt/{beta}-catenin signaling cascade and the activation of the Wnt target genes. Here, we show that homeodomain-interacting protein kinase 2 (HIPK2) acts as a negative regulator of the Wnt/{beta}-catenin pathway. Knock-down of endogenous HIPK2 increases the stability of {beta}-catenin and results in the accumulation of {beta}-catenin in the nucleus, consequently enhancing the expression of Wnt target genes and cell proliferation both in vivo and in cultured cells. HIPK2 inhibits TCF/LEF-mediated target gene activation via degradation of {beta}-catenin. HIPK2 phosphorylates {beta}-catenin at its Ser33 and Ser37 residues without the aid of a priming kinase. Substitutions of Ser33 and Ser37 for alanines abolished the degradation of {beta}-catenin associated with HIPK2. In ex vivo mouse model, HIPK2 knock-down resulted in accumulation of {beta}-catenin, thereby potentiated {beta}-catenin-mediated cell proliferation and tumor formation. Furthermore, the axis duplication induced by the ectopic expression of {beta}-catenin was blocked by co-injection of HIPK2 mRNAs into Xenopus embryos. Taken together, HIPK2 appears to function as a novel negative regulator of {beta}-catenin through its phosphorylation and proteasomal degradation.

  6. Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity

    SciTech Connect

    Kiburu, Irene N.; LaRonde-LeBlanc, Nicole

    2012-10-10

    Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 {angstrom} and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity.

  7. Phosphorylation of the amino-terminus of the AGC kinase Gad8 prevents its interaction with TORC2

    PubMed Central

    Du, Wei; Forte, Gabriella M.; Smith, Duncan; Petersen, Janni

    2016-01-01

    Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKCPck2 expression prevented Gad8–TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress. PMID:26935949

  8. The Fer tyrosine kinase regulates interactions of Rho GDP-Dissociation Inhibitor α with the small GTPase Rac

    PubMed Central

    2010-01-01

    Background RhoGDI proteins are important regulators of the small GTPase Rac, because they shuttle Rac from the cytoplasm to membranes and also protect Rac from activation, deactivation and degradation. How the binding and release of Rac from RhoGDI is regulated is not precisely understood. Results We report that the non-receptor tyrosine kinase Fer is able to phosphorylate RhoGDIα and form a direct protein complex with it. This interaction is mediated by the C-terminal end of RhoGDIα. Activation of Fer by reactive oxygen species caused increased phosphorylation of RhoGDIα and pervanadate treatment further augmented this. Tyrosine phosphorylation of RhoGDIα by Fer prevented subsequent binding of Rac to RhoGDIα, but once a RhoGDIα-Rac complex was formed, the Fer kinase was not able to cause Rac release through tyrosine phosphorylation of preformed RhoGDIα-Rac complexes. Conclusions These results identify tyrosine phosphorylation of RhoGDIα by Fer as a mechanism to regulate binding of RhoGDIα to Rac. PMID:21122136

  9. MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells.

    PubMed

    Mukherjee, P; Tinder, T L; Basu, G D; Gendler, S J

    2005-01-01

    MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex. PMID:15513966

  10. Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2.

    PubMed

    Tapia, Julio C; Bolanos-Garcia, Victor M; Sayed, Muhammed; Allende, Catherine C; Allende, Jorge E

    2004-04-01

    The protein kinase CK2 is constituted by two catalytic (alpha and/or alpha') and two regulatory (beta) subunits. CK2 phosphorylates more than 300 proteins with important functions in the cell cycle. This study has looked at the relation between CK2 and p27(KIP1), which is a regulator of the cell cycle and a known inhibitor of cyclin-dependent kinases (Cdk). We demonstrated that in vitro recombinant Xenopus laevis CK2 can phosphorylate recombinant human p27(KIP1), but this phosphorylation occurs only in the presence of the regulatory beta subunit. The principal site of phosphorylation is serine-83. Analysis using pull down and surface plasmon resonance (SPR) techniques showed that p27(KIP1) interacts with the beta subunit through two domains present in the amino and carboxyl ends, while CD spectra showed that p27(KIP1) phosphorylation by CK2 affects its secondary structure. Altogether, these results suggest that p27(KIP1) phosphorylation by CK2 probably involves a docking event mediated by the CK2beta subunit. The phosphorylation of p27(KIP1) by CK2 may affect its biological activity. PMID:15034923

  11. Gateway Vectors for Simultaneous Detection of Multiple Protein−Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation

    PubMed Central

    Hikino, Kazumi; Goto-Yamada, Shino; Nishimura, Mikio; Nakagawa, Tsuyoshi; Mano, Shoji

    2016-01-01

    Bimolecular fluorescence complementation (BiFC) is widely used to detect protein—protein interactions, because it is technically simple, convenient, and can be adapted for use with conventional fluorescence microscopy. We previously constructed enhanced yellow fluorescent protein (EYFP)-based Gateway cloning technology-compatible vectors. In the current study, we generated new Gateway cloning technology-compatible vectors to detect BiFC-based multiple protein—protein interactions using N- and C-terminal fragments of enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), and monomeric red fluorescent protein (mRFP1). Using a combination of N- and C-terminal fragments from ECFP, EGFP and EYFP, we observed a shift in the emission wavelength, enabling the simultaneous detection of multiple protein—protein interactions. Moreover, we developed these vectors as binary vectors for use in Agrobacterium infiltration and for the generate transgenic plants. We verified that the binary vectors functioned well in tobacco cells. The results demonstrate that the BiFC vectors facilitate the design of various constructions and are convenient for the detection of multiple protein—protein interactions simultaneously in plant cells. PMID:27490375

  12. Protein Kinase CK2 Interacts at the Neuromuscular Synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 Proteins and Phosphorylates the Latter Two*

    PubMed Central

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-01-01

    Previously, we demonstrated that the protein kinase CK2 associates with and phosphorylates the receptor tyrosine kinase MuSK (muscle specific receptor tyrosine kinase) at the neuromuscular junction (NMJ), thereby preventing fragmentation of the NMJs (Cheusova, T., Khan, M. A., Schubert, S. W., Gavin, A. C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H. R., and Hashemolhosseini, S. (2006) Genes Dev. 20, 1800–1816). Here, we asked whether CK2 interacts with other proteins involved in processes at the NMJ, which would be consistent with the previous observation that CK2 appears enriched at the NMJ. We identified the following proteins to interact with protein kinase CK2: (a) the α and β subunits of the nicotinic acetylcholine receptors with weak interaction, (b) dishevelled (Dsh), and (c) another four proteins, Rapsyn, Rac1, 14-3-3γ, and Dok-7, with strong interaction. CK2 phosphorylated 14-3-3γ at serine residue 235 and Dok-7 at several serine residues but does not phosphorylate Rapsyn or Rac1. Furthermore, phosphomimetic Dok-7 mutants aggregated nicotinic acetylcholine receptors in C2C12 myotubes with significantly higher frequency than wild type Dok-7. Additionally, we mapped the interacting epitopes of all four binding partners to CK2 and thereby gained insights into the potential role of the CK2/Rapsyn interaction. PMID:26198629

  13. Identification of protein phosphatase 2A as an interacting protein of leucine-rich repeat kinase 2.

    PubMed

    Athanasopoulos, Panagiotis S; Jacob, Wright; Neumann, Sebastian; Kutsch, Miriam; Wolters, Dirk; Tan, Eng K; Bichler, Zoë; Herrmann, Christian; Heumann, Rolf

    2016-06-01

    Mutations in the gene coding for the multi-domain protein leucine-rich repeat kinase 2 (LRRK2) are the leading cause of genetically inherited Parkinson's disease (PD). Two of the common found mutations are the R1441C and G2019S. In this study we identified protein phosphatase 2A (PP2A) as an interacting partner of LRRK2. We were able to demonstrate that the Ras of complex protein (ROC) domain is sufficient to interact with the three subunits of PP2A in human neuroblastoma SH-SY5Y cells and in HeLa cells. The alpha subunit of PP2A is interacting with LRRK2 in the perinuclear region of HeLa cells. Silencing the catalytic subunit of PP2A by shRNA aggravated cellular degeneration induced by the pathogenic R1441C-LRRK2 mutant expressed in neuroblastoma SH-SY5Y cells. A similar enhancement of apoptotic nuclei was observed by downregulation of the catalytic subunit of PP2A in cultured cortical cells derived from neurons overexpressing the pathogenic mutant G2019S-LRRK2. Conversely, pharmacological activation of PP2A by sodium selenate showed a partial neuroprotection from R1441C-LRRK2-induced cellular degeneration. All these data suggest that PP2A is a new interacting partner of LRRK2 and reveal the importance of PP2A as a potential therapeutic target in PD. PMID:26894577

  14. A Novel Function for p53: Regulation of Growth Cone Motility through Interaction with Rho Kinase

    PubMed Central

    Qin, Qingyu; Baudry, Michel; Liao, Guanghong; Noniyev, Albert; Galeano, James; Bi, Xiaoning

    2009-01-01

    The transcription factor p53 suppresses tumorgenesis by regulating cell proliferation and migration. We investigated whether p53 could also control cell motility in postmitotic neurons. P53 isoforms recognized by phospho-p53-specific (at Ser15) or “mutant” conformation specific antibodies were highly and specifically expressed in axons and axonal growth cones in primary hippocampal neurons. Inhibition of p53 function by inhibitors, siRNAs, or by dominant negative forms, induced axonal growth cone collapse, whereas p53 over-expression led to larger growth cones. Furthermore, deletion of the p53 nuclear export signal blocked its axonal distribution and induced growth cone collapse. P53 inhibition-induced axonal growth cone collapse was significantly reduced by the Rho kinase (ROCK) inhibitor, Y27632. Our results reveal a new function for p53 as a critical regulator of axonal growth cone behavior by suppressing ROCK activity. PMID:19386914

  15. C-terminus of heat shock protein 70– interacting protein facilitates degradation of apoptosis signal-regulating kinase 1 and inhibits apoptosis signal-regulating kinase 1– dependent apoptosis

    PubMed Central

    Hwang, Jae Ryoung; Zhang, Chunlian; Patterson, Cam

    2005-01-01

    Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is regulated under conditions of cellular stress. ASK1 phosphorylates c-Jun N-terminal kinase (JNK) and elicits an apoptotic response. ASK1 activity is regulated at multiple levels, 1 of which is through inhibition by cytosolic chaperones of the heat shock protein (Hsp) 70 family. Among the proteins that determine Hsp70 function, CHIP (C-terminus of Hsp70-interacting protein) is a cochaperone and ubiquitin ligase that interacts with Hsp70 through an amino-terminal tetratricopeptide repeat (TPR) domain. Prominent among the cellular functions mediated by CHIP is protection against physiologic stress. Because ASK1 is known to contain a TPR-acceptor site, we examined the role of CHIP in regulating ASK1 function. CHIP interacted with ASK1 in a TPR-dependent fashion and induced ubiquitylation and proteasome-dependent degradation of ASK1. Targeting of ASK1 by CHIP inhibited JNK activation in response to oxidative challenge and reduced ASK1-dependent apoptosis, whereas short interfering RNA (siRNA)-dependent depletion of CHIP enhanced JNK activation. Consistent with its ability to reduce cytoplasmic ASK1 levels, CHIP triggered the translocation of ASK1 partner protein death-associated protein (Daxx) into the nucleus, where it is known to activate an antiapoptotic response. These results indicate that CHIP regulates ASK1 activity by inducing its ubiquitylation and degradation, which, together with its effects on Daxx localization, provides a mechanism for the antiapoptotic effects of CHIP observed in the face of cellular and physiologic stress. PMID:16038411

  16. A solar type II radio burst from coronal mass ejection-coronal ray interaction: Simultaneous radio and extreme ultraviolet imaging

    SciTech Connect

    Chen, Yao; Du, Guohui; Feng, Shiwei; Kong, Xiangliang; Wang, Bing; Feng, Li; Guo, Fan; Li, Gang

    2014-05-20

    Simultaneous radio and extreme ultraviolet (EUV)/white-light imaging data are examined for a solar type II radio burst occurring on 2010 March 18 to deduce its source location. Using a bow-shock model, we reconstruct the three-dimensional EUV wave front (presumably the type-II-emitting shock) based on the imaging data of the two Solar TErrestrial RElations Observatory spacecraft. It is then combined with the Nançay radio imaging data to infer the three-dimensional position of the type II source. It is found that the type II source coincides with the interface between the coronal mass ejection (CME) EUV wave front and a nearby coronal ray structure, providing evidence that the type II emission is physically related to the CME-ray interaction. This result, consistent with those of previous studies, is based on simultaneous radio and EUV imaging data for the first time.

  17. Identification of extracellular signal-regulated kinase 3 as a new interaction partner of cyclin D3

    SciTech Connect

    Sun Maoyun; Wei Yuanyan; Yao Luyang; Xie Jianhui; Chen Xiaoning; Wang Hanzhou; Jiang Jianhai; Gu Jianxin . E-mail: jxgu@shmu.edu.cn

    2006-02-03

    Cyclin D3, like cyclin D1 and D2 isoforms, is a crucial component of the core cell cycle machinery in mammalian cells. It also exhibits its unique properties in many other physiological processes. In the present study, using yeast two-hybrid screening, we identified ERK3, an atypical mitogen-activated protein kinase (MAPK), as a cyclin D3 binding partner. GST pull-down assays showed that cyclin D3 interacts directly and specifically with ERK3 in vitro. The binding of cyclin D3 and ERK3 was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Moreover, carboxy-terminal extension of ERK3 was responsible for its association with intact cyclin D3. These findings further expand distinct roles of cyclin D3 and suggest the potential activity of ERK3 in cell proliferation.

  18. Activation of focal adhesion kinase through an interaction with β4 integrin contributes to tumorigenicity of colon cancer.

    PubMed

    Tai, Yu-Ling; Lai, I-Rue; Peng, Yu-Ju; Ding, Shih-Torng; Shen, Tang-Long

    2016-06-01

    High expression of either β4 integrin or focal adhesion kinase (FAK) has been reported in human colon cancer. However, it remains unclear how β4 integrin together with FAK contributes to the tumorigenicity of colon cancer. Here, we demonstrate that the co-overexpression of β4 integrin and FAK positively correlates with advanced stages of human colon cancer. Activated β4 integrin interacts with FAK and subsequently induces FAK phosphorylation at Tyr397. Furthermore, ablation of the β4 integrin/FAK complex and/or FAK activation impair colon cancer cell proliferation, anchorage-independent growth, and tumorigenicity. Our data indicate that the β4 integrin/FAK complex and subsequent FAK activation are essential regulators during the tumorigenicity of colon cancer, and we suggest an alternative strategy for colon cancer therapy. PMID:27178753

  19. A Direct Interaction between Leucine-rich Repeat Kinase 2 and Specific β-Tubulin Isoforms Regulates Tubulin Acetylation*

    PubMed Central

    Law, Bernard M. H.; Spain, Victoria A.; Leinster, Veronica H. L.; Chia, Ruth; Beilina, Alexandra; Cho, Hyun J.; Taymans, Jean-Marc; Urban, Mary K.; Sancho, Rosa M.; Ramírez, Marian Blanca; Biskup, Saskia; Baekelandt, Veerle; Cai, Huaibin; Cookson, Mark R.; Berwick, Daniel C.; Harvey, Kirsten

    2014-01-01

    Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease. PMID:24275654

  20. Identification, expression and interaction analyses of calcium-dependent protein kinase (CPK) genes in canola (Brassica napus L.)

    PubMed Central

    2014-01-01

    Background Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a well-known ubiquitous intracellular secondary messenger in plants. Calcium-dependent protein kinases (CPKs) are Ser/Thr protein kinases found only in plants and some protozoans. CPKs are Ca2+ sensors that have both Ca2+ sensing function and kinase activity within a single protein and play crucial roles in plant development and responses to various environmental stresses. Results In this study, we mined the available expressed sequence tags (ESTs) of B. napus and identified a total of 25 CPK genes, among which cDNA sequences of 23 genes were successfully cloned from a double haploid cultivar of canola. Phylogenetic analysis demonstrated that they could be clustered into four subgroups. The subcellular localization of five selected BnaCPKs was determined using green fluorescence protein (GFP) as the reporter. Furthermore, the expression levels of 21 BnaCPK genes in response to salt, drought, cold, heat, abscisic acid (ABA), low potassium (LK) and oxidative stress were studied by quantitative RT-PCR and were found to respond to multiple stimuli, suggesting that canola CPKs may be convergence points of different signaling pathways. We also identified and cloned five and eight Clade A basic leucine zipper (bZIP) and protein phosphatase type 2C (PP2C) genes from canola and, using yeast two-hybrid and bimolecular fluorescence complementation (BiFC), determined the interaction between individual BnaCPKs and BnabZIPs or BnaPP2Cs (Clade A). We identified novel, interesting interaction partners for some of the BnaCPK proteins. Conclusion We present the sequences and characterization of CPK gene family members in canola for the first time. This work provides a foundation for further crop improvement and improved understanding of

  1. Mys protein regulates protein kinase A activity by interacting with regulatory type Ialpha subunit during vertebrate development.

    PubMed

    Kotani, Tomoya; Iemura, Shun-ichiro; Natsume, Tohru; Kawakami, Koichi; Yamashita, Masakane

    2010-02-12

    During embryonic development, protein kinase A (PKA) plays a key role in cell fate specification by antagonizing the Hedgehog (Hh) signaling pathway. However, the mechanism by which PKA activity is regulated remains unknown. Here we show that the Misty somites (Mys) protein regulates the level of PKA activity during embryonic development in zebrafish. We isolate PKA regulatory type Ialpha subunit (Prkar1a) as a protein interacting with Mys by pulldown assay in HEK293 cells followed by mass spectrometry analysis. We show an interaction between endogenous Mys and Prkar1a in the zebrafish embryo. Mys binds to Prkar1a in its C terminus region, termed PRB domain, and activates PKA in vitro. Conversely, knockdown of Mys in zebrafish embryos results in reduction in PKA activity. We also show that knockdown of Mys induces ectopic activation of Hh target genes in the eyes, neural tube, and somites downstream of Smoothened, a protein essential for transduction of Hh signaling activity. The altered patterning of gene expression is rescued by activation of PKA. Together, our results reveal a molecular mechanism of regulation of PKA activity that is dependent on a protein-protein interaction and demonstrate that PKA activity regulated by Mys is indispensable for negative regulation of the Hh signaling pathway in Hh-responsive cells. PMID:20018846

  2. Structural analysis reveals features of the spindle checkpoint kinase Bub1–kinetochore subunit Knl1 interaction

    PubMed Central

    Krenn, Veronica; Wehenkel, Annemarie; Santaguida, Stefano

    2012-01-01

    The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR–KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1. PMID:22331848

  3. Aluminum interaction with human brain tau protein phosphorylation by various kinases

    SciTech Connect

    El-Sebae, A.H.; Zeid, M.M.A.; Saleh, M.A. . Environmental Chemistry and Toxicology Lab.); Abdel-Ghany, M.E.; Shalloway, D. ); Blancato, J. . Environmental Monitoring Systems Lab.)

    1993-01-01

    Phosphorylation is an indispensable process for energy and signal transduction in biological systems. AlCl[sub 3] at 10 nM to 10 uM range activated in-vitro [[gamma]-[sup 32]P] ATP phosphorylation of the brain (tau) [Tau] protein in both normal human or E. coli expressed [Tau] forms; in the presence of the kinases P34, PKP, and PKC to a maximum at 1 mM level. AlCl[sub 3] at 100 uM to 500 uM range induced non-enzymatic phosphorylation of [Tau] with [gamma]-ATP, [gamma]-GTP, and [alpha]-GTP, and [alpha]-GTP. AlCl[sub 3] activated histone phosphorylation by P34 in a similar pattern. The hyperphosphorylation of [Tau] with [gamma]-ATP, [gamma]-GTP, and [alpha]-GTP. AlCl[sub 3] activated histone phosphorylation by P34 in a similar pattern. The hyperphosphorylation of [Tau] by Al[sup 3+] was accompanied by molecular shift and mobility retardation in SDS-PAGE. This may demonstrate the mechanism of the longterm neurological effect of Al[sup 3+] in human brain leading to the formation of the neurofibrillary tangles related to Alzeheimer's disease.

  4. Aluminum interaction with human brain tau protein phosphorylation by various kinases

    SciTech Connect

    El-Sebae; Abou Zeid, M.M.; Saleh, M.A. . Environmental Chemistry and Toxicology Lab.); Abdel-Ghany, M.E.; Shalloway, D. . Section of Biochemistry, Mol, and Cell Biology); Blancato, J. . Environmental Monit. Systems Lab.)

    1993-01-01

    Phosphorylation is an indispensable process for energy and signal transduction in biological systems. AlCl[sub 3] at 10 nM to 10 [mu]M range activated in-vitro [[gamma][sup [minus]32]P]ATP phosphorylation of the brain ([tau]) [Gamma] protein in both normal human or E.coli expressed [Gamma] forms; in the presence of the kinases P34,PKP, and PKC. However, higher concentrations of AlCl[sub 3] inhibited the [Gamma] phosphorylation with P34, PKP, and PKC to a maximum at 1 mM level. AlCl[sub 3] at 100 [mu]M to 500 [mu]M range induced non-enzymatic phosphorylation of [Gamma] with [gamma]-ATP, [gamma]-GTP, and [alpha]-GRP. AlCl[sub 3] activated histone phosphorylation by P34 in a similar pattern. The hyperphosphorylation of [Gamma] by Al[sup 3+] was accompanied in molecular shift and mobility retardation in SDS-PAGE. This may demonstrate the mechanism of the long term neurological effect of Al[sub 3+] in human brain leading to the formation of the neutrofibrillary tangles related to Alzeheimer's disease.

  5. Regulation of genotoxic stress response by homeodomain-interacting protein kinase 2 through phosphorylation of cyclic AMP response element-binding protein at serine 271.

    PubMed

    Sakamoto, Kensuke; Huang, Bo-Wen; Iwasaki, Kenta; Hailemariam, Kiros; Ninomiya-Tsuji, Jun; Tsuji, Yoshiaki

    2010-08-15

    CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2-/- MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2-/- cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress. PMID:20573984

  6. Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

    NASA Technical Reports Server (NTRS)

    Song, W.; Welti, R.; Hafner-Strauss, S.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

  7. An Amino-Terminal Polo Kinase Interaction Motif Acts in the Regulation of Centrosome Formation and Reveals a Novel Function for centrosomin (cnn) in Drosophila.

    PubMed

    Eisman, Robert C; Phelps, Melissa A S; Kaufman, Thomas

    2015-10-01

    The formation of the pericentriolar matrix (PCM) and a fully functional centrosome in syncytial Drosophila melanogaster embryos requires the rapid transport of Cnn during initiation of the centrosome replication cycle. We show a Cnn and Polo kinase interaction is apparently required during embryogenesis and involves the exon 1A-initiating coding exon, suggesting a subset of Cnn splice variants is regulated by Polo kinase. During PCM formation exon 1A Cnn-Long Form proteins likely bind Polo kinase before phosphorylation by Polo for Cnn transport to the centrosome. Loss of either of these interactions in a portion of the total Cnn protein pool is sufficient to remove native Cnn from the pool, thereby altering the normal localization dynamics of Cnn to the PCM. Additionally, Cnn-Short Form proteins are required for polar body formation, a process known to require Polo kinase after the completion of meiosis. Exon 1A Cnn-LF and Cnn-SF proteins, in conjunction with Polo kinase, are required at the completion of meiosis and for the formation of functional centrosomes during early embryogenesis. PMID:26447129

  8. Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

    PubMed Central

    Levitt, James A.; Morton, Penny E.; Fruhwirth, Gilbert O.; Santis, George; Chung, Pei-Hua; Parsons, Maddy; Suhling, Klaus

    2015-01-01

    We present a novel integrated multimodal fluorescence microscopy technique for simultaneous fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging (FAIM). This approach captures a series of polarization-resolved fluorescence lifetime images during a FRAP recovery, maximizing the information available from a limited photon budget. We have applied this method to analyse the behaviour of GFP-labelled coxsackievirus and adenovirus receptor (CAR) in living human epithelial cells. Our data reveal that CAR exists in oligomeric states throughout the cell, and that these complexes occur in conjunction with high immobile fractions of the receptor at cell-cell junctions. These findings shed light on previously unknown molecular associations between CAR receptors in intact cells and demonstrate the power of combined FRAP, FLIM and FAIM microscopy as a robust method to analyse complex multi-component dynamics in living cells. PMID:26504635

  9. Homeodomain-interacting protein kinase 2, a novel autoimmune regulator interaction partner, modulates promiscuous gene expression in medullary thymic epithelial cells.

    PubMed

    Rattay, Kristin; Claude, Janine; Rezavandy, Esmail; Matt, Sonja; Hofmann, Thomas G; Kyewski, Bruno; Derbinski, Jens

    2015-02-01

    Promiscuous expression of a plethora of tissue-restricted Ags (TRAs) by medullary thymic epithelial cells (mTECs) plays an essential role in T cell tolerance. Although the cellular mechanisms by which promiscuous gene expression (pGE) imposes T cell tolerance have been well characterized, the underlying molecular mechanisms remain poorly understood. The autoimmune regulator (AIRE) is to date the only validated molecule known to regulate pGE. AIRE is part of higher-order multiprotein complexes, which promote transcription, elongation, and splicing of a wide range of target genes. How AIRE and its partners mediate these various effects at the molecular level is still largely unclear. Using a yeast two-hybrid screen, we searched for novel AIRE-interacting proteins and identified the homeodomain-interacting protein kinase 2 (HIPK2) as a novel partner. HIPK2 partially colocalized with AIRE in nuclear bodies upon cotransfection and in human mTECs in situ. Moreover, HIPK2 phosphorylated AIRE in vitro and suppressed the coactivator activity of AIRE in a kinase-dependent manner. To evaluate the role of Hipk2 in modulating the function of AIRE in vivo, we compared whole-genome gene signatures of purified mTEC subsets from TEC-specific Hipk2 knockout mice with control mice and identified a small set of differentially expressed genes. Unexpectedly, most differentially expressed genes were confined to the CD80(lo) mTEC subset and preferentially included AIRE-independent TRAs. Thus, although it modulates gene expression in mTECs and in addition affects the size of the medullary compartment, TEC-specific HIPK2 deletion only mildly affects AIRE-directed pGE in vivo. PMID:25552543

  10. Simultaneous prediction of binding free energy and specificity for PDZ domain-peptide interactions

    PubMed Central

    Crivelli, Joseph J.; Lemmon, Gordon; Kaufmann, Kristian W.; Meiler, Jens

    2014-01-01

    Interactions between protein domains and linear peptides underlie many biological processes. Among these interactions, the recognition of C-terminal peptides by PDZ domains is one of the most ubiquitous. In this work, we present a mathematical model for PDZ domain-peptide interactions capable of predicting both affinity and specificity of binding based on x-ray crystal structures and comparative modeling with Rosetta. We developed our mathematical model using a large phage display dataset describing binding specificity for a wild type PDZ domain and 91 single mutants, as well as binding affinity data for a wild type PDZ domain binding to 28 different peptides. Structural refinement was carried out through several Rosetta protocols, the most accurate of which included flexible peptide docking and several iterations of side chain repacking and backbone minimization. Our findings emphasize the importance of backbone flexibility and the energetic contributions of side chain-side chain hydrogen bonds in accurately predicting interactions. We also determined that predicting PDZ domain-peptide interactions became increasingly challenging as the length of the peptide increased in the N-terminal direction. In the training dataset, predicted binding energies correlated with those derived through calorimetry and specificity switches introduced through single mutations at interface positions were recapitulated. In independent tests, our best performing protocol was capable of predicting dissociation constants well within one order of magnitude of the experimental values and specificity profiles at the level of accuracy of previous studies. To our knowledge, this approach represents the first integrated protocol for predicting both affinity and specificity for PDZ domain-peptide interactions. PMID:24305904

  11. Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.

    PubMed Central

    Hu, P; Margolis, B; Skolnik, E Y; Lammers, R; Ullrich, A; Schlessinger, J

    1992-01-01

    One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors. Images PMID:1372091

  12. Expression of sphingosine kinase gene in the interactions between human gastric carcinoma cell and vascular endothelial cell

    PubMed Central

    Ren, Juan; Dong, Lei; Xu, Cang-Bao; Pan, Bo-Rong

    2002-01-01

    AIM: To study the interactions between human gastric carcinoma cell (HGCC) and human vascular endothelial cell (HVEC), and if the expression of sphingosine kinase (SPK) gene was involved in these interactions. METHODS: The specific inhibitor to SPK, dimethyl sphingosine (DMS), was added acting on HGCC and HVEC, then the cell proliferation was measured by MTT. The conditioned mediums (CMs) of HGCC and HVEC were prepared. The CM of one kind of cell was added to the other kind of cell, and the cell proliferation was measured by MTT. After the action of CM, the cellular expression of SPK gene in mRNA level was detected with in situ hybridization (ISH). RESULTS: DMS could almost completely inhibit the proliferation of HGCC and HVEC. The growth inhibitory rates could amount to 97.21%, 83.42%, respectively (P < 0.01). The CM of HGCC could stimulate the growth of HVEC (2.70 ± 0.01, P < 0.01) while the CM of HVEC could inhibit the growth of HGCC (52.97% ± 0.01%, P < 0.01). There was no significant change in the mRNA level of SPK gene in one kind of cell after the action of the CM of the other kind of cell. CONCLUSION: SPK plays a key role in regulating the proliferation of HGCC and HVEC. There exist complicated interactions between HGCC and HVEC. HGCC can significantly stimulate the growth of HVEC while HVEC can significantly inhibit the growth of HGCC. The expression of SPK gene is not involved in the interactions. PMID:12174364

  13. Modulation of kinase-inhibitor interactions by auxiliary protein binding: Crystallography studies on Aurora A interactions with VX-680 and with TPX2

    SciTech Connect

    Zhao, Baoguang; Smallwood, Angela; Yang, Jingsong; Koretke, Kristin; Nurse, Kelvin; Calamari, Amy; Kirkpatrick, Robert B.; Lai, Zhihong

    2008-10-24

    VX-680, also known as MK-0457, is an ATP-competitive small molecule inhibitor of the Aurora kinases that has entered phase II clinical trials for the treatment of cancer. We have solved the cocrystal structure of AurA/TPX2/VX-680 at 2.3 {angstrom} resolution. In the crystal structure, VX-680 binds to the active conformation of AurA. The glycine-rich loop in AurA adopts a unique bent conformation, forming a {pi}-{pi} interaction with the phenyl group of VX-680. In contrast, in the published AurA/VX-680 structure, VX-680 binds to AurA in the inactive conformation, interacting with a hydrophobic pocket only present in the inactive conformation. These data suggest that TPX2, a protein cofactor, can alter the binding mode of VX-680 with AurA. More generally, the presence of physiologically relevant cofactor proteins can alter the kinetics, binding interactions, and inhibition of enzymes, and studies with these multiprotein complexes may be beneficial to the discovery and optimization of enzyme inhibitors as therapeutic agents.

  14. Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells.

    PubMed

    Cherian, Joseph; Nacro, Kassoum; Poh, Zhi Ying; Guo, Samantha; Jeyaraj, Duraiswamy A; Wong, Yun Xuan; Ho, Melvyn; Yang, Hai Yan; Joy, Joma Kanikadu; Kwek, Zekui Perlyn; Liu, Boping; Wee, John Liang Kuan; Ong, Esther H Q; Choong, Meng Ling; Poulsen, Anders; Lee, May Ann; Pendharkar, Vishal; Ding, Li Jun; Manoharan, Vithya; Chew, Yun Shan; Sangthongpitag, Kanda; Lim, Sharon; Ong, S Tiong; Hill, Jeffrey; Keller, Thomas H

    2016-04-14

    Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented. PMID:27011159

  15. Neuropilin 1 directly interacts with Fer kinase to mediate semaphorin 3A-induced death of cortical neurons.

    PubMed

    Jiang, Susan X; Whitehead, Shawn; Aylsworth, Amy; Slinn, Jacqueline; Zurakowski, Bogdan; Chan, Kenneth; Li, Jianjun; Hou, Sheng T

    2010-03-26

    Neuropilins (NRPs) are receptors for the major chemorepulsive axonal guidance cue semaphorins (Sema). The interaction of Sema3A/NRP1 during development leads to the collapse of growth cones. Here we show that Sema3A also induces death of cultured cortical neurons through NRP1. A specific NRP1 inhibitory peptide ameliorated Sema3A-evoked cortical axonal retraction and neuronal death. Moreover, Sema3A was also involved in cerebral ischemia-induced neuronal death. Expression levels of Sema3A and NRP1, but not NRP2, were significantly increased early during brain reperfusion following transient focal cerebral ischemia. NRP1 inhibitory peptide delivered to the ischemic brain was potently neuroprotective and prevented the loss of motor functions in mice. The integrity of the injected NRP1 inhibitory peptide into the brain remained unchanged, and the intact peptide permeated the ischemic hemisphere of the brain as determined using MALDI-MS-based imaging. Mechanistically, NRP1-mediated axonal collapse and neuronal death is through direct and selective interaction with the cytoplasmic tyrosine kinase Fer. Fer RNA interference effectively attenuated Sema3A-induced neurite retraction and neuronal death in cortical neurons. More importantly, down-regulation of Fer expression using Fer-specific RNA interference attenuated cerebral ischemia-induced brain damage. Together, these studies revealed a previously unknown function of NRP1 in signaling Sema3A-evoked neuronal death through Fer in cortical neurons. PMID:20133938

  16. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis*

    PubMed Central

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W.-Y.; Puga, Alvaro; Xia, Ying

    2015-01-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  17. Interactions of Tyrosine Kinase Inhibitors with Organic Cation Transporters, OCTs, and Multidrug and Toxic Compound Extrusion Proteins, MATEs

    PubMed Central

    Minematsu, Tsuyoshi; Giacomini, Kathleen M.

    2011-01-01

    The drug-drug interaction (DDI) potential of tyrosine kinase inhibitors (TKIs) as interacting drugs via transporter inhibition has not been fully assessed. Here, we estimated the half maximal inhibitory concentration (IC50) values for eight small-molecule TKIs (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, sunitinib, lapatinib, and sorafenib) on [14C]metformin transport by human organic cation transporters (OCT1, OCT2, and OCT3), and multidrug and toxic compound extrusion proteins (MATE1 and MATE2-K), using HEK293 cells stably expressing these transporters. We then compared the estimated IC50 values to the maximum clinical concentrations of unbound TKIs in plasma (unbound Cmax,sys,p). Results showed that imatinib, nilotinib, gefitinib, and erlotinib exerted selectively potent inhibitory effects, with unbound Cmax,sys,p/IC50 values ≥ 0.1, on MATE1, OCT3, MATE2-K and OCT1, respectively. In comparison to the common form of OCT1, the OCT1 polymorphism, M420del was more sensitive to drug inhibition by erlotinib. Major metabolites of several TKIs showed IC50 values similar to those for unchanged TKIs. Taken together, these findings suggest the potential of clinical transporter-mediated DDIs between specific TKIs and OCTs and MATEs, which may affect the disposition, efficacy and toxicity of metformin and other drugs that are substrates of these transporters. The study provides the basis for further clinical DDI studies with TKIs. PMID:21252289

  18. Interaction of Hepatitis C Virus Core Protein with Janus Kinase Is Required for Efficient Production of Infectious Viruses

    PubMed Central

    Lee, Choongho

    2013-01-01

    Chronic hepatitis C virus (HCV) infection is responsible for the development of liver cirrhosis and hepatocellular carcinoma. HCV core protein plays not only a structural role in the virion morphogenesis by encapsidating a virus RNA genome but also a non-structural role in HCV-induced pathogenesis by blocking innate immunity. Especially, it has been shown to regulate JAK-STAT signaling pathway through its direct interaction with Janus kinase (JAK) via its proline-rich JAK-binding motif (79PGYPWP84). However, little is known about the physiological significance of this HCV core-JAK association in the context of the virus life cycle. In order to gain an insight, a mutant HCV genome (J6/JFH1-79A82A) was constructed to express the mutant core with a defective JAK-binding motif (79AGYAWP84) using an HCV genotype 2a infectious clone (J6/JFH1). When this mutant HCV genome was introduced into hepatocarcinoma cells, it was found to be severely impaired in its ability to produce infectious viruses in spite of its robust RNA genome replication. Taken together, all these results suggest an essential requirement of HCV core-JAK protein interaction for efficient production of infectious viruses and the potential of using core-JAK blockers as a new anti-HCV therapy. PMID:24009866

  19. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis.

    PubMed

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W-Y; Puga, Alvaro; Xia, Ying

    2015-08-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1(+/-) embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  20. p21-activated kinase 2 regulates HSPC cytoskeleton, migration, and homing via CDC42 activation and interaction with β-Pix.

    PubMed

    Reddy, Pavankumar N G; Radu, Maria; Xu, Ke; Wood, Jenna; Harris, Chad E; Chernoff, Jonathan; Williams, David A

    2016-04-21

    Cytoskeletal remodeling of hematopoietic stem and progenitor cells (HSPCs) is essential for homing to the bone marrow (BM). The Ras-related C3 botulinum toxin substrate (Rac)/cell division control protein 42 homolog (CDC42) effector p21-activated kinase (Pak2) has been implicated in HSPC homing and engraftment. However, the molecular pathways mediating Pak2 functions in HSPCs are unknown. Here, we demonstrate that both Pak2 kinase activity and its interaction with the PAK-interacting exchange factor-β (β-Pix) are required to reconstitute defective ITALIC! Pak2 (ITALIC! Δ/Δ)HSPC homing to the BM. Pak2 serine/threonine kinase activity is required for stromal-derived factor-1 (SDF1α) chemokine-induced HSPC directional migration, whereas Pak2 interaction with β-Pix is required to regulate the velocity of HSPC migration and precise F-actin assembly. Lack of SDF1α-induced filopodia and associated abnormal cell protrusions seen in ITALIC! Pak2 (ITALIC! Δ/Δ)HSPCs were rescued by wild-type (WT) Pak2 but not by a Pak2-kinase dead mutant (KD). Expression of a β-Pix interaction-defective mutant of Pak2 rescued filopodia formation but led to abnormal F-actin bundles. Although CDC42 has previously been considered an upstream regulator of Pak2, we found a paradoxical decrease in baseline activation of CDC42 in ITALIC! Pak2 (ITALIC! Δ/Δ)HSPCs, which was rescued by expression of Pak2-WT but not by Pak2-KD; defective homing of ITALIC! Pak2-deleted HSPCs was rescued by constitutive active CDC42. These data demonstrate that both Pak2 kinase activity and its interaction with β-Pix are essential for HSPC filopodia formation, cytoskeletal integrity, and homing via activation of CDC42. Taken together, we provide mechanistic insights into the role of Pak2 in HSPC migration and homing. PMID:26932803

  1. An approach to simultaneous control of trajectory and interaction forces in dual-arm configurations

    NASA Technical Reports Server (NTRS)

    Yun, Xiaoping; Kumar, Vijay R.

    1991-01-01

    An approach to the control of constrained dynamic systems such as multiple arm systems, multifingered grippers, and walking vehicles is described. The basic philosophy is to utilize a minimal set of inputs to control the trajectory and the surplus input to control the constraint or interaction forces and moments in the closed chain. A dynamic control model for the closed chain is derived that is suitable for designing a controller in which the trajectory and the interaction forces and moments are explicitly controlled. Nonlinear feedback techniques derived from differential geometry are then applied to linearize and decouple the nonlinear model. These ideas are illustrated through a planar example in which two arms are used for cooperative manipulation. Results from a simulation are used to illustrate the efficacy of the method.

  2. Effects of simultaneously fiber transmitted erbium and holmium radiation on the interaction with highly absorbing media

    NASA Astrophysics Data System (ADS)

    Frenz, Martin; Pratisto, Hans S.; Ith, Michael; Koenz, Flurin; Weber, Heinz P.

    1995-05-01

    Erbium and Holmium lasers have both been shown to be suitable for orthopedic surgery performed under water. Erbium lasers emitting in the 3 micrometers wavelength region corresponding to the maximum water absorption peak effectively ablated biological tissues with high precision and minimal thermal damage. Holmium laser radiation at 2 micrometers , due to a lower absorption coefficient, is characterized by a greater extent of thermal damage leading to hemostasis. To combine the special advantages of each system we simultaneously coupled their radiation into a zirconium fluoride fiber (ZrF4) which was protected with a quartz fiber tip. Pressure measurements performed in the liquid using a piezo electrical transducer, transmission measurements and video flash lamp schlieren imaging of the laser induced vapor bubble were used in order to determine optimum laser parameters. The cutting efficiency of the Erbium laser is drastically improved when a low energy Holmium laser pulse is additionally used which is just able to open a vapor channel through which the Erbium laser pulse can be transmitted. The dynamics of the channel formation, geometry and life time are measured as a function of the delay time between the two different laser pulses and the pulse energy applied. The combination of 2 micrometers and 3 micrometers radiation seems to be an ideal instrument for tissue treatment.

  3. Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase.

    PubMed

    Andrews, Rachel E; Galileo, Deni S; Martin-DeLeon, Patricia A

    2015-11-01

    Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca(2+), and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca(2+)/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at

  4. Biophysical and Structural Characterization of the Thioredoxin-binding Domain of Protein Kinase ASK1 and Its Interaction with Reduced Thioredoxin*

    PubMed Central

    Kosek, Dalibor; Kylarova, Salome; Psenakova, Katarina; Rezabkova, Lenka; Herman, Petr; Vecer, Jaroslav; Obsilova, Veronika; Obsil, Tomas

    2014-01-01

    Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp31 being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change. PMID:25037217

  5. Inhibition of the focal adhesion kinase and vascular endothelial growth factor receptor-3 interaction leads to decreased survival in human neuroblastoma cell lines.

    PubMed

    Beierle, Elizabeth A; Ma, Xiaojie; Stewart, Jerry E; Megison, Michael; Cance, William G; Kurenova, Elena V

    2014-03-01

    Neuroblastoma continues to be a devastating childhood solid tumor and is responsible for over 15% of all childhood cancer-related deaths. Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3) are protein tyrosine kinases that are overexpressed in a number of human cancers, including neuroblastoma. These two kinases can directly interact and provide survival signals to cancer cells. In this study, we utilized siRNA to VEGFR-3 to demonstrate the biologic importance of this kinase in neuroblastoma cell survival. We also used confocal microscopy and immunoprecipitation to show that FAK and VEGFR-3 bind in neuroblastoma. Finally, employing a 12-amino-acid peptide (AV3) specific to VEGFR-3, we showed that the colocalization between FAK and VEGFR-3 could be disrupted, and that disruption resulted in decreased neuroblastoma cell survival. These studies provide insight to the FAK-VEGFR-3 interaction in neuroblastoma and demonstrate its importance in this tumor type. Focusing upon the FAK-VEGFR-3 interaction may provide a novel therapeutic target for the development of new strategies for treatment of neuroblastoma. PMID:23065847

  6. Comprehensive analysis of pharmaceutical products using simultaneous mixed-mode (ion-exchange/reversed-phase) and hydrophilic interaction liquid chromatography.

    PubMed

    Kazarian, Artaches A; Nesterenko, Pavel N; Soisungnoen, Phimpha; Burakham, Rodjana; Srijaranai, Supalax; Paull, Brett

    2014-08-01

    Liquid chromatographic assays were developed using a mixed-mode column coupled in sequence with a hydrophilic interaction liquid chromatography column to allow the simultaneous comprehensive analysis of inorganic/organic anions and cations, active pharmaceutical ingredients, and excipients (carbohydrates). The approach utilized dual sample injection and valve-mediated column switching and was based upon a single high-performance liquid chromatography gradient pump. The separation consisted of three distinct sequential separation mechanisms, namely, (i) ion-exchange, (ii) mixed-mode interactions under an applied dual gradient (reversed-phase/ion-exchange), and (iii) hydrophilic interaction chromatography. Upon first injection, the Scherzo SS C18 column (Imtakt) provided resolution of inorganic anions and cations under isocratic conditions, followed by a dual organic/salt gradient to elute active pharmaceutical ingredients and their respective organic counterions and potential degradants. At the top of the mixed-mode gradient (high acetonitrile content), the mobile phase flow was switched to a preconditioned hydrophilic interaction liquid chromatography column, and the standard/sample was reinjected for the separation of hydrophilic carbohydrates, some of which are commonly known excipients in drug formulations. The approach afforded reproducible separation and resolution of up to 23 chemically diverse solutes in a single run. The method was applied to investigate the composition of commercial cough syrups (Robitussin®), allowing resolution and determination of inorganic ions, active pharmaceutical ingredients, excipients, and numerous well-resolved unknown peaks. PMID:24890905

  7. The tRNA-binding moiety in GCN2 contains a dimerization domain that interacts with the kinase domain and is required for tRNA binding and kinase activation

    PubMed Central

    Qiu, Hongfang; Dong, Jinsheng; Hu, Cuihua; Francklyn, Christopher S.; Hinnebusch, Alan G.

    2001-01-01

    GCN2 stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating translation initiation factor 2. GCN2 is activated by binding of uncharged tRNA to a domain related to histidyl-tRNA synthetase (HisRS). The HisRS-like region contains two dimerization domains (HisRS-N and HisRS-C) required for GCN2 function in vivo but dispensable for dimerization by full-length GCN2. Residues corresponding to amino acids at the dimer interface of Escherichia coli HisRS were required for dimerization of recombinant HisRS-N and for tRNA binding by full-length GCN2, suggesting that HisRS-N dimerization promotes tRNA binding and kinase activation. HisRS-N also interacted with the protein kinase (PK) domain, and a deletion impairing this interaction destroyed GCN2 function without reducing tRNA binding; thus, HisRS-N–PK interaction appears to stimulate PK function. The C-terminal domain of GCN2 (C-term) interacted with the PK domain in a manner disrupted by an activating PK mutation (E803V). These results suggest that the C-term is an autoinhibitory domain, counteracted by tRNA binding. We conclude that multiple domain interactions, positive and negative, mediate the activation of GCN2 by uncharged tRNA. PMID:11250908

  8. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  9. Addressing the Glycine-Rich Loop of Protein Kinases by a Multi-Facetted Interaction Network: Inhibition of PKA and a PKB Mimic.

    PubMed

    Lauber, Birgit S; Hardegger, Leo A; Asraful, Alam K; Lund, Bjarte A; Dumele, Oliver; Harder, Michael; Kuhn, Bernd; Engh, Richard A; Diederich, François

    2016-01-01

    Protein kinases continue to be hot targets in drug discovery research, as they are involved in many essential cellular processes and their deregulation can lead to a variety of diseases. A series of 32 enantiomerically pure inhibitors was synthesized and tested towards protein kinase A (PKA) and protein kinase B mimic PKAB3 (PKA triple mutant). The ligands bind to the hinge region, ribose pocket, and glycine-rich loop at the ATP site. Biological assays showed high potency against PKA, with Ki values in the low nanomolar range. The investigation demonstrates the significance of targeting the often neglected glycine-rich loop for gaining high binding potency. X-ray co-crystal structures revealed a multi-facetted network of ligand-loop interactions for the tightest binders, involving orthogonal dipolar contacts, sulfur and other dispersive contacts, amide-π stacking, and H-bonding to organofluorine, besides efficient water replacement. The network was analyzed in a computational approach. PMID:26578105

  10. Selective disruption of rb-raf-1 kinase interaction inhibits pancreatic adenocarcinoma growth irrespective of gemcitabine sensitivity.

    PubMed

    Treviño, José G; Verma, Monika; Singh, Sandeep; Pillai, Smitha; Zhang, Dongyu; Pernazza, Daniele; Sebti, Said M; Lawrence, Nicholas J; Centeno, Barbara A; Chellappan, Srikumar P

    2013-12-01

    Inactivation of the retinoblastoma (Rb) tumor suppressor protein is widespread in human cancers. Inactivation of Rb is thought to be initiated by association with Raf-1 (C-Raf) kinase, and here we determined how RRD-251, a disruptor of the Rb-Raf-1 interaction, affects pancreatic tumor progression. Assessment of phospho-Rb levels in resected human pancreatic tumor specimens by immunohistochemistry (n = 95) showed that increased Rb phosphorylation correlated with increasing grade of resected human pancreatic adenocarcinomas (P = 0.0272), which correlated with reduced overall patient survival (P = 0.0186). To define the antitumor effects of RRD-251 (50 μmol/L), cell-cycle analyses, senescence, cell viability, cell migration, anchorage-independent growth, angiogenic tubule formation and invasion assays were conducted on gemcitabine-sensitive and -resistant pancreatic cancer cells. RRD-251 prevented S-phase entry, induced senescence and apoptosis, and inhibited anchorage-independent growth and invasion (P < 0.01). Drug efficacy on subcutaneous and orthotopic xenograft models was tested by intraperitoneal injections of RRD-251 (50 mg/kg) alone or in combination with gemcitabine (250 mg/kg). RRD-251 significantly reduced tumor growth in vivo accompanied by reduced Rb phosphorylation and lymph node and liver metastasis (P < 0.01). Combination of RRD-251 with gemcitabine showed cooperative effect on tumor growth (P < 0.01). In conclusion, disruption of the Rb-Raf-1 interaction significantly reduces the malignant properties of pancreatic cancer cells irrespective of their gemcitabine sensitivity. Selective targeting of Rb-Raf-1 interaction might be a promising strategy targeting pancreatic cancer. PMID:24107447

  11. Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCF[superscript Cdc4] ubiquitin ligase

    SciTech Connect

    Tang, Xiaojing; Orlicky, Stephen; Mittag, Tanja; Csizmok, Veronika; Pawson, Tony; Forman-Kay, Julie D.; Sicheri, Frank; Tyers, Mike

    2012-05-24

    The ubiquitin ligase SCF{sup Cdc4} (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. Composite dynamic interactions of low affinity sites may be a general mechanism to establish phosphorylation thresholds in biological responses.

  12. Comparison of FDA Approved Kinase Targets to Clinical Trial Ones: Insights from Their System Profiles and Drug-Target Interaction Networks

    PubMed Central

    Xu, Jingyu; Wang, Panpan; Yang, Hong; Li, Yinghong; Yu, Chunyan; Tian, Yubin

    2016-01-01

    Kinase is one of the most productive classes of established targets, but the majority of approved drugs against kinase were developed only for cancer. Intensive efforts were therefore exerted for releasing its therapeutic potential by discovering new therapeutic area. Kinases in clinical trial could provide great opportunities for treating various diseases. However, no systematic comparison between system profiles of established targets and those of clinical trial ones was conducted. The reveal of probable difference or shift of trend would help to identify key factors defining druggability of established targets. In this study, a comparative analysis of system profiles of both types of targets was conducted. Consequently, the systems profiles of the majority of clinical trial kinases were identified to be very similar to those of established ones, but percentages of established targets obeying the system profiles appeared to be slightly but consistently higher than those of clinical trial targets. Moreover, a shift of trend in the system profiles from the clinical trial to the established targets was identified, and popular kinase targets were discovered. In sum, this comparative study may help to facilitate the identification of the druggability of established drug targets by their system profiles and drug-target interaction networks. PMID:27547755

  13. Geminivirus pathogenicity protein C4 interacts with Arabidopsis thaliana shaggy-related protein kinase AtSKeta, a component of the brassinosteroid signalling pathway.

    PubMed

    Piroux, Nathalie; Saunders, Keith; Page, Anthony; Stanley, John

    2007-06-01

    Beet curly top virus (BCTV) C4 interacted with two members of the shaggy-related protein kinase family (AtSKeta and AtSKzeta) and a putative leucine-rich repeat receptor-like kinase (LRR-RLK) in a yeast two-hybrid assay. Tomato golden mosaic virus (TGMV) AC4 also bound with similar efficiency to AtSKeta and AtSKzeta but was unable to interact with the LRR-RLK. BCTV C4 interaction with AtSKeta was confirmed using an in vitro binding assay. The protein kinases were capable of autophosphorylation in vitro and AtSKeta phosphorylated BCTV C4 at threonine and serine residues. AtSKeta phosphorylation of TGMV AC4 was significantly less efficient. The LRR-RLK did not efficiently phosphorylate BCTV C4. BCTV C4 localisation to the cell periphery in Nicotiana benthamiana was dependent on an intact N-terminal myristoylation motif, consistent with plasma membrane targeting. The intact motif was also required to produce the wild-type disease phenotype. Transient expression of BCTV C4 and TGMV AC4 derivatives in N. benthamiana identified additional amino acids within a central domain that contribute to the phenotype. The interaction with AtSKeta indicates that BCTV C4 interacts with the brassinosteroid signalling pathway. PMID:17280695

  14. An interactive effect of simultaneous death of dwarf bamboo, canopy gap, and predatory rodents on beech regeneration.

    PubMed

    Abe, M; Miguchi, H; Nakashizuka, T

    2001-04-01

    To clarify the interactive effect of the simultaneous death of dwarf bamboo (Sasa kurilensis), forest canopy gap formation, and seed predators on beech (Fagus crenata) regeneration, we analyzed beech demography from seed fall until the end of the first growing season of seedlings in an old-growth forest near Lake Towada, northern Japan. The simultaneous death of S. kurilensis took place in 1995. We established four types of sampling site differing in forest canopy conditions (closed or gap) and Sasa status (dead or alive). Beech seed survival and emergence ratio were both highest in gaps with dead Sasa (gap-dead), because rate of predation was lowest. Seedling survival during the first growing season was also highest in the gap-dead treatment, because of less predation and less damping off. As a result, even though density of seed fall was lowest in the gap-dead treatment, the living seedling density there was highest at the end of the first growing season. Predation, which caused the greatest mortality during the seed and seedling stages, was significantly lower at both sites in gaps and sites with dead Sasa. This was probably due to changes in the behavior of rodents in response to the structure of the forest canopy and undergrowth. Both the death of Sasa and canopy gap formation allowed seedlings to avoid damping off because of the high light availability. The indirect effect of the simultaneous death of Sasa and canopy gap formation in reducing predation contributed more to beech regeneration than their direct effect in increasing light for the seedlings. PMID:24577661

  15. Untangling the Diverse Interior and Multiple Exterior Guest Interactions of a Supramolecular Host by the Simultaneous Analysis of Complementary Observables.

    PubMed

    Sgarlata, Carmelo; Raymond, Kenneth N

    2016-07-01

    The entropic and enthalpic driving forces for encapsulation versus sequential exterior guest binding to the [Ga4L6](12-) supramolecular host in solution are very different, which significantly complicates the determination of these thermodynamic parameters. The simultaneous use of complementary techniques, such as NMR, UV-vis, and isothermal titration calorimetry, enables the disentanglement of such multiple host-guest interactions. Indeed, data collected by each technique measure different components of the host-guest equilibria and together provide a complete picture of the solution thermodynamics. Unfortunately, commercially available programs do not allow for global analysis of different physical observables. We thus resorted to a novel procedure for the simultaneous refinement of multiple parameters (ΔG°, ΔH°, and ΔS°) by treating different observables through a weighted nonlinear least-squares analysis of a constrained model. The refinement procedure is discussed for the multiple binding of the Et4N(+) guest, but it is broadly applicable to the deconvolution of other intricate host-guest equilibria. PMID:27244346

  16. Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions

    PubMed Central

    Hussain, Rohanah; Harding, Stephen E.; Hughes, Charlotte S.; Ma, Pikyee; Patching, Simon G.; Edara, Shalini; Siligardi, Giuliano; Henderson, Peter J.F.; Phillips-Jones, Mary K.

    2016-01-01

    This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins–synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs. PMID:27284046

  17. The interaction between Shroom3 and Rho-kinase is required for neural tube morphogenesis in mice.

    PubMed

    Das, Debamitra; Zalewski, Jenna K; Mohan, Swarna; Plageman, Timothy F; VanDemark, Andrew P; Hildebrand, Jeffrey D

    2014-01-01

    Shroom3 is an actin-associated regulator of cell morphology that is required for neural tube closure, formation of the lens placode, and gut morphogenesis in mice and has been linked to chronic kidney disease and directional heart looping in humans. Numerous studies have shown that Shroom3 likely regulates these developmental processes by directly binding to Rho-kinase and facilitating the assembly of apically positioned contractile actomyosin networks. We have characterized the molecular basis for the neural tube defects caused by an ENU-induced mutation that results in an arginine-to-cysteine amino acid substitution at position 1838 of mouse Shroom3. We show that this substitution has no effect on Shroom3 expression or localization but ablates Rock binding and renders Shroom3 non-functional for the ability to regulate cell morphology. Our results indicate that Rock is the major downstream effector of Shroom3 in the process of neural tube morphogenesis. Based on sequence conservation and biochemical analysis, we predict that the Shroom-Rock interaction is highly conserved across animal evolution and represents a signaling module that is utilized in a variety of biological processes. PMID:25171888

  18. Interactions between the nitrogen signal transduction protein PII and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis.

    PubMed

    Burillo, Sergio; Luque, Ignacio; Fuentes, Inmaculada; Contreras, Asunción

    2004-06-01

    PII, one of the most conserved signal transduction proteins, is believed to be a key player in the coordination of nitrogen assimilation and carbon metabolism in bacteria, archaea, and plants. However, the identity of PII receptors remains elusive, particularly in photosynthetic organisms. Here we used yeast two-hybrid approaches to identify new PII receptors and to explore the extent of conservation of PII signaling mechanisms between eubacteria and photosynthetic eukaryotes. Screening of Synechococcus sp. strain PCC 7942 libraries with PII as bait resulted in identification of N-acetyl glutamate kinase (NAGK), a key enzyme in the biosynthesis of arginine. The integrity of Ser49, a residue conserved in PII proteins from organisms that perform oxygenic photosynthesis, appears to be essential for NAGK binding. The effect of glnB mutations on NAGK activity is consistent with positive regulation of NAGK by PII. Phylogenetic and yeast two-hybrid analyses strongly suggest that there was conservation of the NAGK-PII regulatory interaction in the evolution of cyanobacteria and chloroplasts, providing insight into the function of eukaryotic PII-like proteins. PMID:15150219

  19. Clozapine Interaction with Phosphatidyl Inositol 3-Kinase (PI3K)/Insulin Signaling Pathway in Caenorhabditis elegans

    PubMed Central

    Karmacharya, Rakesh; Sliwoski, Gregory R.; Lundy, Miriam Y.; Suckow, Raymond F.; Cohen, Bruce M.; Buttner, Edgar A.

    2012-01-01

    Clozapine has superior and unique effects as an antipsychotic agent, but the mediators of these effects are not known. We studied behavioral and developmental effects of clozapine in Caenorhabditis elegans, as a model system to identify previously undiscovered mechanisms of drug action. Clozapine induced early larval arrest, a phenotype that was also seen with the clozapine metabolite N-desmethyl clozapine but not with any other typical or atypical antipsychotic drug tested. Mutations in the insulin receptor/daf-2 and the phosphatidyl inositol 3-kinase (PI3K)/age-1 suppressed clozapine-induced larval arrest, suggesting that clozapine may activate the insulin signaling pathway. Consistent with this notion, clozapine also increased expression of an age-1::GFP reporter. Activation of the insulin signaling pathway leads to cytoplasmic localization of the fork head transcription factor FOXO/daf-16. Clozapine produced cytoplasmic localization of DAF-16::GFP in arrested L1 larvae, in contrast to stressors such as starvation or high temperature which produce nuclear localization of DAF-16::GFP in arrested L1 larvae. Clozapine also inhibited pharyngeal pumping in C. elegans, an effect that may contribute to but did not explain clozapine-induced larval arrest. Our findings demonstrate a drug-specific interaction between clozapine and the PI3K/insulin signaling pathway in C. elegans. As this pathway is conserved across species, the results may have implications for understanding the unique effects of clozapine in humans. PMID:19322168

  20. Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions.

    PubMed

    Hussain, Rohanah; Harding, Stephen E; Hughes, Charlotte S; Ma, Pikyee; Patching, Simon G; Edara, Shalini; Siligardi, Giuliano; Henderson, Peter J F; Phillips-Jones, Mary K

    2016-06-15

    This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins-synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs. PMID:27284046

  1. Genetic Interactions among AMPK Catalytic Subunit Ssp2 and Glycogen Synthase Kinases Gsk3 and Gsk31 in Schizosaccharomyces Pombe.

    PubMed

    Qingyun; Ma, Yan; Kato, Toshiaki; Furuyashiki, Tomoyuki

    2016-01-01

    In Schizosaccharomyces pombe, Ssp2, an ortholog of AMP-activated protein kinase (AMPK), is critical for cell growth at restrictive temperatures and under glucose depletion as well as sexual differentiation under nitrogen depletion. To identify genes genetically related to Ssp2, we performed a genetic screening to search for the genes whose overexpression rescued the growth defects in Δssp2 cells at restrictive temperatures, and identified 35 cosmids as multicopy suppressor genes. In Southern blot analyses, 22 out of these cosmids were hybridized to an ssp2+ probe. Using nucleotide sequencing, we identified the gsk3+ gene in one of the cosmids, and the remaining 12 cosmids were hybridized to a gsk3+ probe. Overexpression of the gsk3+ gene or the gsk31+ gene, another GSK3 member, rescues defective growth of Δssp2 cells at restrictive temperatures and under glucose depletion as well as sexual differentiation under nitrogen depletion. Δgsk3Δgsk31 double knockout cells, but neither Δgsk3 nor Δgsk31 single knockout cells, phenocopy Δssp2 cells. The deletion of the gsk3+ or gsk31+ gene augments the phenotypes of Δssp2 cells. These findings suggest that Gsk3 and Gsk31 are critical and interact with Ssp2 in multiple cellular functions. PMID:27604537

  2. Las1 interacts with Grc3 polynucleotide kinase and is required for ribosome synthesis in Saccharomyces cerevisiae

    PubMed Central

    Castle, Christopher D.; Sardana, Richa; Dandekar, Varada; Borgianini, Victoria; Johnson, Arlen W.; Denicourt, Catherine

    2013-01-01

    Ribosome biogenesis is a multi-step process that couples cell growth with cell proliferation. Although several large-scale analysis of pre-ribosomal particles have identified numerous trans-acting factors involved in this process, many proteins involved in pre-rRNA processing and ribosomal subunit maturation have yet to be identified. Las1 was originally identified in Saccharomyces cerevisiae as a protein involved in cell morphogenesis. We previously demonstrated that the human homolog, Las1L, is required for efficient ITS2 rRNA processing and synthesis of the 60S ribosomal subunit. Here, we report that the functions of Las1 in ribosome biogenesis are also conserved in S. cerevisiae. Depletion of Las1 led to the accumulation of both the 27S and 7S rRNA intermediates and impaired the synthesis of the 60S subunit. We show that Las1 co-precipitates mainly with the 27S rRNA and associates with an Nsa1 and Rix1-containing pre-60S particle. We further identify Grc3 as a major Las1-interacting protein. We demonstrate that the kinase activity of Grc3 is required for efficient pre-rRNA processing and that depletion of Grc3 leads to rRNA processing defects similar to the ones observed in Las1-depleted cells. We propose that Las1 and Grc3 function together in a conserved mechanism to modulate rRNA processing and eukaryotic ribosome biogenesis. PMID:23175604

  3. Receptor-interacting protein kinase 3-mediated programmed cell necrosis in rats subjected to focal cerebral ischemia-reperfusion injury

    PubMed Central

    DONG, YANRU; BAO, CUIFEN; YU, JINGWEI; LIU, XIA

    2016-01-01

    In the current study, the activation of tumor necrosis factor-α receptor 1 (TNFR1) and receptor-interacting protein kinase 3 (RIP3) were investigated following cerebral ischemia-reperfusion injury (CIRI). Healthy SD rats were randomly divided into 3 groups: Sham operation group, model group and inhibitor group. The model group and inhibitor group were further divided into 4 subgroups of 6, 12, 24 and 72 h following CIRI. Using right middle cerebral artery embolization, the CIRI model was generated. To confirm that the CIRI model was established, neurological scores, TTC staining and brain water content measurements were conducted. Immunohistochemistry and western blotting were conducted to investigate the expression of TNFR1 and RIP3 in the cerebral cortex. It was observed that nerve cell necrosis occurred following 6 h of CIRI. The appearance of necrotic cells was gradually increased with increasing CIRI duration. TNFR1 and RIP3 were positively expressed following 6 h of CIRI. With increasing durations of CIRI, the protein expression levels of TNFR1 and RIP3 were significantly increased. Pre-administration with Z-VAD-FMK (zVAD) significantly increased the protein level of RIP3, however, had no effect on the levels of TNFR1, and was accompanied by a reduction in necrosis. In conclusion, RIP3-mediated cell necrosis was enhanced by caspase blockade zVAD and the function of zVAD was independent of TNFR1 signaling following IR. PMID:27220678

  4. Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo.

    PubMed

    Lageix, Sebastien; Zhang, Jinwei; Rothenburg, Stefan; Hinnebusch, Alan G

    2015-02-01

    The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction. PMID:25695491

  5. Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

    PubMed Central

    Lageix, Sebastien; Zhang, Jinwei; Rothenburg, Stefan; Hinnebusch, Alan G.

    2015-01-01

    The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction. PMID:25695491

  6. Traf2- and Nck-interacting kinase (TNIK) is involved in the anti-cancer mechanism of dovitinib in human multiple myeloma IM-9 cells.

    PubMed

    Chon, Hae Jung; Lee, Yura; Bae, Kyoung Jun; Byun, Byung Jin; Kim, Soon Ae; Kim, Jiyeon

    2016-07-01

    Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family. TNIK was first identified as a kinase that is involved in regulating cytoskeletal organization in many types of cells, and it was recently proposed as a novel therapeutic target in several types of human cancers. Although previous studies suggest that TNIK plays a pivotal role in cancer cell survival and prognosis, its function in hematological cancer cell survival has not been investigated. Here we investigated the relationship between TNIK function and cell viability in multiple myeloma IM-9 cells using TNIK small interfering RNA (siRNA) transfection and dovitinib treatment. Treatment of IM-9 cells with TNIK siRNA and dovitinib treatment reduced cell proliferation. The ATP competing kinase assay and western blot analysis showed that dovitinib strongly inhibited both the interaction of TNIK with ATP (K i, 13 nM) and the activation of Wnt signaling effectors such as β-catenin and TCF4. Dovitinib also induced caspase-dependent apoptosis in IM-9 cells without significant cytotoxicity in PBMCs. Our results provide new evidence that TNIK may be involved in the proliferation of multiple myeloma IM-9 cells and in the anti-cancer activity of dovitinib via inhibition of the endogenous Wnt signaling pathway. PMID:26995282

  7. Early closure of a long loop in the refolding of adenylate kinase: a possible key role of non-local interactions in the initial folding steps.

    PubMed

    Orevi, Tomer; Ben Ishay, Eldad; Pirchi, Menachem; Jacob, Maik H; Amir, Dan; Haas, Elisha

    2009-01-30

    Most globular protein chains, when transferred from high to low denaturant concentrations, collapse instantly before they refold to their native state. The initial compaction of the protein molecule is assumed to have a key effect on the folding pathway, but it is not known whether the earliest structures formed during or instantly after collapse are defined by local or by non-local interactions--that is, by secondary structural elements or by loop closure of long segments of the protein chain. Stable closure of one or several long loops can reduce the chain entropy at a very early stage and can prevent the protein from following non-productive pathways whose number grows exponentially with the length of the protein chain. In Escherichia coli adenylate kinase (AK), about seven long loops define the topology of the native structure. We selected four loop-forming sections of the chain and probed the time course of loop formation during refolding of AK. We labeled the termini of the loop segments with tryptophan and cysteine-5-amidosalicylic acid. This donor-acceptor pair of probes used with fluorescence resonance excitation energy transfer spectroscopy (FRET) is suitable for detecting very short distances and thus is able to distinguish between random and specific compactions. Refolding of AK was initiated by stopped-flow mixing, followed simultaneously by donor and acceptor fluorescence, and analyzed in terms of energy transfer efficiency and distance. In the collapsed state of AK, observed after the 5-ms dead time of the instrument, one of the selected segments shows a native-like separation of its termini; it forms a loop already in the collapsed state. A second segment that includes the first but is longer by 15 residues shows an almost native-like separation of its termini. In contrast, a segment that is shorter but part of the second segment shows a distance separation of its termini as high as a segment that spans almost the whole protein chain. We conclude

  8. Tyrosine kinase receptor Axl enhances entry of Zaire ebolavirus without direct interactions with the viral glycoprotein

    PubMed Central

    Brindley, Melinda A.; Hunt, Catherine L.; Kondratowicz, Andrew S.; Bowman, Jill; Sinn, Patrick L.; McCray, Paul B.; Quinn, Kathrina; Weller, Melodie L.; Chiorini, John A.; Maury, Wendy

    2011-01-01

    In a bioinformatics-based screen for cellular genes that enhance Zaire ebolavirus (ZEBOV) transduction, AXL mRNA expression strongly correlated with ZEBOV infection. A series of cell lines and primary cells were identified that require Axl for optimal ZEBOV entry. Using one of these cell lines, we identified ZEBOV entry events that are Axl-dependent. Interactions between ZEBOV-GP and the Axl ectodomain were not detected in immunoprecipitations and reduction of surface expressed Axl by RNAi did not alter ZEBOV-GP binding, providing evidence that Axl does not serve as a receptor for the virus. However, RNAi knock down of Axl reduced ZEBOV pseudovirion internalization and α-Axl antisera inhibited pseudovirion fusion with cellular membranes. Consistent with the importance of Axl for ZEBOV transduction, Axl transiently co-localized on the surface of cells with ZEBOV virus particles and was internalized during virion transduction. In total, these findings indicate that endosomal uptake of filoviruses is facilitated by Axl. PMID:21529875

  9. Tyrosine kinase receptor Axl enhances entry of Zaire ebolavirus without direct interactions with the viral glycoprotein

    SciTech Connect

    Brindley, Melinda A.; Hunt, Catherine L.; Kondratowicz, Andrew S.; Bowman, Jill; Sinn, Patrick L.; McCray, Paul B.; Quinn, Kathrina; Weller, Melodie L.; Chiorini, John A.; Maury, Wendy

    2011-07-05

    In a bioinformatics-based screen for cellular genes that enhance Zaire ebolavirus (ZEBOV) transduction, AXL mRNA expression strongly correlated with ZEBOV infection. A series of cell lines and primary cells were identified that require Axl for optimal ZEBOV entry. Using one of these cell lines, we identified ZEBOV entry events that are Axl-dependent. Interactions between ZEBOV-GP and the Axl ectodomain were not detected in immunoprecipitations and reduction of surface-expressed Axl by RNAi did not alter ZEBOV-GP binding, providing evidence that Axl does not serve as a receptor for the virus. However, RNAi knock down of Axl reduced ZEBOV pseudovirion internalization and {alpha}-Axl antisera inhibited pseudovirion fusion with cellular membranes. Consistent with the importance of Axl for ZEBOV transduction, Axl transiently co-localized on the surface of cells with ZEBOV virus particles and was internalized during virion transduction. In total, these findings indicate that endosomal uptake of filoviruses is facilitated by Axl.

  10. TaCIPK29, a CBL-Interacting Protein Kinase Gene from Wheat, Confers Salt Stress Tolerance in Transgenic Tobacco

    PubMed Central

    Zhou, Shiyi; Zhang, Fan; Han, Jiapeng; Chen, Lihong; Li, Yin; Feng, Jialu; Fang, Bin; Luo, Qingchen; Li, Shasha; Liu, Yunyi; Yang, Guangxiao; He, Guangyuan

    2013-01-01

    Calcineurin B-like protein-interacting protein kinases (CIPKs) have been found to be responsive to abiotic stress. However, their precise functions and the related molecular mechanisms in abiotic stress tolerance are not completely understood, especially in wheat. In the present study, TaCIPK29 was identified as a new member of CIPK gene family in wheat. TaCIPK29 transcript increased after NaCl, cold, methyl viologen (MV), abscisic acid (ABA) and ethylene treatments. Over-expression of TaCIPK29 in tobacco resulted in increased salt tolerance, which was demonstrated by higher germination rates, longer root lengths and better growth status of transgenic tobacco plants compared to controls when both were treated with salt stress. Physiological measurements indicated that transgenic tobacco seedlings retained high K+/Na+ ratios and Ca2+ content by up-regulating some transporter genes expression and also possessed lower H2O2 levels and reduced membrane injury by increasing the expression and activities of catalase (CAT) and peroxidase (POD) under salt stress. Moreover, transgenic lines conferred tolerance to oxidative stress by increasing the activity and expression of CAT. Finally, TaCIPK29 was located throughout cells and it preferentially interacted with TaCBL2, TaCBL3, NtCBL2, NtCBL3 and NtCAT1. Taken together, our results showed that TaCIPK29 functions as a positive factor under salt stress and is involved in regulating cations and reactive oxygen species (ROS) homeostasis. PMID:23922838

  11. Interaction of a tyrosine kinase inhibitor, vandetanib with human serum albumin as studied by fluorescence quenching and molecular docking.

    PubMed

    Kabir, Md Zahirul; Feroz, Shevin R; Mukarram, Abdul Kadir; Alias, Zazali; Mohamad, Saharuddin B; Tayyab, Saad

    2016-08-01

    Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB-HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92-6.89 × 10(3 )M(-1) at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB-HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J mol(-1) K(-1)) and negative ΔH (-6.57 kJ mol(-1)) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB-HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow's site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca(2+), Zn(2+), Cu(2+), Ba(2+), Mg(2+), and Mn(2+) in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA. PMID:26331959

  12. Brain-type creatine kinase BB-CK interacts with the Golgi Matrix Protein GM130 in early prophase.

    PubMed

    Bürklen, Tanja S; Hirschy, Alain; Wallimann, Theo

    2007-03-01

    Creatine kinase (CK) isoenzymes are essential for storing, buffering and intracellular transport of "energy-rich" phosphate compounds in tissues with fluctuating high energy demand such as muscle, brain and other tissues and cells where CK is expressed. In brain and many non-muscle cells, ubiquitous cytosolic "brain-type" BB-CK and ubiquitous mitochondrial CK (uMtCK) act as components of a phosphocreatine shuttle to maintain cellular energy pools and distribute energy flux. To date, still relatively little is known about direct coupling of functional dimeric BB-CK with other partner proteins or enzymes that are important for cell function. Using a global yeast two-hybrid (Y2H) screen with monomeric B-CK as bait and a representative brain cDNA library to search for interaction partners of B-CK with proteins of the brain, we repeatedly identified the cis-Golgi Matrix protein (GM130) as recurrent interacting partner of B-CK. Since HeLa cells also express both BB-CK and GM130, we subsequently used this cellular model system to verify and characterize the BB-CK-GM130 complex by GST-pulldown experiments, as well as by in vivo co-localization studies with confocal microscopy. Using dividing HeLa cells, we report here for the first time that GM130 and BB-CK co-localize specifically in a transient fashion during early prophase of mitosis, when GM130 plays an important role in Golgi fragmentation that starts also at early prophase. These data may shed new light on BB-CK function for energy provision for Golgi-fragmentation that is initiated by cell signalling cascades in the early phases of mitosis. PMID:17036164

  13. Evidence that the Rhodopsin Kinase (GRK1) N-Terminus and the Transducin Gα C-Terminus Interact with the Same "Hydrophobic Patch" on Rhodopsin TM5.

    PubMed

    Jones Brunette, Amber M; Sinha, Abhinav; David, Larry; Farrens, David L

    2016-06-01

    Phosphorylation of G protein-coupled receptors (GPCRs) terminates their ability to couple with and activate G proteins by increasing their affinity for arrestins. Unfortunately, detailed information regarding how GPCRs interact with the kinases responsible for their phosphorylation is still limited. Here, we purified fully functional GPCR kinase 1 (GRK1) using a rapid method and used it to gain insights into how this important kinase interacts with the GPCR rhodopsin. Specifically, we find that GRK1 uses the same site on rhodopsin as the transducin (Gt) Gtα C-terminal tail and the arrestin "finger loop", a cleft formed in the cytoplasmic face of the receptor upon activation. Our studies also show GRK1 requires two conserved residues located in this cleft (L226 and V230) that have been shown to be required for Gt activation due to their direct interactions with hydrophobic residues on the Gα C-terminal tail. Our data and modeling studies are consistent with the idea that all three proteins (Gt, GRK1, and visual arrestin) bind, at least in part, in the same site on rhodopsin and interact with the receptor through a similar hydrophobic contact-driven mechanism. PMID:27078130

  14. Calculation of simultaneous isotopic and trace element variations during water-rock interaction with applications to carbonate diagenesis

    SciTech Connect

    Banner, J.L. ); Hanson, G.N. )

    1990-11-01

    Equations are developed for the calculation of the simultaneous variations in trace element concentrations and radiogenic and stable isotopic compositions that occur during water-rock interaction. The equations are of general use for modeling chemical and isotopic variations in porous media and are applied here to the diagenesis of carbonate rocks and sediments. Owing to the extreme differences in the concentrations of oxygen and carbon in diagenetic fluids, carbonate minerals equilibrate with fluid {delta}{sup 18}O values at three orders of magnitude lower waterrock ratios (< 10) than the waterrock ratios at which they equilibrate with fluid {delta}{sup 13}C values (10{sup 3}). {sup 87}Sr/{sup 86}Sr ratios are affected at variable rates. In order to reset the {sup 87}Sr/{sup 86}Sr value of a marine limestone during freshwater diagenesis, waterrock ratios similar to those calculated for equilibration of {delta}{sup 13}C values are required, while Sr-Ca-rich brines can reset the {sup 87}Sr/{sup 86}Sr value of a limestone at low waterrock ratios, similar to those calculated for equilibration of {delta}{sup 18}O values. These large, relative differences in the response of different isotopic systems to water-rock interaction translate into characteristic trends on isotope and trace element covariation diagrams that can be used to distinguish between (1) different models for water-rock interaction and (2) different processes such as water-rock interaction, mixing of fluids, and mixing of mineral endmembers. These modeling approaches are applied to the regionally extensive dolomites of the Mississippian Burlington-Keokuk Formation.

  15. Characterization and interactions of anodic isolates in microbial fuel cells explored for simultaneous electricity generation and Congo red decolorization.

    PubMed

    Xu, Qian; Sun, Jian; Hu, Yong-You; Chen, Jie; Li, Wan-Jun

    2013-08-01

    To investigate functions and interactions of predominant microorganisms in microbial fuel cells (MFCs) for simultaneous electricity generation and Congo red decolorization, four strains were isolated from the anodic biofilm, and identified as Pseudomonas (M-P and I-P), Bacillus (M-B) and Aquamicrobium (I-A). Higher maximum power density (by 158.2% and 58.1%) but lower Congo red decolorization rate (by 3.2% and 5.9%) were achieved in MFCs using pure cultures I-P and M-P as inoculums than those using I-A and M-B, respectively. By comparing MFCs using co-cultures with those using pure cultures (M-P&B versus M-B and M-P, I-P&A versus I-A and I-P), the maximum power density of MFCs using co-cultures increased 82.0%, 15.1%, 94.6% and -24.6% (minus meant decreased), but decolorization rate decreased 33.3%, 29.4%, 7.9% and 5.0%, respectively. The results indicated specific interaction could enhance the performance of MFCs and might benefit the development of bio-process controlling. PMID:23735792

  16. Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

    PubMed Central

    Holt, K H; Olson, L; Moye-Rowley, W S; Pessin, J E

    1994-01-01

    Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110. Images PMID:8264609

  17. NUP214-ABL1-mediated cell proliferation in T-cell acute lymphoblastic leukemia is dependent on the LCK kinase and various interacting proteins

    PubMed Central

    De Keersmaecker, Kim; Porcu, Michaël; Cox, Luk; Girardi, Tiziana; Vandepoel, Roel; de Beeck, Joyce Op; Gielen, Olga; Mentens, Nicole; Bennett, Keiryn L.; Hantschel, Oliver

    2014-01-01

    The NUP214-ABL1 fusion protein is a constitutively active protein tyrosine kinase that is found in 6% of patients with T-cell acute lymphoblastic leukemia and that promotes proliferation and survival of T-lymphoblasts. Although NUP214-ABL1 is sensitive to ABL1 kinase inhibitors, development of resistance to these compounds is a major clinical problem, underlining the need for additional drug targets in the sparsely studied NUP214-ABL1 signaling network. In this work, we identify and validate the SRC family kinase LCK as a protein whose activity is absolutely required for the proliferation and survival of T-cell acute lymphoblastic leukemia cells that depend on NUP214-ABL1 activity. These findings underscore the potential of SRC kinase inhibitors and of the dual ABL1/SRC kinase inhibitors dasatinib and bosutinib for the treatment of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia. In addition, we used mass spectrometry to identify protein interaction partners of NUP214-ABL1. Our results strongly support that the signaling network of NUP214-ABL1 is distinct from that previously reported for BCR-ABL1. Moreover, we found that three NUP214-ABL1-interacting proteins, MAD2L1, NUP155, and SMC4, are strictly required for the proliferation and survival of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia cells. In conclusion, this work identifies LCK, MAD2L1, NUP155 and SMC4 as four new potential drug targets in NUP214-ABL1-positive T-cell acute lymphoblastic leukemia. PMID:23872305

  18. A search for weakly interacting dark matter particles with low temperature detectors capable of simultaneously measuring ionization and heat

    NASA Astrophysics Data System (ADS)

    Sonnenschein, Andrew Harry

    1999-01-01

    The nature and extent of the mysterious ``dark matter'' has been one of the central puzzles in cosmology over the last decade. Current evidence suggests that the universe has a matter content of about 0.3 of the critical density. The theory of Big Bang nucleosynthesis allows only 20% of this matter to be made of protons and neutrons, hinting that the remaining 80% of the mass is in the form of a new elementary particle. One possibility is that this dark matter is made of the lightest neutralino suggested by supersymmetry, which should be stable and have a mass in the range 30-10000 GeV/c 2. These particles would have only weak interactions with ordinary matter, with the typical cross section for scattering on a nucleon below 10 -41 cm2. The particles would constitute a ``dark halo'' in our galaxy and could be discovered through their interactions with detectors on Earth. Since the typical interaction rate would be below 0.1 event/kg-day, the main experimental problem is to reduce the interference from environmental background radiation. The most troublesome interference comes from photons produced by the decay of radioactive elements present at trace levels in the detectors and other apparatus. One way to discriminate between signal events from WIMP- nucleus scattering and background events from photon interactions is to measure the ratio of ionization to heat deposited in a semiconductor detector. This differs by a factor of ~3 between the two cases. The Cryogenic Dark Matter Search (CDMS) experiment is based on the simultaneous measurement of ionization and phonons in germanium and silicon crystals cooled to 25 mK. This measurement allows >99% discrimination against photon interactions above 10 keV of deposited energy. We present data from two 165 g germanium detectors operated at the Stanford Underground Facility, with a total exposure equivalent to 6.4 kg-days. From this data set, we derive limits on the cross sections and masses of weakly interacting dark

  19. Myotubularin-related Proteins 3 and 4 Interact with Polo-like Kinase 1 and Centrosomal Protein of 55 kDa to Ensure Proper Abscission*

    PubMed Central

    St-Denis, Nicole; Gupta, Gagan D.; Lin, Zhen Yuan; Gonzalez-Badillo, Beatriz; Pelletier, Laurence; Gingras, Anne-Claude

    2015-01-01

    The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein–protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to

  20. Impaired Angiogenesis during Fracture Healing in GPCR Kinase 2 Interacting Protein-1 (GIT1) Knock Out Mice

    PubMed Central

    Menon, Prashanthi; Pang, Jinjiang; Ho, Hsin-Chiu; Shi, Shanshan; Xie, Chao; Smolock, Elaine; Yan, Chen; Zuscik, Michael J.; Berk, Bradford C.

    2014-01-01

    G protein coupled receptor kinase 2 (GRK2) interacting protein-1 (GIT1), is a scaffold protein that plays an important role in angiogenesis and osteoclast activity. We have previously demonstrated that GIT1 knockout (GIT1 KO) mice have impaired angiogenesis and dysregulated osteoclast podosome formation leading to a reduction in the bone resorbing ability of these cells. Since both angiogenesis and osteoclast-mediated bone remodeling are involved in the fracture healing process, we hypothesized that GIT1 participates in the normal progression of repair following bone injury. In the present study, comparison of fracture healing in wild type (WT) and GIT1 KO mice revealed altered healing in mice with loss of GIT1 function. Alcian blue staining of fracture callus indicated a persistence of cartilagenous matrix in day 21 callus samples from GIT1 KO mice which was temporally correlated with increased type 2 collagen immunostaining. GIT1 KO mice also showed a decrease in chondrocyte proliferation and apoptosis at days 7 and 14, as determined by PCNA and TUNEL staining. Vascular microcomputed tomography analysis of callus samples at days 7, 14 and 21 revealed decreased blood vessel volume, number, and connection density in GIT1 KO mice compared to WT controls. Correlating with this, VEGF-A, phospho-VEGFR2 and PECAM1 (CD31) were decreased in GIT1 KO mice, indicating reduced angiogenesis with loss of GIT1. Finally, calluses from GIT1 KO mice displayed a reduced number of tartrate resistant acid phosphatase-positive osteoclasts at days 14 and 21. Collectively, these results indicate that GIT1 is an important signaling participant in fracture healing, with gene ablation leading to reduced callus vascularity and reduced osteoclast number in the healing callus. PMID:24586541

  1. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes

    PubMed Central

    Bellance, Catherine; Khan, Junaid A.; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-01-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(–) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl­amino)­phenyl-17β-hydroxy-17-(1-propynyl)­estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes. PMID:23485561

  2. Drosophila homeodomain-interacting protein kinase inhibits the Skp1-Cul1-F-box E3 ligase complex to dually promote Wingless and Hedgehog signaling.

    PubMed

    Swarup, Sharan; Verheyen, Esther M

    2011-06-14

    Drosophila Homeodomain-interacting protein kinase (Hipk) has been shown to regulate in vivo, the stability of Armadillo, the transcriptional effector of Wingless signaling. The Wingless pathway culminates in the stabilization of Armadillo that, in the absence of signaling, is sequentially phosphorylated, polyubiquitinated and degraded. Loss-of-function clones for hipk result in reduced stabilized Armadillo, whereas overexpression of hipk elevates Armadillo levels to promote Wingless-responsive target gene expression. Here, we show that overexpression of hipk can suppress the effects of negative regulators of Armadillo to prevent its degradation in the wing imaginal disc. Hipk acts to stabilize Armadillo by impeding the function of the E3 ubiquitin ligase Skp1-Cul1-F-box (SCF)(Slimb), thereby inhibiting Armadillo ubiquitination and subsequent degradation. Vertebrate Hipk2 displays a similar ability to prevent β-catenin ubiquitination in a functionally conserved mechanism. We find that Hipk's ability to inhibit SCF(Slimb)-mediated ubiquitination is not restricted to Armadillo and extends to other substrates of SCF(Slimb), including the Hedgehog signaling effector Ci. Thus, similar to casein kinase 1 and glycogen synthase kinase 3, Hipk dually regulates both Wingless and Hedgehog signaling by controlling the stability of their respective signaling effectors, but it is the first kinase to our knowledge identified that promotes the stability of both Armadillo and Ci. PMID:21628596

  3. KEA: kinase enrichment analysis

    PubMed Central

    Lachmann, Alexander; Ma'ayan, Avi

    2009-01-01

    Motivation: Multivariate experiments applied to mammalian cells often produce lists of proteins/genes altered under treatment versus control conditions. Such lists can be projected onto prior knowledge of kinase–substrate interactions to infer the list of kinases associated with a specific protein list. By computing how the proportion of kinases, associated with a specific list of proteins/genes, deviates from an expected distribution, we can rank kinases and kinase families based on the likelihood that these kinases are functionally associated with regulating the cell under specific experimental conditions. Such analysis can assist in producing hypotheses that can explain how the kinome is involved in the maintenance of different cellular states and can be manipulated to modulate cells towards a desired phenotype. Summary: Kinase enrichment analysis (KEA) is a web-based tool with an underlying database providing users with the ability to link lists of mammalian proteins/genes with the kinases that phosphorylate them. The system draws from several available kinase–substrate databases to compute kinase enrichment probability based on the distribution of kinase–substrate proportions in the background kinase–substrate database compared with kinases found to be associated with an input list of genes/proteins. Availability: The KEA system is freely available at http://amp.pharm.mssm.edu/lib/kea.jsp Contact: avi.maayan@mssm.edu PMID:19176546

  4. Picomolar platelet-activating factor mobilizes Ca to change platelet shape without activating phospholipase C or protein kinase C; simultaneous fluorometric measurement of intracellular free Ca concentration and aggregation.

    PubMed

    James-Kracke, M R; Sexe, R B; Shukla, S D

    1994-11-01

    The purpose of this study was to investigate signal transduction mechanisms activated by low and high concentrations of platelet-activating factor (PAF) in rabbit platelets and to contrast the responses to those induced by thrombin. We measured changes in intracellular free calcium ([Ca++]i) with fura2, while monitoring light scatter simultaneously as a measure of shape change and aggregation in a dual-excitation dual-emission spectrofluorometer. An abrupt 20% fall in light scatter, coincident with the peak of the [Ca++]i, indicated shape change in Ca-containing or Ca-free medium and was blocked by BAPTA loading and 10 microM cytochalasin B. A secondary decline in light scatter, indicating aggregation, occurred only in Ca-containing medium and only under conditions favoring protein kinase C (PKC) activation. PAF at 10(-12) M did not increase 1,4,5-inositol triphosphate content, which suggested PKC would not be activated. However, PAF at 10(-12) rapidly increased [Ca++]i to 900 nM in 7 sec seemingly by Ca influx through receptor-operated channels inducing shape change. PAF at 10(-9) and 10(-8) M increased [Ca++]i to 2 microM in 12 sec and induced both shape change and aggregation. However, in platelets pretreated with 100 nM staurosporine to inhibit protein kinases, 10(-9) M PAF did not cause aggregation even though [Ca++]i still rose to 2 microM, which indicated that PKC plays a role in aggregation but not in Ca++ mobilization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7965802

  5. Dimer of arfaptin 2 regulates NF-κB signaling by interacting with IKKβ/NEMO and inhibiting IKKβ kinase activity.

    PubMed

    You, Dong-Joo; Park, Cho Rong; Furlong, Michael; Koo, Okjae; Lee, Cheolju; Ahn, Curie; Seong, Jae Young; Hwang, Jong-Ik

    2015-11-01

    IκB kinases (IKKs) are a therapeutic target due to their crucial roles in various biological processes, including the immune response, the stress response, and tumor development. IKKs integrate various upstream signals that activate NF-κB by phosphorylating IκB and also regulate many proteins related to cell growth and metabolism. Although they function as a heteromeric complex comprised of kinase subunits and an adaptor, these kinases produce distinct cellular responses by phosphorylating different target molecules, suggesting that they may also be regulated in a subtype-specific manner. In this study, arfaptin 2 was identified as an IKKβ-specific binding partner. Interestingly, arfaptin 2 also interacted with NEMO. Domain mapping studies revealed that the C-terminal region, including the IKKβ HLH domain and the first coiled-coil NEMO region were respectively required for interactions with the arfaptin 2 N-terminal flexible region. Overexpression of arfaptin 2 inhibited tumor necrosis factor (TNF)-α-stimulated nuclear factor-κB (NF-κB) signaling, whereas downregulation of arfaptin 2 by small interfering RNA enhanced NF-κB activity. Dimerization of arfaptin 2 through the Bin-Amphiphysin-Rvs domain may be essential to inhibit activation of NF-κB through multimodal interactions with IKKβs or IKKβ/NEMO, as ectopic expression of the arfaptin 2 fragment responsible for IKK interactions did not change TNFα-stimulated NF-κB activation. These data indicate that arfaptin 2 is the first molecule to regulate NF-κB signaling by interacting with the functional IKK complex but not by direct inhibiting IKKβ phosphorylation. PMID:26296658

  6. Homeodomain-interacting protein kinases (Hipks) promote Wnt/Wg signaling through stabilization of beta-catenin/Arm and stimulation of target gene expression.

    PubMed

    Lee, Wendy; Swarup, Sharan; Chen, Joanna; Ishitani, Tohru; Verheyen, Esther M

    2009-01-01

    The Wnt/Wingless (Wg) pathway represents a conserved signaling cascade involved in diverse biological processes. Misregulation of Wnt/Wg signal transduction has profound effects on development. Homeodomain-interacting protein kinases (Hipks) represent a novel family of serine/threonine kinases. Members of this group (in particular Hipk2) are implicated as important factors in transcriptional regulation to control cell growth, apoptosis and development. Here, we provide genetic and phenotypic evidence that the sole Drosophila member of this family, Hipk, functions as a positive regulator in the Wg pathway. Expression of hipk in the wing rescues loss of the Wg signal, whereas loss of hipk can enhance decreased wg signaling phenotypes. Furthermore, loss of hipk leads to diminished Arm protein levels, whereas overexpression of hipk promotes the Wg signal by stabilizing Arm, resulting in activation of Wg responsive targets. In Wg transcriptional assays, Hipk enhanced Tcf/Arm-mediated gene expression in a kinase-dependent manner. In addition, Hipk can bind to Arm and Drosophila Tcf, and phosphorylate Arm. Using both in vitro and in vivo assays, Hipk was found to promote the stabilization of Arm. We observe similar molecular interactions between Lef1/beta-catenin and vertebrate Hipk2, suggesting a direct and conserved role for Hipk proteins in promoting Wnt signaling. PMID:19088090

  7. Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions.

    PubMed

    Villarino, A; Duran, R; Wehenkel, A; Fernandez, P; England, P; Brodin, P; Cole, S T; Zimny-Arndt, U; Jungblut, P R; Cerveñansky, C; Alzari, P M

    2005-07-29

    Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops. PMID:15978616

  8. Effect of process variables interaction on simultaneous adsorption of phenol and 4-chlorophenol: statistical modeling and optimization using RSM

    NASA Astrophysics Data System (ADS)

    Leong, Kwok-Yii; See, Sylvia; Lim, Jun-Wei; Bashir, Mohammed J. K.; Ng, Choon-Aun; Tham, Leony

    2016-02-01

    Results of the interaction of process variables and the consequential mixture of phenolic compounds adsorption study are expected to shed brighter light on the wastewater treatment applications. Accordingly, the aims of this research are to model and optimize the process variables which impinged on the simultaneous adsorption of phenol and 4-chlorophenol (4-CP) in the binary solution by spherical activated carbon (SAC). Batch assessments were designed using response surface methodology software. The process variables, namely SAC dosage and pH were varied over the 1.50-3.50 g/L and 4.00-9.00 g/L ranges, respectively, were experimented. The analysis of variance results showed the significant models could precisely predict the percentage removals of phenol and 4-CP, indicating models reliability. The interaction of process variables was inconspicuous for the case of phenol adsorption. However, increasing the pH would deteriorate the 4-CP adsorption which was partially offset by raising the SAC dosage. Considering the environmental benefits, optimization taken place at the SAC dosage and pH of 3.50 g/L and 7.60 g/L, respectively, was selected. By employing the optimized conditions of SAC dosage of 3.50 g/L at pH 7.60 for the adsorption process, the predicted phenol and 4-CP removal percentages were found to be 85.4 % (73.1 mg/g) and 96.2 % (82.6 mg/g), respectively, which were in agreement with the experimental runs.

  9. Fragment-Based Screening Maps Inhibitor Interactions in the ATP-Binding Site of Checkpoint Kinase 2

    PubMed Central

    Silva-Santisteban, M. Cris; Westwood, Isaac M.; Boxall, Kathy; Brown, Nathan; Peacock, Sam; McAndrew, Craig; Barrie, Elaine; Richards, Meirion; Mirza, Amin; Oliver, Antony W.; Burke, Rosemary; Hoelder, Swen; Jones, Keith; Aherne, G. Wynne; Blagg, Julian; Collins, Ian; Garrett, Michelle D.; van Montfort, Rob L. M.

    2013-01-01

    Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragment-based screening campaign using a combination of a high-concentration AlphaScreen™ kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors. PMID:23776527

  10. rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways.

    PubMed Central

    Shamloula, Hoda K; Mbogho, Mkajuma P; Pimentel, Angel C; Chrzanowska-Lightowlers, Zosia M A; Hyatt, Vanneta; Okano, Hideyuki; Venkatesh, Tadmiri R

    2002-01-01

    In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways. PMID:12072466

  11. Knockdown of Sec8 enhances the binding affinity of c-Jun N-terminal kinase (JNK)-interacting protein 4 for mitogen-activated protein kinase kinase 4 (MKK4) and suppresses the phosphorylation of MKK4, p38, and JNK, thereby inhibiting apoptosis.

    PubMed

    Tanaka, Toshiaki; Iino, Mitsuyoshi; Goto, Kaoru

    2014-12-01

    The exocyst complex, also called the Sec6/8 complex, is important for targeting exocytic vesicles to specific docking sites on the plasma membrane in yeast and mammalian cells. In addition to these original findings, recent results of studies suggest that Sec8 is also involved in oncogenesis, although the functional implications of Sec8 in cancer cells are not well understood. c-Jun N-terminal kinase-interacting protein 4 (JIP4) is a scaffold protein that plays a crucial role in the regulation of mitogen-activated protein kinase (MAPK) signaling cascades. The present study examined how Sec8 is involved in JIP4-mediated MAPK signaling under apoptotic conditions. It was found that Sec8 binds to and regulates JIP4, and that knockdown of Sec8 enhances the binding of JIP4 to MAPK kinase 4, thereby decreasing the phosphorylation of MAPK kinase 4, JNK, and p38. These results raise the possibility that Sec8 serves as an important regulator of MAPK signaling cascades. PMID:25244576

  12. Interaction of Omega, Sigma, and Theta Glutathione Transferases with p38b Mitogen-Activated Protein Kinase from the Fruit Fly, Drosophila melanogaster

    PubMed Central

    Wongtrakul, J.; Janphen, K.; Saisawang, C.; Ketterman, A.J.

    2014-01-01

    Glutathione S-transferases (GSTs) are a diverse family of phase II detoxification enzymes found in almost all organisms. Besides playing a major role in the detoxification of xenobiotic and toxic compounds, GSTs are also involved in the regulation of mitogen activated protein (MAP) kinase signal transduction by interaction with proteins in the pathway. An in vitro study was performed for Theta, Omega, Sigma GSTs and their interaction with MAP kinase p38b protein from the fruit fly Drosophila melanogaster Meigen (Diptera: Drosophilidae). The study included the effects of all five Omega class GSTs (DmGSTO1, DmGSTO2a, DmGSTO2b, DmGSTO3, DmGSTO4), all five Theta class GSTs (DmGSTT1, DmGSTT2, DmGSTT3a, DmGSTT3b, DmGSTT4), and one Sigma class glutathione transferase on the activity of Drosophila p38b, including the reciprocal effect of this kinase protein on glutathione transferase activity. It was found that DmGSTT2, DmGSTT3b, DmGSTO1, and DmGSTO3 activated p38b significantly. Substrate specificities of GSTs were also altered after co-incubation with p38b. Although p38b activated DmGSTO1, DmGSTO2a, and DmGSTT2, it inhibited DmGSTT3b and DmGSTO3 activity toward xenobiotic and physiological substrates tested. These results suggest a novel link between Omega and Theta GSTs with the p38b MAP kinase pathway. PMID:25373207

  13. The Tomato Calcium Sensor Cbl10 and Its Interacting Protein Kinase Cipk6 Define a Signaling Pathway in Plant Immunity[C][W

    PubMed Central

    de la Torre, Fernando; Gutiérrez-Beltrán, Emilio; Pareja-Jaime, Yolanda; Chakravarthy, Suma; Martin, Gregory B.; del Pozo, Olga

    2013-01-01

    Ca2+ signaling is an early and necessary event in plant immunity. The tomato (Solanum lycopersicum) kinase Pto triggers localized programmed cell death (PCD) upon recognition of Pseudomonas syringae effectors AvrPto or AvrPtoB. In a virus-induced gene silencing screen in Nicotiana benthamiana, we independently identified two components of a Ca2+-signaling system, Cbl10 (for calcineurin B-like protein) and Cipk6 (for calcineurin B-like interacting protein kinase), as their silencing inhibited Pto/AvrPto-elicited PCD. N. benthamiana Cbl10 and Cipk6 are also required for PCD triggered by other plant resistance genes and virus, oomycete, and nematode effectors and for host susceptibility to two P. syringae pathogens. Tomato Cipk6 interacts with Cbl10 and its in vitro kinase activity is enhanced in the presence of Cbl10 and Ca2+, suggesting that tomato Cbl10 and Cipk6 constitute a Ca2+-regulated signaling module. Overexpression of tomato Cipk6 in N. benthamiana leaves causes accumulation of reactive oxygen species (ROS), which requires the respiratory burst homolog RbohB. Tomato Cbl10 and Cipk6 interact with RbohB at the plasma membrane. Finally, Cbl10 and Cipk6 contribute to ROS generated during effector-triggered immunity in the interaction of P. syringae pv tomato DC3000 and N. benthamiana. We identify a role for the Cbl/Cipk signaling module in PCD, establishing a mechanistic link between Ca2+ and ROS signaling in plant immunity. PMID:23903322

  14. A CYSTEINE-RICH EXTRACELLULAR PROTEIN, LAT52, INTERACTS WITH THE EXTRACVELLULAR DOMAIN OF THE POLLEN RECEPTOR KINASE LEPRK2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pollen germination and pollen tube growth are thought to require extracellular cues, but how these cues are perceived and transduced remains largely unknown. Pollen receptor kinases are plausible candidates for this role; they might bind extracellular ligands and thereby mediate cytoplasmic events r...

  15. Homeodomain-Interacting Protein Kinase (HIPK)-1 Is Required for Splenic B Cell Homeostasis and Optimal T-Independent Type 2 Humoral Response

    PubMed Central

    Guerra, Fiona M.; Gommerman, Jennifer L.; Corfe, Steven A.; Paige, Christopher J.; Rottapel, Robert

    2012-01-01

    The homeodomain-interacting protein kinase (HIPK) family is comprised of four highly related serine/threonine kinases originally identified as co-repressors for various homeodomain-containing transcription factors. The HIPKs have been shown to be involved in growth regulation and apoptosis, with numerous studies highlighting HIPK regulation of the tumor suppressor p53. In this study, we have discovered a B cell homeostatic defect in HIPK1-deficient (HIPK1−/−) mice. Lymphopoietic populations within the thymus and bone marrow of HIPK1−/− mice appeared normal based upon FACS analysis; however, the spleen exhibited a reduced number of total B cells with a significant loss of transitional-1 and follicular B cell populations. Interestingly, the marginal zone B cell population was expanded in HIPK1−/− mice, yielding an increased frequency of these cells. HIPK1−/− B cells exhibited impaired cell division in response to B cell receptor cross-linking in vitro based upon thymidine incorporation or CFSE dilution; however, the addition of CD40L rescued HIPK1−/− proliferation to wild-type levels. Despite the expanded MZ B cell population in the HIPK1−/− mice, the T-independent type 2 humoral response was impaired. These data identify HIPK1 as a novel kinase required for optimal B cell function in mice. PMID:22545114

  16. DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4-ones as Highly Potent and Monoselective Receptor Interacting Protein 1 Kinase Inhibitors.

    PubMed

    Harris, Philip A; King, Bryan W; Bandyopadhyay, Deepak; Berger, Scott B; Campobasso, Nino; Capriotti, Carol A; Cox, Julie A; Dare, Lauren; Dong, Xiaoyang; Finger, Joshua N; Grady, LaShadric C; Hoffman, Sandra J; Jeong, Jae U; Kang, James; Kasparcova, Viera; Lakdawala, Ami S; Lehr, Ruth; McNulty, Dean E; Nagilla, Rakesh; Ouellette, Michael T; Pao, Christina S; Rendina, Alan R; Schaeffer, Michelle C; Summerfield, Jennifer D; Swift, Barbara A; Totoritis, Rachel D; Ward, Paris; Zhang, Aming; Zhang, Daohua; Marquis, Robert W; Bertin, John; Gough, Peter J

    2016-03-10

    The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate. PMID:26854747

  17. The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

    PubMed Central

    Brzoska, P M; Chen, H; Zhu, Y; Levin, N A; Disatnik, M H; Mochly-Rosen, D; Murnane, J P; Christman, M F

    1995-01-01

    Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C. Images Fig. 3 Fig. 4 Fig. 5 PMID:7644499

  18. Interactive exploration of the vulnerability of the human infrastructure: an approach using simultaneous display of similar locations

    NASA Astrophysics Data System (ADS)

    Ceré, Raphaël; Kaiser, Christian

    2015-04-01

    Currently, three quarters of the Swiss population is living in urban areas. The total population is still increasing, and urbanized space is increasing event faster. Consequently, the intensity of use has decreased but the exposure of the urban space to natural events has grown along with the cost related to the impact of hazards. In line with this fact, during the 20th century there has been a noticeable increase of natural disasters accompanied by the rapid increase of the world population, leading to higher costs. Additionally to the fact that more people are exposed to natural hazards, the value of goods globally has increased more than proportionally. Consequently, the vulnerability of urban space is, more than ever before, a major issue for socio-economic development. Here, vulnerability is defined as the potential human loss or loss of infrastructure caused by a hazardous event. It encompasses factors of urban infrastructure, population and the environment, which increase the susceptibility of a location to the impact of hazards. This paper describes a novel method for improving the interactive use of exploratory data analysis in the context of minimizing vulnerability and disaster risk by prevention or mitigation. This method is used to assess the similarity between different locations with respect to several characteristics relevant to vulnerability at different scales, allowing for automatic display of multiple locations similar to the one under investigation by an expert. Visualizing vulnerability simultaneously for several locations allows for analyzing and comparing of metric characteristics between multiple places and at different scales. The interactivity aspect is also useful for understanding vulnerability patterns and it facilitates disaster risk management and decisions on global preventive measures in urban spaces. Metrics for vulnerability assessment can be extracted from extensive geospatial datasets such as high-resolution digital elevation

  19. Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.

    PubMed

    Sui, Dexin; Xu, Xinjing; Ye, Xuemei; Liu, Mengyu; Mianecki, Maxwell; Rattanasinchai, Chotirat; Buehl, Christopher; Deng, Xiexiong; Kuo, Min-Hao

    2015-01-01

    Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research. PMID:25385071

  20. Tobacco Translationally Controlled Tumor Protein Interacts with Ethylene Receptor Tobacco Histidine Kinase1 and Enhances Plant Growth through Promotion of Cell Proliferation1[OPEN

    PubMed Central

    Tao, Jian-Jun; Cao, Yang-Rong; Chen, Hao-Wei; Wei, Wei; Li, Qing-Tian; Ma, Biao; Zhang, Wan-Ke; Chen, Shou-Yi; Zhang, Jin-Song

    2015-01-01

    Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation. PMID:25941315

  1. BcIqg1, a fungal IQGAP homolog, interacts with NADPH oxidase, MAP kinase and calcium signaling proteins and regulates virulence and development in Botrytis cinerea.

    PubMed

    Marschall, Robert; Tudzynski, Paul

    2016-07-01

    NADPH oxidases (Nox) produce reactive oxygen species (ROS) in multicellular eukaryotic organisms. They trigger defense reactions ('oxidative burst') - in phagocytes and plant cells -, and are involved in a broad range of differentiation processes. Fungal Nox-complexes play a central role in vegetative, sexual and pathogenic processes. In contrast to mammalian systems, knowledge is limited about composition, localisation and connection to major signaling cascades in fungi. Here, we characterize a fungal homolog of the RasGAP scaffold protein IQGAP, which links several major signaling processes, including Nox in mammalian cell lines. We show that BcIqg1 interacts directly with a cytosolic, regulatory component (BcRac) and a membrane-associated subunit (BcNoxD) of a Nox-complex in the pathogen Botrytis cinerea. Thus, this protein may be a scaffold that mediates interaction of the catalytic subunits with the regulator BcNoxR. The protein interacts with modules of the MAP kinase- and calcium-dependent signaling pathways. Functional analysis of BcIqg1 substantiated its involvement in different signaling pathways. It mediates the Ca(2+) -triggered nuclear translocation of - BcCRZ1 and the MAP kinase BcBmp1. BcIqg1 is involved in resistance against oxidative and membrane stress and is required for several developmental processes including formation of sclerotia, conidial anastomosis tubes and infection cushions as well as for virulence. PMID:27062300

  2. N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points

    PubMed Central

    Islam, Md Ariful; Sharif, Syeda Ridita; Lee, HyunSook; Seog, Dae-Hyun; Moon, Il Soo

    2015-01-01

    N-acetylglucosamine kinase (GlcNAc kinase or NAGK) is a ubiquitously expressed enzyme in mammalian cells. Recent studies have shown that NAGK has an essential structural, non-enzymatic role in the upregulation of dendritogenesis. In this study, we conducted yeast two-hybrid screening to search for NAGK-binding proteins and found a specific interaction between NAGK and dynein light-chain roadblock type 1 (DYNLRB1). Immunocytochemistry (ICC) on hippocampal neurons using antibodies against NAGK and DYNLRB1 or dynein heavy chain showed some colocalization, which was increased by treating the live cells with a crosslinker. A proximity ligation assay (PLA) of NAGK-dynein followed by tubulin ICC showed the localization of PLA signals on microtubule fibers at dendritic branch points. NAGK-dynein PLA combined with Golgi ICC showed the colocalization of PLA signals with somal Golgi facing the apical dendrite and with Golgi outposts in dendritic branch points and distensions. NAGK-Golgi PLA followed by tubulin or DYNLRB1 ICC showed that PLA signals colocalize with DYNLRB1 at dendritic branch points and at somal Golgi, indicating a tripartite interaction between NAGK, dynein and Golgi. Finally, the ectopic introduction of a small peptide derived from the C-terminal amino acids 74–96 of DYNLRB1 resulted in the stunting of hippocampal neuron dendrites in culture. Our data indicate that the NAGK-dynein-Golgi tripartite interaction at dendritic branch points functions to regulate dendritic growth and/or branching. PMID:26272270

  3. Interaction of ribosomal protein L22 with casein kinase 2α: a novel mechanism for understanding the biology of non-small cell lung cancer.

    PubMed

    Yang, Mingxia; Sun, Haibo; He, Ji; Wang, Hong; Yu, Xiaowei; Ma, Lei; Zhu, Changliang

    2014-07-01

    Dysfunction of ribosomal proteins (RPs) may play an important role in molecular tumorigenesis, such as lung cancer, acting in extraribosomal functions. Many protein-protein interaction studies and genetic screens have confirmed the extraribosomal capacity of RPs. As reported, ribosomal protein L22 (RPL22) dysfunction could increase cancer risk. In the present study, we examined RPL22-protein complexes in lung cancer cells. Tandem affinity purification (TAP) was used to screen the RPL22-protein complexes, and GST pull-down experiments and confocal microscopy were used to assess the protein-protein interaction. The experiment of kinase assay was used to study the function of the RPL22-protein complexes. The results showed that several differentially expressed proteins were isolated and identified by LC-MS/MS, which revealed that one of the protein complexes included casein kinase 2α (CK2α). RPL22 and CK2α interact in vitro. RPL22 also inhibited CK2α substrate phosphorylation in vitro. This is the first report of the RPL22-CK2α relationship in lung cancer. Dysregulated CK2 may impact cell proliferation and apoptosis, key features of cancer cell biology. Our results indicate that RPL22 may be a candidate anticancer agent due to its CK2α-binding and -inhibitory functions in human lung cancer. PMID:24840952

  4. The Abi-domain protein Abx1 interacts with the CovS histidine kinase to control virulence gene expression in group B Streptococcus.

    PubMed

    Firon, Arnaud; Tazi, Asmaa; Da Cunha, Violette; Brinster, Sophie; Sauvage, Elisabeth; Dramsi, Shaynoor; Golenbock, Douglas T; Glaser, Philippe; Poyart, Claire; Trieu-Cuot, Patrick

    2013-02-01

    Group B Streptococcus (GBS), a common commensal of the female genital tract, is the leading cause of invasive infections in neonates. Expression of major GBS virulence factors, such as the hemolysin operon cyl, is regulated directly at the transcriptional level by the CovSR two-component system. Using a random genetic approach, we identified a multi-spanning transmembrane protein, Abx1, essential for the production of the GBS hemolysin. Despite its similarity to eukaryotic CaaX proteases, the Abx1 function is not involved in a post-translational modification of the GBS hemolysin. Instead, we demonstrate that Abx1 regulates transcription of several virulence genes, including those comprising the hemolysin operon, by a CovSR-dependent mechanism. By combining genetic analyses, transcriptome profiling, and site-directed mutagenesis, we showed that Abx1 is a regulator of the histidine kinase CovS. Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS, overcoming the need for an additional signal. Conversely, the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state. Using a bacterial two-hybrid system, direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing. We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1. In conclusion, our study reports a regulatory function for Abx1, a member of a large protein family with a characteristic Abi-domain, which forms a signaling complex with the histidine kinase CovS in GBS. PMID:23436996

  5. A surface plasmon resonance study of the interactions between the component subunits of protein kinase CK2 and two protein substrates, casein and calmodulin.

    PubMed

    Benítez, M J; Cochet, C; Jiménez, J S

    2001-11-01

    Surface plasmon resonance has been used to study the interaction between the subunits composing protein kinase CK2 (two catalytic, alpha-subunits, and two regulatory, beta-subunits), as well as the interaction of each subunit with two types of protein substrates, casein, the phosphorylation of which is activated by the regulatory subunit, and calmodulin, which belongs to the kind of substrates on which the catalytic subunit is downregulated by the regulatory subunit. The interaction of casein with the catalytic subunit differs from the interaction with the holoenzyme. Similarly to the interaction with the regulatory subunit, the catalytic subunit interacts with the protein substrate forming a very stable, irreversible complex. The reconstituted holoenzyme, however, binds casein reversibly, displaying a binding mode similar to that displayed by the regulatory subunit. The interaction of calmodulin with the catalytic subunit gives place, like in the case of casein, to an irreversible complex. The interactions with the regulatory subunit and with the holoenzyme were practically negligible, and the interaction with the regulatory subunit disappeared upon increasing the temperature value to close to 30 degrees C. The presence of polylysine induced a high increase in the extent of calmodulin binding to the holoenzyme. The results obtained suggest that CK2beta subunit and protein substrates share a common, or at least an overlapping, site of interaction on the catalytic subunit. The interaction between both subunits would prevent substrates from binding irreversibly to alpha subunit, and, at the same time, it would generate a new and milder site of interaction between the whole holoenzyme and the protein substrate. The main difference between casein and calmodulin would consist in the lower affinity display by the last for the new site generated upon the binding of the regulatory subunit, in the absence of polycations like polylysine. PMID:11827172

  6. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

    PubMed Central

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

    2015-01-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

  7. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses.

    PubMed

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A; Kay, Steve A; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R; Schroeder, Julian I

    2015-09-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

  8. Interaction with Pyruvate Kinase M2 Destabilizes Tristetraprolin by Proteasome Degradation and Regulates Cell Proliferation in Breast Cancer

    PubMed Central

    Huang, Liangqian; Yu, Zhenhai; Zhang, Zhenchao; Ma, Wenjing; Song, Shaoli; Huang, Gang

    2016-01-01

    Pyruvate kinase M2 (PKM2), which is predominantly expressed in most cancers, plays a key role in the Warburg effect. However, how PKM2 functions as a tumor supportive protein has not been fully elucidated. Here, we identified tristetraprolin (TTP), an AU-rich, element-binding protein that regulates mRNA stability, as a new binding partner of PKM2. Our data reveal that PKM2 suppresses TTP protein levels by promoting its phosphorylation, ubiquitination, and proteasome degradation, reducing its mRNA turnover ability and ultimately impairing cell viability in breast cancer cells. The p38/mitogen-activated protein kinase (MAPK) pathway might be involved in PKM2-mediated TTP degradation, while treatment with the p38 inhibitor or siRNA abolished PKM2-induced TTP protein degradation. These findings demonstrate that PKM2–TTP association is crucial for regulating breast cancer cell proliferation and is therefore a potential therapeutic target in cancer. PMID:26926077

  9. Escherichia coli K1 internalization via caveolae requires caveolin-1 and protein kinase Calpha interaction in human brain microvascular endothelial cells.

    PubMed

    Sukumaran, Sunil K; Quon, Michael J; Prasadarao, Nemani V

    2002-12-27

    The morbidity and mortality associated with Escherichia coli K1 meningitis during the neonatal period have remained significant over the last decade and are once again on the rise. Transcytosis of brain microvascular endothelial cells (BMEC) by E. coli within an endosome to avoid lysosomal fusion is crucial for dissemination into the central nervous system. Central to E. coli internalization of BMEC is the expression of OmpA (outer membrane protein A), which interacts with its receptor for the actin reorganization that leads to invasion. However, nothing is known about the nature of the signaling events for the formation of endosomes containing E. coli K1. We show here that E. coli K1 infection of human BMEC (HBMEC) results in activation of caveolin-1 for bacterial uptake via caveolae. The interaction of caveolin-1 with phosphorylated protein kinase Calpha (PKCalpha) at the E. coli attachment site is critical for the invasion of HBMEC. Optical sectioning of confocal images of infected HBMEC indicates continuing association of caveolin-1 with E. coli during transcytosis. Overexpression of a dominant-negative form of caveolin-1 containing mutations in the scaffolding domain blocked the interaction of phospho-PKCalpha with caveolin-1 and the E. coli invasion of HBMEC, but not actin cytoskeleton rearrangement or the phosphorylation of PKCalpha. The interaction of caveolin-1 with phospho-PKCalpha was completely abrogated in HBMEC overexpressing dominant-negative forms of either focal adhesion kinase or PKCalpha. Treatment of HBMEC with a cell-permeable peptide that represents the scaffolding domain, which was coupled to an antennapedia motif of a Drosophila transcription factor significantly blocked the interaction of caveolin-1 with phospho-PKCalpha and E. coli invasion. These results show that E. coli K1 internalizes HBMEC via caveolae and that the scaffolding domain of caveolin-1 plays a significant role in the formation of endosomes. PMID:12386163

  10. Simultaneous determination of erlotinib and tamoxifen in rat plasma using UPLC-MS/MS: Application to pharmacokinetic interaction studies.

    PubMed

    Maher, Hadir M; Alzoman, Nourah Z; Shehata, Shereen M

    2016-08-15

    Tamoxifen (TAM) is a non-steroidal estrogen receptor antagonist that enhances erlotinib (ERL)-induced cytotoxicity in the treatment of NSCLC. ERL and TAM are metabolized by CYP3A4 enzymes. In addition, both drugs have the potential of altering the enzymatic activity through either inhibition (ERL) or induction (TAM). Thus it was expected that pharmacokinetics (PK) drug-drug interactions (DDIs) could be encountered following their co-administration. In this respect, a bioanalytical UPLC-MS/MS method has been developed and validated for the simultaneous determination of ERL and TAM in rat plasma samples, using ondansetron (OND) as an internal standard (IS). Plasma samples were prepared using mixed mode cationic solid phase extraction (SPE) STRATA™ -X-C 33μm cartridges with good extraction recovery of both drugs from rat plasma (Er% from -13.92 to -3.32). The drugs were separated on a Waters BEH™ C18 column with an isocratic elution using a mobile phase composed of a mixture of acetonitrile and water, each with 0.15% formic acid, in the ratio of 80: 20, v/v. Quantitation was carried out using the positive ionization mode with multiple reaction monitoring (MRM) at m/z 394.20>278.04 (ERL), m/z 372.25>72.01 (TAM), and m/z 294.18>170.16 (OND). The method was fully validated as per the FDA guidelines over the concentration range of 0.2-50ng/mL with very low lower limit of quantification (LLOQ) of 0.2ng/mL for both ERL and TAM. The intra- and inter-day assay precision (in terms of relative standard deviation, RSD) and accuracy (in terms of percentage relative error, % Er) were evaluated for both drugs and the calculated values evaluated at four different concentration levels were within the acceptable limits (<15%) for concentrations other than LLOQ and 20% for LLOQ. The method was successfully applied to the study of possible PK-DDI following the oral administration of ERL and TAM in a combination, compared to their single administration. PMID:27336702

  11. Simultaneous determination of four local anesthetics by CE with ECL and study on interaction between procainamide and human serum albumin.

    PubMed

    Duan, Hong-Bing; Cao, Jun-Tao; Yang, Jiu-Jun; Wang, Hui; Liu, Yan-Ming

    2016-07-01

    A new method of capillary electrophoresis (CE) coupled with tris(2, 2'-bipyridyl) ruthenium(II) electrochemiluminescence (ECL) detection has been developed to detect four local anesthetics procainamide (PAH), tetracaine (TCH), proparacaine (PCH) and cinchocaine (CIN) simultaneously. An europium (III)-doped prussian blue analogue film (Eu-PB) modified platinum electrode was prepared and applied to improve the detection sensitivity. The parameters including additives, concentration and pH of the running buffer, separation voltage and detection potential that affect CE separation and ECL detection were optimized in detail. The four local anesthetics were baseline separated and detected within 10min under the optimized conditions. The detection limits (LOD) of PAH, TCH, PCH and CIN are 5.5×10(-8), 9.6×10(-8), 2.5×10(-8) and 3.5×10(-8)molL(-1) (S/N=3), respectively. RSDs of the migration time for four analytes range from 1.2% to 2.5% within intraday and from 2.4% to 4.9% in interday, RSDs of the peak area for four analytes are from 1.7% to 3.3% within intraday and from 2.2% to 5.6% in interday, respectively. The limits of quantitation (LOQ) (S/N=10) for PAH, TCH, PCH and CIN in human urine sample are 5.9×10(-7), 9.2×10(-7), 8.3×10(-7) and 5.0×10(-7)molL(-1), separately. The recoveries (n=3) of four analytes in human urine are from 87.6% to 107.7% with less than 5.9% in RSDs. The developed method was used to determine four local anesthetics in human urine samples and investigate the interaction between PAH and human serum albumin (HSA). The number of binding sites and the binding constant of PAH with HSA were calculated to be 1.03 and 2.4×10(4)Lmol(-1), respectively. PMID:27154684

  12. Identification of Potential Plk1 Targets in a Cell-Cycle Specific Proteome through Structural Dynamics of Kinase and Polo Box-Mediated Interactions

    PubMed Central

    Bibi, Nousheen; Parveen, Zahida; Rashid, Sajid

    2013-01-01

    Polo like kinase 1 (Plk1) is a key player in orchestrating the wide variety of cell-cycle events ranging from centrosome maturation, mitotic entry, checkpoint recovery, transcriptional control, spindle assembly, mitotic progression, cytokinesis and DNA damage checkpoints recovery. Due to its versatile nature, Plk1 is considered an imperative regulator to tightly control the diverse aspects of the cell cycle network. Interactions among Plk1 polo box domain (PBD) and its putative binding proteins are crucial for the activation of Plk1 kinase domain (KD). To date, only a few substrate candidates have been characterized through the inclusion of both polo box and kinase domain-mediated interactions. Thus it became compelling to explore precise and specific Plk1 substrates through reassessment and extension of the structure-function paradigm. To narrow this apparently wide gap in knowledge, here we employed a thorough sequence search of Plk1 phosphorylation signature containing proteins and explored their structure-based features like conceptual PBD-binding capabilities and subsequent recruitment of KD directed phosphorylation to dissect novel targets of Plk1. Collectively, we identified 4,521 phosphodependent proteins sharing similarity to the consensus phosphorylation and PBD recognition motifs. Subsequent application of filters including similarity index, Gene Ontology enrichment and protein localization resulted in stringent pre-filtering of irrelevant candidates and isolated unique targets with well-defined roles in cell-cycle machinery and carcinogenesis. These candidates were further refined structurally using molecular docking and dynamic simulation assays. Overall, our screening approach enables the identification of several undefined cell-cycle associated functions of Plk1 by uncovering novel phosphorylation targets. PMID:23967120

  13. Complex Drug Interactions of the HIV Protease Inhibitors 3: Effect of Simultaneous or Staggered Dosing of Digoxin and Ritonavir, Nelfinavir, Rifampin, or Bupropion

    PubMed Central

    Kirby, Brian J.; Collier, Ann C.; Kharasch, Evan D.; Whittington, Dale; Thummel, Kenneth E.

    2012-01-01

    As part of a larger clinical drug-drug interaction (DDI) study aimed at in vitro to in vivo prediction of HIV protease inhibitor metabolic and transporter-based DDIs, we measured the inductive (staggered administration) and inductive plus inhibitory (simultaneously administered) effect of multiple dose ritonavir (RTV), nelfinavir (NFV), or rifampin (RIF) on the pharmacokinetics of the P-glycoprotein probe, digoxin (DIG), when administered simultaneously or staggered with the protease inhibitors or RIF. In both cases, NFV did not significantly affect DIG disposition. RTV decreased DIG renal clearance (Clrenal) when administered simultaneously or staggered but significantly increased DIG area under the curve from time zero to 24 h (AUC0–24 h) only when administered simultaneously. RIF decreased DIG AUC0–24 h only when RIF and DIG administration was staggered. When RIF and DIG were administered simultaneously, DIG maximal observed plasma concentration and area under the curve from time zero to 4 h were significantly increased, and DIG Clrenal was decreased. An unexpected and potentially clinically significant DDI was observed between DIG and the CYP2B6 probe, bupropion, which decreased DIG AUC0–24 h 1.6-fold and increased Clrenal 1.8-fold. Because this was an unexpected DDI and our studies were not specifically designed to quantify this interaction, further studies are required to confirm the interaction and understand the mechanistic basis of the DDI. In summary, RTV or NFV do not induce P-glycoprotein activity measured with DIG, and RIF does so only under staggered administration. PMID:22190694

  14. MAP kinase cascades: scaffolding signal specificity.

    PubMed

    van Drogen, Frank; Peter, Matthias

    2002-01-22

    Scaffold proteins organize many MAP kinase pathways by interacting with several components of these cascades. Recent studies suggest that scaffold proteins provide local activation platforms that contribute to signal specificity by insulating different MAP kinase pathways. PMID:11818078

  15. Zscan4 is regulated by PI3-kinase and DNA-damaging agents and directly interacts with the transcriptional repressors LSD1 and CtBP2 in mouse embryonic stem cells.

    PubMed

    Storm, Michael P; Kumpfmueller, Benjamin; Bone, Heather K; Buchholz, Michael; Sanchez Ripoll, Yolanda; Chaudhuri, Julian B; Niwa, Hitoshi; Tosh, David; Welham, Melanie J

    2014-01-01

    The Zscan4 family of genes, encoding SCAN-domain and zinc finger-containing proteins, has been implicated in the control of early mammalian embryogenesis as well as the regulation of pluripotency and maintenance of genome integrity in mouse embryonic stem cells. However, many features of this enigmatic family of genes are poorly understood. Here we show that undifferentiated mouse embryonic stem cell (ESC) lines simultaneously express multiple members of the Zscan4 gene family, with Zscan4c, Zscan4f and Zscan4-ps2 consistently being the most abundant. Despite this, between only 0.1 and 0.7% of undifferentiated mouse pluripotent stem cells express Zscan4 protein at a given time, consistent with a very restricted pattern of Zscan4 transcripts reported previously. Herein we demonstrate that Zscan4 expression is regulated by the p110α catalytic isoform of phosphoinositide 3-kinases and is induced following exposure to a sub-class of DNA-damage-inducing agents, including Zeocin and Cisplatin. Furthermore, we observe that Zscan4 protein expression peaks during the G2 phase of the cell cycle, suggesting that it may play a critical role at this checkpoint. Studies with GAL4-fusion proteins suggest a role for Zscan4 in transcriptional regulation, further supported by the fact that protein interaction analyses demonstrate that Zscan4 interacts with both LSD1 and CtBP2 in ESC nuclei. This study advances and extends our understanding of Zscan4 expression, regulation and mechanism of action. Based on our data we propose that Zscan4 may regulate gene transcription in mouse ES cells through interaction with LSD1 and CtBP2. PMID:24594919

  16. Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2.

    PubMed

    Janeček, Matej; Rossmann, Maxim; Sharma, Pooja; Emery, Amy; Huggins, David J; Stockwell, Simon R; Stokes, Jamie E; Tan, Yaw S; Almeida, Estrella Guarino; Hardwick, Bryn; Narvaez, Ana J; Hyvönen, Marko; Spring, David R; McKenzie, Grahame J; Venkitaraman, Ashok R

    2016-01-01

    The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the 'Y pocket') that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases. PMID:27339427

  17. Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ.

    PubMed

    Maisonneuve, Pierre; Caillet-Saguy, Célia; Vaney, Marie-Christine; Bibi-Zainab, Edoo; Sawyer, Kristi; Raynal, Bertrand; Haouz, Ahmed; Delepierre, Muriel; Lafon, Monique; Cordier, Florence; Wolff, Nicolas

    2016-08-01

    The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction. PMID:27246854

  18. Focal Adhesion Kinase Directly Interacts with TSC2 Through Its FAT Domain and Regulates Cell Proliferation in Cashmere Goat Fetal Fibroblasts.

    PubMed

    Zheng, Xu; Bao, Wenlei; Yang, Jiaofu; Zhang, Tao; Sun, Dongsheng; Liang, Yan; Li, Shuyu; Wang, Yanfeng; Feng, Xue; Hao, Huifang; Wang, Zhigang

    2016-09-01

    Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor tyrosine kinase that senses a variety of extracellular signals, such as growth factors and integrins, to control the process of cell proliferation and metabolism. We cloned three goat FAK transcript variants (KM655805, KM658268, and KM658269) that encode 1052, 1006, and 962 amino-acid residue proteins. Bioinformatics analysis indicated that the putative FAK protein contains an FERM domain, a PTK domain, two Proline-rich regions, and a focal adhesion-targeting (FAT) domain. All the three transcript variants of FAK were detected in seven different goat tissues, and variant 1 had the most accumulation whereas variant 2 and variant 3 had lower accumulation. Treatment of goat fetal fibroblasts (GFbs) with a specific FAK inhibitor, TAE226, inhibited cell proliferation (p < 0.05) and induced damage to the cell morphology in a dose- and time-dependent manner. Further research demonstrated that FAK directly interacted with TSC2 (Tuberous sclerosis 2) tuberin domain through its C-terminus, which contains the complete FAT domain. In conclusion, our results indicated that FAK may be widely expressed in Cashmere goat tissues and its products participate in the mammalian target of rapamycin signaling pathway and cell proliferation through a direct interaction with TSC2 in GFBs. PMID:27380318

  19. Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2

    PubMed Central

    Janeček, Matej; Rossmann, Maxim; Sharma, Pooja; Emery, Amy; Huggins, David J.; Stockwell, Simon R.; Stokes, Jamie E.; Tan, Yaw S.; Almeida, Estrella Guarino; Hardwick, Bryn; Narvaez, Ana J.; Hyvönen, Marko; Spring, David R.; McKenzie, Grahame J.; Venkitaraman, Ashok R.

    2016-01-01

    The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the ‘Y pocket’) that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases. PMID:27339427

  20. Downregulation of microRNA‑146a inhibits ovarian granulosa cell apoptosis by simultaneously targeting interleukin‑1 receptor‑associated kinase and tumor necrosis factor receptor‑associated factor 6.

    PubMed

    Chen, Xi; Xie, Mingxuan; Liu, Da; Shi, Ke

    2015-10-01

    Premature ovarian failure (POF), an ovarian disorder of multifactorial origin, is defined as the occurrence of amenorrhea, hypergonadotropism and hypoestrogenism in females <40 years old. Apoptosis of ovarian granulosa cells is important in POF and understanding the regulatory mechanism underlying ovarian granulosa cell apoptosis may be beneficial for the management of POF. Increasing evidence suggests that microRNAs (miRs) have a regulatory function in oocyte maturation and ovarian follicular development. In the present study, the expression of miR‑146a in plasma and ovarian granulosa cells obtained from patients with POF, its effect on the apoptosis of ovarian granulosa cells and the possible underlying mechanisms were examined. The present study demonstrated that compared with the control groups, the expression of miR‑146a in the plasma and in ovarian granulosa cells of patients with POF was significantly upregulated. Furthermore, it was found that miR‑146a simultaneously targeted interleukin‑1 receptor‑associated kinase (IRAK1) and tumor necrosis factor receptor‑associated factor 6 (TRAF6), which regulated the activity of nuclear factor‑κB and IκBα. In addition, the results demonstrated that inhibition of the caspase cascade by caspase inhibitors attenuated the effects of miR‑146a on ovarian granulosa cell apoptosis. Taken together, these results suggest that miR‑146a has an important promoting effect on the apoptosis of granulosa cells by targeting IRAK1 and TRAF6 via the caspase cascade pathway. These results may be useful for the management of POF. PMID:26151128

  1. Sak1 kinase interacts with Pso2 nuclease in response to DNA damage induced by interstrand crosslink-inducing agents in Saccharomyces cerevisiae.

    PubMed

    Munari, Fernanda M; Revers, Luis F; Cardone, Jacqueline M; Immich, Bruna F; Moura, Dinara J; Guecheva, Temenouga N; Bonatto, Diego; Laurino, Jomar P; Saffi, Jenifer; Brendel, Martin; Henriques, João A P

    2014-01-01

    By isolating putative binding partners through the two-hybrid system (THS) we further extended the characterization of the specific interstrand cross-link (ICL) repair gene PSO2 of Saccharomyces cerevisiae. Nine fusion protein products were isolated for Pso2p using THS, among them the Sak1 kinase, which interacted with the C-terminal β-CASP domain of Pso2p. Comparison of mutagen-sensitivity phenotypes of pso2Δ, sak1Δ and pso2Δsak1Δ disruptants revealed that SAK1 is necessary for complete WT-like repair. The epistatic interaction of both mutant alleles suggests that Sak1p and Pso2p act in the same pathway of controlling sensitivity to DNA-damaging agents. We also observed that Pso2p is phosphorylated by Sak1 kinase in vitro and co-immunoprecipitates with Sak1p after 8-MOP+UVA treatment. Survival data after treatment of pso2Δ, yku70Δ and yku70Δpso2Δ with nitrogen mustard, PSO2 and SAK1 with YKU70 or DNL4 single-, double- and triple mutants with 8-MOP+UVA indicated that ICL repair is independent of YKu70p and DNL4p in S. cerevisiae. Furthermore, a non-epistatic interaction was observed between MRE11, PSO2 and SAK1 genes after ICL induction, indicating that their encoded proteins act on the same substrate, but in distinct repair pathways. In contrast, an epistatic interaction was observed for PSO2 and RAD52, PSO2 and RAD50, PSO2 and XRS2 genes in 8-MOP+UVA treated exponentially growing cells. PMID:24362320

  2. Phosphorylation of bovine adrenodoxin by protein kinase CK2 affects the interaction with its redox partner cytochrome P450scc (CYP11A1).

    PubMed

    Bureik, Matthias; Zöllner, Andy; Schuster, Norbert; Montenarh, Mathias; Bernhardt, Rita

    2005-03-15

    Adrenodoxin (Adx), a [2Fe-2S] vertebrate-type ferredoxin, transfers electrons from the NADPH-dependent flavoprotein Adx reductase (AdR) to mitochondrial cytochrome P450 enzymes of the CYP11A and CYP11B families, which catalyze key reactions in steroid hormone biosynthesis. Adx is a known phosphoprotein, but the kinases that phosphorylate Adx have remained mostly obscure. The aim of this study was to identify previously unknown Adx phosphorylating kinases and to acquire a deeper insight into the functional consequences of such a modification. Here, we show for the first time that bovine Adx is a substrate of protein kinase CK2, whereas bovine CYP11A1, CYP11B1, and AdR are not phosphorylated by this kinase. CK2 phosphorylation of mature Adx requires the presence of both the catalytic alpha-subunit and the regulatory beta-subunit of CK2 and takes place exclusively at residue Thr-71, which is located within the redox partner interaction domain of the protein. We created two Adx mutants, Adx-T71E (imitating a phosphorylation) and Adx-T71V (which cannot be phosphorylated at this site), respectively, and investigated how these mutations affected the interaction of Adx with its redox partners. These data were supplemented with detailed spectroscopic and functional assays using the phosphorylated protein. All Adx species behaved like wild type (Adx-WT) with respect to their redox potential, iron-sulfur cluster symmetry, and overall backbone structure. Substrate conversion assays catalyzed by CYP11A1 showed an increase in product formation when Adx-T71E or CK2-phosphorylated Adx were used as electron carrier instead of Adx-WT, whereas the activity toward CYP11B1 was not altered using these Adx species. Additionally, Adx-T71E represents the only full-length Adx mutant which leads to an increase in CYP11A1 product formation. Therefore, characterizing this full-length mutant helps to improve our knowledge on the functional effects of phosphorylations on complex redox systems

  3. Mutations in CIT, encoding citron rho-interacting serine/threonine kinase, cause severe primary microcephaly in humans.

    PubMed

    Shaheen, Ranad; Hashem, Amal; Abdel-Salam, Ghada M H; Al-Fadhli, Fatima; Ewida, Nour; Alkuraya, Fowzan S

    2016-10-01

    Primary microcephaly is a clinical phenotype in which the head circumference is significantly reduced at birth due to abnormal brain development, primarily at the cortical level. Despite the marked genetic heterogeneity, most primary microcephaly-linked genes converge on mitosis regulation. Two consanguineous families segregating the phenotype of severe primary microcephaly, spasticity and failure to thrive had overlapping autozygomes in which exome sequencing identified homozygous splicing variants in CIT that segregate with the phenotype within each family. CIT encodes citron, an effector of the Rho signaling that is required for cytokinesis specifically in proliferating neuroprogenitors, as well as for postnatal brain development. In agreement with the critical role assigned to the kinase domain in effecting these biological roles, we show that both splicing variants predict variable disruption of this domain. The striking phenotypic overlap between CIT-mutated individuals and the knockout mice and rats that are specifically deficient in the kinase domain supports the proposed causal link between CIT mutation and primary microcephaly in humans. PMID:27503289

  4. Interaction of new kinase inhibitors cabozantinib and tofacitinib with human serum alpha-1 acid glycoprotein. A comprehensive spectroscopic and molecular Docking approach.

    PubMed

    Ajmal, Mohammad Rehan; Abdelhameed, Ali Saber; Alam, Parvez; Khan, Rizwan Hasan

    2016-04-15

    In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 10(4). With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300 K was calculated as -5.234 kcal mol(-1) for CBZ-AAG interaction and -6.237 kcal mol(-1) for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are -9.553 kcal mol(-1) and -14.618 cal mol(-1) K(-1) respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol(-1) and 7.206 cal mol(-1) K(-1) respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results. PMID:26851488

  5. Interaction of new kinase inhibitors cabozantinib and tofacitinib with human serum alpha-1 acid glycoprotein. A comprehensive spectroscopic and molecular Docking approach

    NASA Astrophysics Data System (ADS)

    Ajmal, Mohammad Rehan; Abdelhameed, Ali Saber; Alam, Parvez; Khan, Rizwan Hasan

    2016-04-01

    In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 104. With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300K was calculated as - 5.234 kcal mol- 1 for CBZ-AAG interaction and - 6.237 kcal mol- 1 for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are - 9.553 kcal mol- 1 and - 14.618 cal mol- 1K- 1 respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol- 1 and 7.206 cal mol- 1K- 1 respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.

  6. Elucidation of the Interaction Loci of the Human Pyruvate Dehydrogenase Complex E2·E3BP Core with Pyruvate Dehydrogenase Kinase 1 and Kinase 2 by H/D Exchange Mass Spectrometry and Nuclear Magnetic Resonance

    PubMed Central

    2015-01-01

    The human pyruvate dehydrogenase complex (PDC) comprises three principal catalytic components for its mission: E1, E2, and E3. The core of the complex is a strong subcomplex between E2 and an E3-binding protein (E3BP). The PDC is subject to regulation at E1 by serine phosphorylation by four kinases (PDK1–4), an inactivation reversed by the action of two phosphatases (PDP1 and -2). We report H/D exchange mass spectrometric (HDX-MS) and nuclear magnetic resonance (NMR) studies in the first attempt to define the interaction loci between PDK1 and PDK2 with the intact E2·E3BP core and their C-terminally truncated proteins. While the three lipoyl domains (L1 and L2 on E2 and L3 on E3BP) lend themselves to NMR studies and determination of interaction maps with PDK1 and PDK2 at the individual residue level, HDX-MS allowed studies of interaction loci on both partners in the complexes, PDKs, and other regions of the E2·E3BP core, as well, at the peptide level. HDX-MS suggested that the intact E2·E3BP core enhances the binding specificity of L2 for PDK2 over PDK1, while NMR studies detected lipoyl domain residues unique to interaction with PDK1 and PDK2. The E2·E3BP core induced more changes on PDKs than any C-terminally truncated protein, with clear evidence of greater plasticity of PDK1 than of PDK2. The effect of L1L2S paralleled HDX-MS results obtained with the intact E2·E3BP core; hence, L1L2S is an excellent candidate with which to define interaction loci with these two PDKs. Surprisingly, L3S′ induced moderate interaction with both PDKs according to both methods. PMID:25436986

  7. Homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and modulates its DNA binding affinity.

    PubMed

    Zhang, Qingchun; Wang, Yinsheng

    2007-12-01

    The chromosomal high-mobility group A (HMGA) proteins, composed of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation, and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated, HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook. PMID:17960875

  8. Disruption of focal adhesion kinase and p53 interaction with small molecule compound R2 reactivated p53 and blocked tumor growth

    PubMed Central

    2013-01-01

    Background Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor kinase that plays a major role in cancer cell survival and metastasis. Methods We performed computer modeling of the p53 peptide containing the site of interaction with FAK, predicted the peptide structure and docked it into the three-dimensional structure of the N-terminal domain of FAK involved in the complex with p53. We screened small molecule compounds that targeted the site of the FAK-p53 interaction and identified compounds (called Roslins, or R compounds) docked in silico to this site. Results By different assays in isogenic HCT116p53+/+ and HCT116 p53-/- cells we identified a small molecule compound called Roslin 2 (R2) that bound FAK, disrupted the binding of FAK and p53 and decreased cancer cell viability and clonogenicity in a p53-dependent manner. In addition, dual-luciferase assays demonstrated that the R2 compound increased p53 transcriptional activity that was inhibited by FAK using p21, Mdm-2, and Bax-promoter targets. R2 also caused increased expression of p53 targets: p21, Mdm-2 and Bax proteins. Furthermore, R2 significantly decreased tumor growth, disrupted the complex of FAK and p53, and up-regulated p21 in HCT116 p53+/+ but not in HCT116 p53-/- xenografts in vivo. In addition, R2 sensitized HCT116p53+/+ cells to doxorubicin and 5-fluorouracil. Conclusions Thus, disruption of the FAK and p53 interaction with a novel small molecule reactivated p53 in cancer cells in vitro and in vivo and can be effectively used for development of FAK-p53 targeted cancer therapy approaches. PMID:23841915

  9. Repression of the antiapoptotic molecule galectin-3 by homeodomain-interacting protein kinase 2-activated p53 is required for p53-induced apoptosis.

    PubMed

    Cecchinelli, Barbara; Lavra, Luca; Rinaldo, Cinzia; Iacovelli, Stefano; Gurtner, Aymone; Gasbarri, Alessandra; Ulivieri, Alessandra; Del Prete, Fabrizio; Trovato, Maria; Piaggio, Giulia; Bartolazzi, Armando; Soddu, Silvia; Sciacchitano, Salvatore

    2006-06-01

    Galectin 3 (Gal-3), a member of the beta-galactoside binding lectin family, exhibits antiapoptotic functions, and its aberrant expression is involved in various aspects of tumor progression. Here we show that p53-induced apoptosis is associated with transcriptional repression of Gal-3. Previously, it has been reported that phosphorylation of p53 at Ser46 is important for transcription of proapoptotic genes and induction of apoptosis and that homeodomain-interacting protein kinase 2 (HIPK2) is specifically involved in these functions. We show that HIPK2 cooperates with p53 in Gal-3 repression and that this cooperation requires HIPK2 kinase activity. Gene-specific RNA interference demonstrates that HIPK2 is essential for repression of Gal-3 upon induction of p53-dependent apoptosis. Furthermore, expression of a nonrepressible Gal-3 prevents HIPK2- and p53-induced apoptosis. These results reveal a new apoptotic pathway induced by HIPK2-activated p53 and requiring repression of the antiapoptotic factor Gal-3. PMID:16738336

  10. CDC2L5, a Cdk-like kinase with RS domain, interacts with the ASF/SF2-associated protein p32 and affects splicing in vivo.

    PubMed

    Even, Yasmine; Durieux, Sandrine; Escande, Marie-Line; Lozano, Jean Claude; Peaucellier, Gérard; Weil, Dominique; Genevière, Anne-Marie

    2006-10-15

    The human CDC2L5 gene encodes a protein of unknown physiological function. This protein is closely related to the cyclin-dependent kinase (Cdks) family and contains an arginine/serine-rich (RS) domain. The Cdks were first identified as crucial regulators of cell-cycle progression, more recently they were found to be involved in transcription and mRNA processing. RS domains are mainly present in proteins regulating pre-mRNA splicing, suggesting CDC2L5 having a possible role in this process. In this study, we demonstrate that CDC2L5 is located in the nucleoplasm, at a higher concentration in speckles, the storage sites for splicing factors. Furthermore, this localization is dependent on the presence of the N-terminal sequence including the RS domain. Then, we report that CDC2L5 directly interacts with the ASF/SF2-associated protein p32, a protein involved in splicing regulation. Overexpression of CDC2L5 constructs disturbs constitutive splicing and switches alternative splice site selection in vivo. These results argue in favor of a functional role of the CDC2L5 kinase in splicing regulation. PMID:16721827

  11. p300/β-Catenin Interactions Regulate Adult Progenitor Cell Differentiation Downstream of WNT5a/Protein Kinase C (PKC).

    PubMed

    Rieger, Megan E; Zhou, Beiyun; Solomon, Nicola; Sunohara, Mitsuhiro; Li, Changgong; Nguyen, Cu; Liu, Yixin; Pan, Jie-Hong; Minoo, Parviz; Crandall, Edward D; Brody, Steven L; Kahn, Michael; Borok, Zea

    2016-03-18

    Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of β-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential β-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific β-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/β-catenin but not CBP/β-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/β-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/β-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/β-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting β-catenin to modulate adult progenitor cell

  12. Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase.

    PubMed Central

    Bertrand, L; Vertommen, D; Depiereux, E; Hue, L; Rider, M H; Feytmans, E

    1997-01-01

    Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted. PMID:9032445

  13. The Interaction between Cancer Stem Cell Marker CD133 and Src Protein Promotes Focal Adhesion Kinase (FAK) Phosphorylation and Cell Migration.

    PubMed

    Liu, Chanjuan; Li, Yinan; Xing, Yang; Cao, Benjin; Yang, Fan; Yang, Tianxiao; Ai, Zhilong; Wei, Yuanyan; Jiang, Jianhai

    2016-07-22

    CD133, a widely known cancer stem cell marker, has been proved to promote tumor metastasis. However, the mechanism by which CD133 regulates metastasis remains largely unknown. Here, we report that CD133 knockdown inhibits cancer cell migration, and CD133 overexpression promotes cell migration. CD133 expression is beneficial to activate the Src-focal adhesion kinase (FAK) signaling pathway. Further studies show that CD133 could interact with Src, and the region between amino acids 845 and 857 in the CD133 C-terminal domain is indispensable for its interaction with Src. The interaction activates Src to phosphorylate its substrate FAK and to promote cell migration. Likewise, a Src binding-deficient CD133 mutant loses the abilities to increase Src and FAK phosphorylation and to promote cell migration. Inhibition of Src activity by PP2, a known Src activity inhibitor, could block the activation of FAK phosphorylation and cell migration induced by CD133. In summary, our data suggest that activation of FAK by the interaction between CD133 and Src promotes cell migration, providing clues to understand the migratory mechanism of CD133(+) tumor cells. PMID:27226554

  14. MAP kinase dynamics in yeast.

    PubMed

    van Drogen, F; Peter, M

    2001-09-01

    MAP kinase pathways play key roles in cellular responses towards extracellular signals. In several cases, the three core kinases interact with a scaffold molecule, but the function of these scaffolds is poorly understood. They have been proposed to contribute to signal specificity, signal amplification, or subcellular localization of MAP kinases. Several MAP kinases translocate to the nucleus in response to their activation, suggesting that nuclear transport may provide a regulatory mechanism. Here we describe new applications for Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP), to study dynamic translocations of MAPKs between different subcellular compartments. We have used these methods to measure the nuclear/cytoplasmic dynamics of several yeast MAP kinases, and in particular to address the role of scaffold proteins for MAP-kinase signaling. PMID:11730324

  15. EGFR and EGFRvIII interact with PUMA to inhibit mitochondrial translocalization of PUMA and PUMA-mediated apoptosis independent of EGFR kinase activity

    PubMed Central

    Zhu, Hu; Cao, Xinyu; Ali-Osman, Francis; Keir, Stephen; Lo, Hui-Wen

    2010-01-01

    EGFR and its constitutively activated variant EGFRvIII are linked to glioblastoma resistance to therapy, the mechanisms underlying this association, however, are still unclear. We report that in glioblastoma, EGFR/EGFRvIII paradoxically co-expresses with p53-upregulated modulator of apoptosis (PUMA), a proapoptotic member of the Bcl-2 family of proteins primarily located on the mitochondria. EGFR/EGFRvIII binds to PUMA constitutively and under apoptotic stress, and subsequently sequesters PUMA in the cytoplasm. The EGFR-PUMA interaction is independent of EGFR activation and is sustained under EGFR inhibition. A Bcl-2/Bcl-xL inhibitor that mimics PUMA activity sensitizes EGFR/EGFRvIII-expressing glioblastoma cells to Iressa. Collectively, we uncovered a novel kinase-independent function of EGFR/EGFRvIII that leads to tumor drug resistance. PMID:20153921

  16. Specificity Rendering ‘Hot-Spots’ for Aurora Kinase Inhibitor Design: The Role of Non-Covalent Interactions and Conformational Transitions

    PubMed Central

    Badrinarayan, Preethi; Sastry, G. Narahari

    2014-01-01

    The present study examines the conformational transitions occurring among the major structural motifs of Aurora kinase (AK) concomitant with the DFG-flip and deciphers the role of non-covalent interactions in rendering specificity. Multiple sequence alignment, docking and structural analysis of a repertoire of 56 crystal structures of AK from Protein Data Bank (PDB) has been carried out. The crystal structures were systematically categorized based on the conformational disposition of the DFG-loop [in (DI) 42, out (DO) 5 and out-up (DOU) 9], G-loop [extended (GE) 53 and folded (GF) 3] and αC-helix [in (CI) 42 and out (CO) 14]. The overlapping subsets on categorization show the inter-dependency among structural motifs. Therefore, the four distinct possibilities a) 2W1C (DI, CI, GE) b) 3E5A (DI, CI, GF) c) 3DJ6 (DI, CO, GF) d) 3UNZ (DOU, CO, GF) along with their co-crystals and apo-forms were subjected to molecular dynamics simulations of 40 ns each to evaluate the variations of individual residues and their impact on forming interactions. The non-covalent interactions formed by the 157 AK co-crystals with different regions of the binding site were initially studied with the docked complexes and structure interaction fingerprints. The frequency of the most prominent interactions was gauged in the AK inhibitors from PDB and the four representative conformations during 40 ns. Based on this study, seven major non-covalent interactions and their complementary sites in AK capable of rendering specificity have been prioritized for the design of different classes of inhibitors. PMID:25485544

  17. Simultaneous determination of a novel KDR kinase inhibitor and its N-oxide metabolite in human plasma using 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometry.

    PubMed

    Xu, Yang; Du, Lihong; Soli, Eric D; Braun, Matthew P; Dean, Dennis C; Musson, Donald G

    2005-03-25

    To support pharmacokinetic studies, a selective and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of a novel KDR kinase inhibitor (1) and its active metabolite (2) in human plasma. The method is fully automated using a Packard MultiPROBE II system and a TomTec Quadra 96 liquid handling workstation to perform sample preparation and solid-phase extraction (SPE). Following the extraction on a mixed-mode SPE using Oasis MCX 96-well plate, the analytes were separated on a Aquasil C18 column (50 mm x 2.1 mm, i.d., 3 microm) with a mobile phase consisting of acetonitrile/ammonium acetate buffer (5 mM, pH 5.0) (60/40, v/v). The run time for each injection was 4.5 min with the retention times of approximately 2.0 and 2.7 min for 1 and 2 respectively, at a flow rate of 0.25 mL/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) under the positive ion mode with a turbo ion-spray interface. The linear ranges of the calibration curves were 0.05-400 ng/mL for 1 and 0.1-400 ng/mL for 2 on a PE Sciex API 4000 LC-MS/MS system. The lower limits of quantitation (LLOQ) of the assay were 0.05 and 0.1 ng/mL for 1 and 2 respectively, when 0.4 mL of plasma was processed. Intra-day assay precision (using five standard curves prepared by spiking compounds to five lots of plasma) was less than 4.9% for 1 and less than 9.6% for 2 on each concentration. Assay accuracy was found to be 95.1-104.6% of nominal for 1 standards and 93.5-105.6% for 2 standards. QC samples were stable when kept at room temperature for 4 h, at -70 degrees C for 10 days, and after three freeze-thaw cycles. The extraction recoveries were 80%, 83% and 84% for 1 and 2 and I.S. respectively, and no significant matrix effects were observed. The method was successfully applied to plasma samples from clinical studies after oral administration of compound 1. PMID:15686997

  18. Contribution of the interaction between the rabies virus P protein and I-kappa B kinase ϵ to the inhibition of type I IFN induction signalling.

    PubMed

    Masatani, Tatsunori; Ozawa, Makoto; Yamada, Kentaro; Ito, Naoto; Horie, Masayuki; Matsuu, Aya; Okuya, Kosuke; Tsukiyama-Kohara, Kyoko; Sugiyama, Makoto; Nishizono, Akira

    2016-02-01

    The P protein of rabies virus (RABV) is known to interfere with the phosphorylation of the host IFN regulatory factor 3 (IRF-3) and to consequently inhibit type I IFN induction. Previous studies, however, have only tested P proteins from laboratory-adapted fixed virus strains, and to the best of our knowledge there is no report about the effect of P proteins from street RABV strains or other lyssaviruses on the IRF-3-mediated type I IFN induction system. In this study, we evaluated the inhibitory effect of P proteins from several RABV strains, including fixed and street virus strains and other lyssaviruses (Lagos bat, Mokola and Duvenhage viruses), on IRF-3 signalling. All P proteins tested inhibited retinoic acid-inducible gene-1 (RIG-I)- and TANK binding kinase 1 (TBK1)-mediated IRF-3-dependent IFN-β promoter activities. On the other hand, the P proteins from the RABV street strains 1088 and HCM-9, but not from fixed strains Nishigahara (Ni) and CVS-11 and other lyssaviruses tested, significantly inhibited I-kappa B kinase ϵ (IKKϵ)-inducible IRF-3-dependent IFN-β promoter activity. Importantly, we revealed that the P proteins from the 1088 and HCM-9 strains, but not from the remaining viruses, interacted with IKKϵ. By using expression plasmids encoding chimeric P proteins from the 1088 strain and Ni strain, we found that the C-terminal region of the P protein is important for the interaction with IKKϵ. These findings suggest that the P protein of RABV street strains may contribute to efficient evasion of host innate immunity. PMID:26647356

  19. Carboxyl terminus of HSC70-interacting protein (CHIP) down-regulates NF-κB-inducing kinase (NIK) and suppresses NIK-induced liver injury.

    PubMed

    Jiang, Bijie; Shen, Hong; Chen, Zheng; Yin, Lei; Zan, Linsen; Rui, Liangyou

    2015-05-01

    Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease. PMID:25792747

  20. Serum- and Glucocorticoid-Inducible Kinase 1 Delay the Onset of Endothelial Senescence by Directly Interacting with Human Telomerase Reverse Transcriptase.

    PubMed

    Basello, Katia; Pacifici, Francesca; Capuani, Barbara; Pastore, Donatella; Lombardo, Marco F; Ferrelli, Francesca; Coppola, Andrea; Donadel, Giulia; Arriga, Roberto; Sconocchia, Giuseppe; Bellia, Alfonso; Rogliani, Paola; Federici, Massimo; Sbraccia, Paolo; Lauro, Davide; Della-Morte, David

    2016-02-01

    Endothelial senescence is characteristic of vascular aging. Serum- and glucocorticoid-inducible kinase (SGK)1 belongs to a family of serine/threonine kinases regulated by various external stimuli. SGK1 has been shown to be protective against reactive oxygen species (ROS) production and to be involved in processes regulating aging. However, data on the direct relationship between SGK1 and senescence are sparse. In the present study, we sought to investigate the role of SGK1 in cellular aging by using human umbilical vein endothelial cells (HUVECs) infected with different constructs. Senescence was measured at different cellular stages by senescence-associated β-galactosidase (SA-β-gal) activity, human telomerase reverse transcriptase (hTERT) activity, p21 protein levels, and ROS production. HUVECs over-expressing full-length SGK1 (wild-type SGK1 [SGK1WT]) showed a decrease in SA-β-gal and p21 expression and a corresponding increase in hTERT activity in the early stages of aging. Moreover, SGK1WT presented lower levels of ROS production. A direct interaction between SGK1WT and hTERT was also shown by co-immunoprecipitation. The SGK1Δ60 isoform, lacking the amino-terminal 60 amino acids, did not show interaction with hTERT, suggesting a pivotal role of this protein site for the SGK1 anti-aging function. The results from this study may be of particular importance, because SGK1WT over-expression by activating telomerase and reducing ROS levels may delay the processes of endothelial senescence. PMID:26230157

  1. Protein Interacting C-Kinase 1 Modulates Surface Expression of P2Y6 Purinoreceptor, Actin Polymerization and Phagocytosis in Microglia.

    PubMed

    Zhu, Jia; Wang, Zhen; Zhang, Nan; Ma, Jiao; Xu, Shui-Lin; Wang, Yin; Shen, Ying; Li, Yun-Hong

    2016-04-01

    Microglia clean up dead cells and debris through phagocytosis in the central nervous system. UDP-activated P2Y6 receptors (P2Y6Rs) induce the formation of phagocytic cup-like structure and P2Y6R expression is increased during the phagocytosis. However, it remains unclear how surface expression of P2Y6R is increased. PICK1 (protein interacting with C-kinase-1) interacts with various neurotransmitter receptors, transporters, and enzymes. We here report that PICK1 might interact with P2Y6R. Surface P2Y6R was reduced in microglia from PICK1-knockout mice and PICK1-knockdown BV2 cells, which was also confirmed by electrophysiological recordings, showing that P2Y6R-mediated current was increased by PICK1 overexpression but was reduced by PICK1-knockdown in BV2 microglia. Finally, PICK1 was sufficient to affect cytoskeletal aggregation and phagocytosis both in primary microglia and BV2 cells. These results indicate that PICK1 is an important regulator of P2Y6R expression and microglial phagocytosis. PMID:26566795

  2. Modulation of Pantothenate Kinase 3 Activity by Small Molecules that Interact with the Substrate/Allosteric Regulatory Domain

    SciTech Connect

    Leonardi, Roberta; Zhang, Yong-Mei; Yun, Mi-Kyung; Zhou, Ruobing; Zeng, Fu-Yue; Lin, Wenwei; Cui, Jimmy; Chen, Taosheng; Rock, Charles O.; White, Stephen W.; Jackowski, Suzanne

    2010-09-27

    Pantothenate kinase (PanK) catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. PanK3 is stringently regulated by acetyl-CoA and uses an ordered kinetic mechanism with ATP as the leading substrate. Biochemical analysis of site-directed mutants indicates that pantothenate binds in a tunnel adjacent to the active site that is occupied by the pantothenate moiety of the acetyl-CoA regulator in the PanK3 acetyl-CoA binary complex. A high-throughput screen for PanK3 inhibitors and activators was applied to a bioactive compound library. Thiazolidinediones, sulfonylureas and steroids were inhibitors, and fatty acyl-amides and tamoxifen were activators. The PanK3 activators and inhibitors either stimulated or repressed CoA biosynthesis in HepG2/C3A cells. The flexible allosteric acetyl-CoA regulatory domain of PanK3 also binds the substrates, pantothenate and pantetheine, and small molecule inhibitors and activators to modulate PanK3 activity.

  3. Hepatocyte Growth Factor Modulates MET Receptor Tyrosine Kinase and β-Catenin Functional Interactions to Enhance Synapse Formation.

    PubMed

    Xie, Zhihui; Eagleson, Kathie L; Wu, Hsiao-Huei; Levitt, Pat

    2016-01-01

    MET, a pleiotropic receptor tyrosine kinase implicated in autism risk, influences multiple neurodevelopmental processes. There is a knowledge gap, however, in the molecular mechanism through which MET mediates developmental events related to disorder risk. In the neocortex, MET is expressed transiently during periods of peak dendritic outgrowth and synaptogenesis, with expression enriched at developing synapses, consistent with demonstrated roles in dendritic morphogenesis, modulation of spine volume, and excitatory synapse development. In a recent coimmunoprecipitation/mass spectrometry screen, β-catenin was identified as part of the MET interactome in developing neocortical synaptosomes. Here, we investigated the influence of the MET/β-catenin complex in mouse neocortical synaptogenesis. Western blot analysis confirms that MET and β-catenin coimmunoprecipitate, but N-cadherin is not associated with the MET complex. Following stimulation with hepatocyte growth factor (HGF), β-catenin is phosphorylated at tyrosine(142) (Y142) and dissociates from MET, accompanied by an increase in β-catenin/N-cadherin and MET/synapsin 1 protein complexes. In neocortical neurons in vitro, proximity ligation assays confirmed the close proximity of these proteins. Moreover, in neurons transfected with synaptophysin-GFP, HGF stimulation increases the density of synaptophysin/bassoon (a presynaptic marker) and synaptophysin/PSD-95 (a postsynaptic marker) clusters. Mutation of β-catenin at Y142 disrupts the dissociation of the MET/β-catenin complex and prevents the increase in clusters in response to HGF. The data demonstrate a new mechanism for the modulation of synapse formation, whereby MET activation induces an alignment of presynaptic and postsynaptic elements that are necessary for assembly and formation of functional synapses by subsets of neocortical neurons that express MET/β-catenin complex. PMID:27595133

  4. Hepatocyte Growth Factor Modulates MET Receptor Tyrosine Kinase and β-Catenin Functional Interactions to Enhance Synapse Formation

    PubMed Central

    Xie, Zhihui; Eagleson, Kathie L.

    2016-01-01

    MET, a pleiotropic receptor tyrosine kinase implicated in autism risk, influences multiple neurodevelopmental processes. There is a knowledge gap, however, in the molecular mechanism through which MET mediates developmental events related to disorder risk. In the neocortex, MET is expressed transiently during periods of peak dendritic outgrowth and synaptogenesis, with expression enriched at developing synapses, consistent with demonstrated roles in dendritic morphogenesis, modulation of spine volume, and excitatory synapse development. In a recent coimmunoprecipitation/mass spectrometry screen, β-catenin was identified as part of the MET interactome in developing neocortical synaptosomes. Here, we investigated the influence of the MET/β-catenin complex in mouse neocortical synaptogenesis. Western blot analysis confirms that MET and β-catenin coimmunoprecipitate, but N-cadherin is not associated with the MET complex. Following stimulation with hepatocyte growth factor (HGF), β-catenin is phosphorylated at tyrosine142 (Y142) and dissociates from MET, accompanied by an increase in β-catenin/N-cadherin and MET/synapsin 1 protein complexes. In neocortical neurons in vitro, proximity ligation assays confirmed the close proximity of these proteins. Moreover, in neurons transfected with synaptophysin-GFP, HGF stimulation increases the density of synaptophysin/bassoon (a presynaptic marker) and synaptophysin/PSD-95 (a postsynaptic marker) clusters. Mutation of β-catenin at Y142 disrupts the dissociation of the MET/β-catenin complex and prevents the increase in clusters in response to HGF. The data demonstrate a new mechanism for the modulation of synapse formation, whereby MET activation induces an alignment of presynaptic and postsynaptic elements that are necessary for assembly and formation of functional synapses by subsets of neocortical neurons that express MET/β-catenin complex. PMID:27595133

  5. A robust quasi-simultaneous interaction method for a full potential flow with a boundary layer with application to wing/body configurations

    NASA Technical Reports Server (NTRS)

    Vanderwees, A. J.; Vanmuijden, J.

    1992-01-01

    The MATRICS flow solver calculates the inviscid transonic potential flow about a wing/body semi-configuration. At present, work is in progress to extend MATRICS to take viscous effects into account through coupling with a boundary layer solver. This solver, MATRICS-V, is based on robust calculation methods for the boundary layer, the outer wing flow and their interaction. MATRICS-V is intended for (inverse) design purposes. The boundary layer and wake are based on an integral formulation of the unsteady first order boundary layer equations, the inviscid method is the existing MATRICS potential flow solver, and the interaction algorithm is of the quasi-simultaneous type. The paper gives a progress report on the coupled potential-flow boundary-layer method for transonic wing/body configurations.

  6. Combined SAXS/EM Based Models of the S. elongatus Post-Translational Circadian Oscillator and its Interactions with the Output His-Kinase SasA

    SciTech Connect

    Pattanayek, Rekha; Williams, Dewight R.; Rossi, Gian; Weigand, Steven; Mori, Tetsuya; Johnson, Carl H.; Stewart, Phoebe L.; Egli, Martin

    2012-03-15

    The circadian clock in the cyanobacterium Synechococcus elongatus is composed of a post-translational oscillator (PTO) that can be reconstituted in vitro from three different proteins in the presence of ATP and a transcription-translation feedback loop (TTFL). The homo-hexameric KaiC kinase, phosphatase and ATPase alternates between hypo- and hyper-phosphorylated states over the 24-h cycle, with KaiA enhancing phosphorylation, and KaiB antagonizing KaiA and promoting KaiC subunit exchange. SasA is a His kinase that relays output signals from the PTO formed by the three Kai proteins to the TTFL. Although the crystal structures for all three Kai proteins are known, atomic resolution structures of Kai and Kai/SasA protein complexes have remained elusive. Here, we present models of the KaiAC and KaiBC complexes derived from solution small angle X-ray scattering (SAXS), which are consistent with previous EM based models. We also present a combined SAXS/EM model of the KaiC/SasA complex, which has two N-terminal SasA sensory domains occupying positions on the C-terminal KaiC ring reminiscent of the orientations adopted by KaiB dimers. Using EM we demonstrate that KaiB and SasA compete for similar binding sites on KaiC. We also propose an EM based model of the ternary KaiABC complex that is consistent with the sequestering of KaiA by KaiB on KaiC during the PTO dephosphorylation phase. This work provides the first 3D-catalogue of protein-protein interactions in the KaiABC PTO and the output pathway mediated by SasA.

  7. Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.

    PubMed

    Pattanayek, Rekha; Williams, Dewight R; Rossi, Gian; Weigand, Steven; Mori, Tetsuya; Johnson, Carl H; Stewart, Phoebe L; Egli, Martin

    2011-01-01

    The circadian clock in the cyanobacterium Synechococcus elongatus is composed of a post-translational oscillator (PTO) that can be reconstituted in vitro from three different proteins in the presence of ATP and a transcription-translation feedback loop (TTFL). The homo-hexameric KaiC kinase, phosphatase and ATPase alternates between hypo- and hyper-phosphorylated states over the 24-h cycle, with KaiA enhancing phosphorylation, and KaiB antagonizing KaiA and promoting KaiC subunit exchange. SasA is a His kinase that relays output signals from the PTO formed by the three Kai proteins to the TTFL. Although the crystal structures for all three Kai proteins are known, atomic resolution structures of Kai and Kai/SasA protein complexes have remained elusive. Here, we present models of the KaiAC and KaiBC complexes derived from solution small angle X-ray scattering (SAXS), which are consistent with previous EM based models. We also present a combined SAXS/EM model of the KaiC/SasA complex, which has two N-terminal SasA sensory domains occupying positions on the C-terminal KaiC ring reminiscent of the orientations adopted by KaiB dimers. Using EM we demonstrate that KaiB and SasA compete for similar binding sites on KaiC. We also propose an EM based model of the ternary KaiABC complex that is consistent with the sequestering of KaiA by KaiB on KaiC during the PTO dephosphorylation phase. This work provides the first 3D-catalogue of protein-protein interactions in the KaiABC PTO and the output pathway mediated by SasA. PMID:21887298

  8. Combined SAXS/EM Based Models of the S. elongatus Post-Translational Circadian Oscillator and its Interactions with the Output His-Kinase SasA

    PubMed Central

    Pattanayek, Rekha; Williams, Dewight R.; Rossi, Gian; Weigand, Steven; Mori, Tetsuya; Johnson, Carl H.; Stewart, Phoebe L.; Egli, Martin

    2011-01-01

    The circadian clock in the cyanobacterium Synechococcus elongatus is composed of a post-translational oscillator (PTO) that can be reconstituted in vitro from three different proteins in the presence of ATP and a transcription-translation feedback loop (TTFL). The homo-hexameric KaiC kinase, phosphatase and ATPase alternates between hypo- and hyper-phosphorylated states over the 24-h cycle, with KaiA enhancing phosphorylation, and KaiB antagonizing KaiA and promoting KaiC subunit exchange. SasA is a His kinase that relays output signals from the PTO formed by the three Kai proteins to the TTFL. Although the crystal structures for all three Kai proteins are known, atomic resolution structures of Kai and Kai/SasA protein complexes have remained elusive. Here, we present models of the KaiAC and KaiBC complexes derived from solution small angle X-ray scattering (SAXS), which are consistent with previous EM based models. We also present a combined SAXS/EM model of the KaiC/SasA complex, which has two N-terminal SasA sensory domains occupying positions on the C-terminal KaiC ring reminiscent of the orientations adopted by KaiB dimers. Using EM we demonstrate that KaiB and SasA compete for similar binding sites on KaiC. We also propose an EM based model of the ternary KaiABC complex that is consistent with the sequestering of KaiA by KaiB on KaiC during the PTO dephosphorylation phase. This work provides the first 3D-catalogue of protein-protein interactions in the KaiABC PTO and the output pathway mediated by SasA. PMID:21887298

  9. Oral epidermal growth factor receptor tyrosine kinase inhibitors for the treatment of non-small cell lung cancer: comparative pharmacokinetics and drug-drug interactions.

    PubMed

    Peters, Solange; Zimmermann, Stefan; Adjei, Alex A

    2014-09-01

    The development of orally active small molecule inhibitors of the epidermal growth factor receptor (EGFR) has led to new treatment options for non-small cell lung cancer (NSCLC). Patients with activating mutations of the EGFR gene show sensitivity to, and clinical benefit from, treatment with EGFR tyrosine kinase inhibitors (EGFR-TKls). First generation reversible ATP-competitive EGFR-TKls, gefitinib and erlotinib, are effective as first, second-line or maintenance therapy. Despite initial benefit, most patients develop resistance within a year, 50-60% of cases being related to the appearance of a T790M gatekeeper mutation. Newer, irreversible EGFR-TKls - afatinib and dacomitinib - covalently bind to and inhibit multiple receptors in the ErbB family (EGFR, HER2 and HER4). These agents have been mainly evaluated for first-line treatment but also in the setting of acquired resistance to first-generation EGFR-TKls. Afatinib is the first ErbB family blocker approved for patients with NSCLC with activating EGFR mutations; dacomitinib is in late stage clinical development. Mutant-selective EGFR inhibitors (AZD9291, CO-1686, HM61713) that specifically target the T790M resistance mutation are in early development. The EGFR-TKIs differ in their spectrum of target kinases, reversibility of binding to EGFR receptor, pharmacokinetics and potential for drug-drug interactions, as discussed in this review. For the clinician, these differences are relevant in the setting of polymedicated patients with NSCLC, as well as from the perspective of innovative anticancer drug combination strategies. PMID:25027951

  10. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury

    PubMed Central

    Wang, Shaogui; Ni, Hong-Min; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Nawabi, Atta; Komatsu, Masaaki; Huang, Heqing; Ding, Wen-Xing

    2016-01-01

    Chronic alcohol exposure increased hepatic receptor-interacting protein kinase (RIP) 3 expression and necroptosis in the liver but its mechanisms are unclear. In the present study, we demonstrated that chronic alcohol feeding plus binge (Gao-binge) increased RIP3 but not RIP1 protein levels in mouse livers. RIP3 knockout mice had decreased serum alanine amino transferase activity and hepatic steatosis but had no effect on hepatic neutrophil infiltration compared with wild type mice after Gao-binge alcohol treatment. The hepatic mRNA levels of RIP3 did not change between Gao-binge and control mice, suggesting that alcohol-induced hepatic RIP3 proteins are regulated at the posttranslational level. We found that Gao-binge treatment decreased the levels of proteasome subunit alpha type-2 (PSMA2) and proteasome 26S subunit, ATPase 1 (PSMC1) and impaired hepatic proteasome function. Pharmacological or genetic inhibition of proteasome resulted in the accumulation of RIP3 in mouse livers. More importantly, human alcoholics had decreased expression of PSMA2 and PSMC1 but increased protein levels of RIP3 compared with healthy human livers. Moreover, pharmacological inhibition of RIP1 decreased Gao-binge-induced hepatic inflammation, neutrophil infiltration and NF-κB subunit (p65) nuclear translocation but failed to protect against steatosis and liver injury induced by Gao-binge alcohol. In conclusion, results from this study suggest that impaired hepatic proteasome function by alcohol exposure may contribute to hepatic accumulation of RIP3 resulting in necroptosis and steatosis while RIP1 kinase activity is important for alcohol-induced inflammation. PMID:26769846

  11. Isoform-specific interactions between meprin metalloproteases and the catalytic subunit of protein kinase A: significance in acute and chronic kidney injury

    PubMed Central

    Niyitegeka, Jean-Marie V.; Bastidas, Adam C.; Newman, Robert H.; Taylor, Susan S.

    2014-01-01

    Meprin metalloproteases are abundantly expressed in the brush-border membranes of kidney proximal tubules. Meprins are implicated in ischemia-reperfusion (IR)-induced renal injury and diabetic nephropathy. The protein kinase A (PKA) signaling pathway modulates extracellular matrix metabolism in diabetic kidneys. The present study evaluated isoform-specific interactions between the catalytic subunit of PKA (PKA C) and meprins. To this end, cytosolic-enriched kidney proteins from meprin αβ double knockout mice, and purified forms of recombinant mouse PKA Cα, Cβ1, and Cβ2, were incubated with activated forms of either homomeric meprin A or meprin B. The cleaved protein products were subjected to SDS-PAGE and analyzed by Coomassie staining and Western blot analysis. While meprin A only cleaved PKA Cβ1, meprin B cleaved all three PKA C isoforms. Analysis of the proteolytic fragments by mass spectrometry revealed that meprin A and B cleave the PKA C isoforms at defined sites, resulting in unique cleavage products. Michaelis-Menten enzyme kinetics demonstrated that meprin B-mediated cleavage of PKA Cα occurs at a rate consistent with that of other physiologically relevant meprin substrates. Meprin cleavage decreased the kinase activity of PKA Cα, Cβ1, and Cβ2. PKA C levels were higher in diabetic kidneys, with evidence of in vivo fragmentation in wild-type diabetic kidneys. Confocal microscopy showed localization of meprin A in the glomeruli of diabetic kidneys. At 3 h post-IR, PKA C levels in proximal tubules decreased compared with distal tubules, which lack meprins. These data suggest that meprins may impact kidney injury, in part, via modulation of PKA signaling pathways. PMID:25354939

  12. Simultaneous characterization of protein-material and cell-protein interactions using dynamic QCM-D analysis on SAM surfaces.

    PubMed

    Kushiro, Keiichiro; Lee, Chih-Hao; Takai, Madoka

    2016-05-24

    Understanding the interactions among materials, proteins and cells is critical for the development of novel biomaterials, and establishing a highly sensitive and quantitative method to standardize these interactions is desired. In this study, quartz crystal microbalance with dissipation (QCM-D) combined with microscopy was utilized to quantitatively monitor the entirety of the cell adhesion processes, starting from the protein adsorption, on various self-assembled monolayer (SAM) surfaces. Although the resulting cell adhesion morphologies were similar on most of the surfaces, the dynamic QCM-D signal patterns were unique on each surface, suggesting different forms of material-protein-cell interactions. The viscoelasticity and the density of the surface-adsorbed fibronectin (FN), as well as the relative exposure of the cell adhesive arginine-glycine-aspartic acid (RGD) motifs, were correlated to the different cell adhesion dynamics and mechanics. Some surfaces exhibited complicated behaviors alluding to the detachment/rearrangement of surface proteins or highly sparse but bioactive proteins that promote a slow adhesion process. This study underscores the potential use of the QCM-D signal pattern as a rule of thumb for delineating different protein-material and cell-protein interactions, and offers a rapid in vitro platform for the dynamic evaluation of protein and cell behaviors on novel biomaterials. PMID:27127807

  13. Performing two different actions simultaneously: The critical role of interhemispheric interactions during the preparation of bimanual movement.

    PubMed

    Fujiyama, Hakuei; Van Soom, Jago; Rens, Guy; Cuypers, Koen; Heise, Kirstin-Friederike; Levin, Oron; Swinnen, Stephan P

    2016-04-01

    Even though it has been suggested that the dorsolateral prefrontal cortex (DLPFC) and dorsal premotor cortex (PMd) are highly involved in the planning of bimanual movements, the exact nature (facilitatory or inhibitory) of their role is not well understood. Using a dual-site transcranial magnetic stimulation (TMS) paradigm, we examined the functional influence from DLPFC and PMd to the contralateral primary cortex (M1) during the preparation of a complex bimanual coordination task in which inter-hand movement frequency was manipulated. Only the left PMd showed inter-hand frequency-specific modulations in the interaction with the contralateral M1. Left PMd-right M1 interaction became facilitatory during the preparation phase when the left hand had to move faster than the right hand, while inhibitory modulation was observed when the movement frequency arrangement was reversed. Interestingly, bilateral DLPFC showed a facilitatory interaction with the contralateral M1s during the preparation period only in difficult conditions, irrespective of the inter-hand frequency ratio, suggesting a less task-specific role in the organization of complex bimanual actions. Observed task-related modulations in DLPFC-M1 and left PMd-right M1 interactions during preparation were significantly correlated with up-coming performance, predicting successful bimanual movements. These observations highlight the distinct roles of DLPFC and left PMd in the preparation of bimanual movements that require a differential contribution of each limb. PMID:26963084

  14. Ultraviolet Radiation and 12-O-Tetradecanoylphorbol-13-Acetate-Induced Interaction of Mouse Epidermal Protein Kinase Cε With Stat3 Involve Integration With Erk1/2

    PubMed Central

    Sand, Jordan Marshall; Hafeez, Bilal Bin; Aziz, Moammir Hasan; Siebers, Emily Marie; Dreckschmidt, Nancy Ellen; Verma, Ajit Kumar

    2012-01-01

    We have reported that protein kinase C epsilon (PKCε) expression level in epidermis dictates the susceptibility of mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by the DMBA-TPA tumor promotion protocol. To find clues about the mechanism by which PKCε mediates susceptibility to UVR-induced development of SCC, we found that PKCε-over-expressing transgenic mice, as compared to their wild-type littermates, when exposed to UVR, elicit enhanced phosphorylation of Stat3 at Ser727 residues. Stat3 is constitutively activated in SCC and UVR fails to induce SCC in Stat3 mutant mice. Stat3Ser727 phosphorylation is essential for Stat3 transcriptional activity (Cancer Res. 67: 1385, 2007). We now present severa novel findings including that PKCε integrates with its downstream partner ERK1/2 to phosphorylate Stat3Ser727. In these experiments, mice were either exposed to UVR (2 kJ/m2/dose) emitted by Kodacel-filtered FS-40 sun lamps or treated with TPA (5 nmol). Both UVR and TPA treatment stimulated PKCε-Stat3 interaction, Stat3Ser727 phosphorylation and Stat3-regulated gene COX-2 expression. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was also observed in SCC elicited by repeated UVR exposures of mice. PKCε-Stat3 interaction was PKCε specific. UVR or TPA-stimulated Stat3Ser727 phosphorylation accompanied interaction of PKCε with ERK1/2 in intact mouse skin in vivo. Deletion of PKCε in wild-type mice attenuated both TPA and UVR-induced expression of phosphoforms of ERK1/2 and Stat3Ser727. These results indicate that PKCε integrates with ERK1/2 to mediate both TPA and UVR-induced epidermal Stat3Ser727 phosphorylation. PKCε and Stat3 may be potential molecular targets for SCC prevention. PMID:21480396

  15. Ultraviolet radiation and 12-O-tetradecanoylphorbol-13-acetate-induced interaction of mouse epidermal protein kinase Cε with Stat3 involve integration with ERK1/2.

    PubMed

    Sand, Jordan Marshall; Bin Hafeez, Bilal; Aziz, Moammir Hasan; Siebers, Emily Marie; Dreckschmidt, Nancy Ellen; Verma, Ajit Kumar

    2012-04-01

    We have reported that protein kinase C epsilon (PKCε) expression level in epidermis dictates the susceptibility of mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by the DMBA-TPA tumor promotion protocol. To find clues about the mechanism by which PKCε mediates susceptibility to UVR-induced development of SCC, we found that PKCε-over-expressing transgenic mice, as compared to their wild-type littermates, when exposed to UVR, elicit enhanced phosphorylation of Stat3 at Ser727 residues. Stat3 is constitutively activated in SCC and UVR fails to induce SCC in Stat3 mutant mice. Stat3Ser727 phosphorylation is essential for Stat3 transcriptional activity (Cancer Res. 67: 1385, 2007). We now present several novel findings including that PKCε integrates with its downstream partner ERK1/2 to phosphorylate Stat3Ser727. In these experiments, mice were either exposed to UVR (2 kJ/m(2)/dose) emitted by Kodacel-filtered FS-40 sun lamps or treated with TPA (5 nmol). Both UVR and TPA treatment stimulated PKCε-Stat3 interaction, Stat3Ser727 phosphorylation and Stat3-regulated gene COX-2 expression. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was also observed in SCC elicited by repeated UVR exposures of mice. PKCε-Stat3 interaction was PKCε specific. UVR or TPA-stimulated Stat3Ser727 phosphorylation accompanied interaction of PKCε with ERK1/2 in intact mouse skin in vivo. Deletion of PKCε in wild-type mice attenuated both TPA and UVR-induced expression of phosphoforms of ERK1/2 and Stat3Ser727. These results indicate that PKCε integrates with ERK1/2 to mediate both TPA and UVR-induced epidermal Stat3Ser727 phosphorylation. PKCε and Stat3 may be potential molecular targets for SCC prevention. PMID:21480396

  16. Simultaneous Enrichment of Plasma Soluble and Extracellular Vesicular Glycoproteins Using Prolonged Ultracentrifugation-Electrostatic Repulsion-hydrophilic Interaction Chromatography (PUC-ERLIC) Approach*

    PubMed Central

    Sok Hwee Cheow, Esther; Hwan Sim, Kae; de Kleijn, Dominique; Neng Lee, Chuen; Sorokin, Vitaly; Sze, Siu Kwan

    2015-01-01

    Plasma glycoproteins and extracellular vesicles represent excellent sources of disease biomarkers, but laboratory detection of these circulating structures are limited by their relatively low abundance in complex biological fluids. Although intensive research has led to the development of effective methods for the enrichment and isolation of either plasma glycoproteins or extracellular vesicles from clinical materials, at present it is not possible to enrich both structures simultaneously from individual patient sample, a method that affords the identification of biomarker combinations from both entities for the prediction of clinical outcomes will be clinically useful. We have therefore developed an enrichment method for use in mass spectrometry-based proteomic profiling that couples prolonged ultracentrifugation with electrostatic repulsion-hydrophilic interaction chromatography, to facilitate the recovery of both glycoproteins and extracellular vesicles from nondepleted human plasma. Following prolonged ultracentrifugation, plasma glycoproteins and extracellular vesicles were concentrated as a yellow suspension, and simultaneous analyses of low abundant secretory and vesicular glycoproteins was achieved in a single LC-MS/MS run. Using this systematic prolonged ultracentrifugation-electrostatic repulsion-hydrophilic interaction chromatography approach, we identified a total of 127 plasma glycoproteins at a high level of confidence (FDR ≤ 1%), including 48 glycoproteins with concentrations ranging from pg to ng/ml. The novel enrichment method we report should facilitate future human plasma-based proteome and glycoproteome that will identify novel biomarkers, or combinations of secreted and vesicle-derived biomarkers, that can be used to predict clinical outcomes in human patients. PMID:25862729

  17. Structure-Based Network Analysis of Activation Mechanisms in the ErbB Family of Receptor Tyrosine Kinases: The Regulatory Spine Residues Are Global Mediators of Structural Stability and Allosteric Interactions

    PubMed Central

    James, Kevin A.; Verkhivker, Gennady M.

    2014-01-01

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

  18. Piperlongumine chemosensitizes tumor cells through interaction with cysteine 179 of IκBα kinase, leading to suppression of NF-κB-regulated gene products.

    PubMed

    Han, Jia Gang; Gupta, Subash C; Prasad, Sahdeo; Aggarwal, Bharat B

    2014-10-01

    Recently, two different reports appeared in prominent journals suggesting a mechanism by which piperlongumine, a pyridine alkaloid, mediates anticancer effects. In the current report, we describe another novel mechanism by which this alkaloid mediates its anticancer effects. We found that piperlongumine blocked NF-κB activated by TNFα and various other cancer promoters. This downregulation was accompanied by inhibition of phosphorylation and degradation of IκBα. Further investigation revealed that this pyridine alkaloid directly interacts with IκBα kinase (IKK) and inhibits its activity. Inhibition of IKK occurred through interaction with its cysteine 179 as the mutation of this residue to alanine abolished the activity of piperlongumine. Inhibition in NF-κB activity downregulated the expression of proteins involved in cell survival (Bcl-2, Bcl-xL, c-IAP-1, c-IAP-2, survivin), proliferation (c-Myc, cyclin D1), inflammation (COX-2, IL6), and invasion (ICAM-1, -9, CXCR-4, VEGF). Overall, our results reveal a novel mechanism by which piperlongumine can exhibit antitumor activity through downmodulation of proinflammatory pathway. PMID:25082961

  19. Kindlin-3 Mediates Integrin αLβ2 Outside-in Signaling, and It Interacts with Scaffold Protein Receptor for Activated-C Kinase 1 (RACK1)*

    PubMed Central

    Feng, Chen; Li, Yan-Feng; Yau, Yin-Hoe; Lee, Hui-Shan; Tang, Xiao-Yan; Xue, Zhi-Hong; Zhou, Yi-Chao; Lim, Wei-Min; Cornvik, Tobias C.; Ruedl, Christiane; Shochat, Susana G.; Tan, Suet-Mien

    2012-01-01

    Integrins are heterodimeric type I membrane cell adhesion molecules that are involved in many biological processes. Integrins are bidirectional signal transducers because their cytoplasmic tails are docking sites for cytoskeletal and signaling molecules. Kindlins are cytoplasmic molecules that mediate inside-out signaling and activation of the integrins. The three kindlin paralogs in humans are kindlin-1, -2, and -3. Each of these contains a 4.1-ezrin-radixin-moesin (FERM) domain and a pleckstrin homology domain. Kindlin-3 is expressed in platelets, hematopoietic cells, and endothelial cells. Here we show that kindlin-3 is involved in integrin αLβ2 outside-in signaling. It also promotes micro-clustering of integrin αLβ2. We provide evidence that kindlin-3 interacts with the receptor for activated-C kinase 1 (RACK1), a scaffold protein that folds into a seven-blade propeller. This interaction involves the pleckstrin homology domain of kindlin-3 and blades 5–7 of RACK1. Using the SKW3 human T lymphoma cells, we show that integrin αLβ2 engagement by its ligand ICAM-1 promotes the association of kindlin-3 with RACK1. We also show that kindlin-3 co-localizes with RACK1 in polarized SKW3 cells and human T lymphoblasts. Our findings suggest that kindlin-3 plays an important role in integrin αLβ2 outside-in signaling. PMID:22334666

  20. NMR analysis of interactions of a phosphatidylinositol 3'-kinase SH2 domain with phosphotyrosine peptides reveals interdependence of major binding sites.

    PubMed

    Günther, U L; Liu, Y; Sanford, D; Bachovchin, W W; Schaffhausen, B

    1996-12-01

    The interactions of the N-terminal src homology (SH2) domain (N-SH2) of the 85 kDa subunit of phosphatidylinositol 3'-kinase (PI-3K) with phosphotyrosine (ptyr) and a series of ptyr-containing peptides have been examined by NMR spectroscopy. HSQC (heteronuclear single-quantum coherence) NMR spectra of 15N-labeled SH2 were used to evaluate its interactions with ptyr-containing ligands. The ability of ligands to cause chemical shift changes was compared to their potency as competitors in in vitro binding experiments using polyoma virus middle T antigen (MT). The results suggest the interdependence of SH2 binding elements. Chemical shifts of residues involved in the ptyr binding were altered by variations of the sequence of the bound peptide, suggesting that the ptyr fit can be adjusted by the peptide sequence. Perturbations of chemical shifts of residues coordinating the methionine three residues C-terminal to the ptyr (the +3 residue) were affected by substitution in the binding peptide at +1 and vice versa. Such results show synergistic interplay between regions of the SH2 binding residues C-terminal to the ptyr. PMID:8952511

  1. LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis1[OPEN

    PubMed Central

    Min, Ling; Hu, Qin; Li, Yaoyao; Xu, Jiao; Ma, Yizan; Zhu, Longfu; Yang, Xiyan; Zhang, Xianlong

    2015-01-01

    Somatic embryogenesis (SE) is an efficient tool for the propagation of plant species and also, a useful model for studying the regulatory networks in embryo development. However, the regulatory networks underlying the transition from nonembryogenic callus to somatic embryos during SE remain poorly understood. Here, we describe an upland cotton (Gossypium hirsutum) CASEIN KINASE I gene, GhCKI, which is a unique key regulatory factor that strongly affects SE. Overexpressing GhCKI halted the formation of embryoids and plant regeneration because of a block in the transition from nonembryogenic callus to somatic embryos. In contrast, defective GhCKI in plants facilitated SE. To better understand the mechanism by which GhCKI regulates SE, the regulatory network was analyzed. A direct upstream negative regulator protein, cotton LEAFY COTYLEDON1, was identified to be targeted to a cis-element, CTTTTC, in the promoter of GhCKI. Moreover, GhCKI interacted with and phosphorylated cotton CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF transcription factor15 by coordinately regulating the expression of cotton PHYTOCHROME INTERACTING FACTOR4, finally disrupting auxin homeostasis, which led to increased cell proliferation and aborted somatic embryo formation in GhCKI-overexpressing somatic cells. Our results show a complex process of SE that is negatively regulated by GhCKI through a complex regulatory network. PMID:26491146

  2. Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy*

    PubMed Central

    Bagnéris, Claire; Rogala, Kacper B.; Baratchian, Mehdi; Zamfir, Vlad; Kunze, Micha B. A.; Dagless, Selina; Pirker, Katharina F.; Collins, Mary K.; Hall, Benjamin A.; Barrett, Tracey E.; Kay, Christopher W. M.

    2015-01-01

    Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site. PMID:25979343

  3. Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy.

    PubMed

    Bagnéris, Claire; Rogala, Kacper B; Baratchian, Mehdi; Zamfir, Vlad; Kunze, Micha B A; Dagless, Selina; Pirker, Katharina F; Collins, Mary K; Hall, Benjamin A; Barrett, Tracey E; Kay, Christopher W M

    2015-07-01

    Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site. PMID:25979343

  4. Simultaneous analysis of proteome, phospho- and glycoproteome of rat kidney tissue with electrostatic repulsion hydrophilic interaction chromatography.

    PubMed

    Hao, Piliang; Guo, Tiannan; Sze, Siu Kwan

    2011-01-01

    Protein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation. PMID:21373199

  5. Interaction Potential of the Multitargeted Receptor Tyrosine Kinase Inhibitor Dovitinib with Drug Transporters and Drug Metabolising Enzymes Assessed in Vitro

    PubMed Central

    Weiss, Johanna; Theile, Dirk; Dvorak, Zdenek; Haefeli, Walter Emil

    2014-01-01

    Dovitinib (TKI-258) is under development for the treatment of diverse cancer entities. No published information on its pharmacokinetic drug interaction potential is available. Thus, we assessed its interaction with important drug metabolising enzymes and drug transporters and its efficacy in multidrug resistant cells in vitro. P-glycoprotein (P-gp, MDR1, ABCB1) inhibition was evaluated by calcein assay, inhibition of breast cancer resistance protein (BCRP, ABCG2) by pheophorbide A efflux, and inhibition of organic anion transporting polypeptides (OATPs) by 8-fluorescein-cAMP uptake. Inhibition of cytochrome P450 3A4, 2C19, and 2D6 was assessed by using commercial kits. Induction of transporters and enzymes was quantified by real-time RT-PCR. Possible aryl hydrocarbon receptor (AhR) activating properties were assessed by a reporter gene assay. Substrate characteristics were evaluated by growth inhibition assays in cells over-expressing P-gp or BCRP. Dovitinib weakly inhibited CYP2C19, CYP3A4, P-gp and OATPs. The strongest inhibition was observed for BCRP (IC50 = 10.3 ± 4.5 μM). Among the genes investigated, dovitinib only induced mRNA expression of CYP1A1, CYP1A2, ABCC3 (coding for multidrug resistance-associated protein 3), and ABCG2 and suppressed mRNA expression of some transporters and drug metabolising enzymes. AhR reporter gene assay demonstrated that dovitinib is an activator of this nuclear receptor. Dovitinib retained its efficacy in cell lines over-expressing P-gp or BCRP. Our analysis indicates that dovitinib will most likely retain its efficacy in tumours over-expressing P-gp or BCRP and gives first evidence that dovitinib might act as a perpetrator drug in pharmacokinetic drug–drug interactions. PMID:25521244

  6. Liberation of an interaction domain from the phosphotransfer region of CheA, a signaling kinase of Escherichia coli.

    PubMed Central

    Morrison, T B; Parkinson, J S

    1994-01-01

    The CheA protein of Escherichia coli is a histidine autokinase that donates its phosphate groups to two target proteins, CheY and CheB, to regulate flagellar rotation and sensory adaptation during chemotactic responses. The amino-terminal third of CheA contains the autophosphorylation site, determinants needed to interact with the catalytic center of the molecule, and determinants needed for specific recognition of its phosphorylation targets. To understand the structural basis for these activities, we examined the domain organization of the CheA phosphotransfer region by using DNA sequence analysis, limited proteolytic digestion, and a genetic technique called domain liberation. Comparison of the functionally interchangeable CheA proteins of E. coli and Salmonella typhimurium revealed two extensively mismatched segments within the phosphotransfer region, 22 and 25 aa long, with sequences characteristic of domain linkers. Both segments were readily susceptible to proteases, implying that they have an extended, flexible structure. In contrast, the intervening segments of the phosphotransfer region, designated P1 and P2 (roughly 140 and 65 aa, respectively), were relatively insensitive, suggesting they correspond to more compactly folded structural domains. Their functional properties were explored by identifying portions of the cheA coding region capable of interfering with chemotactic behavior when "liberated" and expressed as polypeptides. P1 fragments were not inhibitory, but P2 fragments blocked the interaction of CheY with the rotational switch at the flagellar motor, leading to incessant forward swimming. These results suggest that P2 contains CheY-binding determinants which are normally responsible for phosphotransfer specificity. Domain-liberation approaches should prove generally useful for analyzing multidomain proteins and their interaction targets. Images PMID:8202513

  7. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  8. Leucine-rich repeat kinase 2 binds to neuronal vesicles through protein interactions mediated by its C-terminal WD40 domain.

    PubMed

    Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J O; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius; Gloeckner, Christian Johannes

    2014-06-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

  9. Crystal structure of the N-terminal SH3 domain of mouse {beta}PIX, p21-activated kinase-interacting exchange factor

    SciTech Connect

    Li Xiaofeng; Liu Xueqi; Sun Fei; Gao Jia; Zhou Hongwei; Gao, George F.; Bartlam, Mark; Rao Zihe . E-mail: raozh@xtal.tsinghua.edu.cn

    2006-01-06

    The mouse {beta}PIX-SH3 domain, residues 8-63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3{sub 2}21 diffracted to 2.0 A and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact {beta}-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse {beta}PIX-SH3 domain binding the way in which the {beta}PIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse {beta}PIX-SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse {beta}PIX-SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of {beta}PIX-SH3 domain resulting in its multiple biological functions.

  10. Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

    PubMed Central

    Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J. O.; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius

    2014-01-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

  11. Molecular cloning and functional characterization of a novel CBL-interacting protein kinase NtCIPK2 in the halophyte Nitraria tangutorum.

    PubMed

    Zheng, L L; Gao, Z; Wang, J; Zhang, H R; Wang, Y C

    2014-01-01

    CBL-interacting protein kinases (CIPKs) mediate many plant responses to abiotic stress. However, their functions are poorly understood in halophytes. In this study, we isolated a CIPK gene, NtCIPK2, from the halophyte Nitraria tangutorum. By sequence alignment and the construction of a phylogenetic tree, we found that NtCIPK2 is similar to CIPK2 proteins from other plants, and contains conserved domains and motifs. The promoter of NtCIPK2 harbors many cis-acting elements that might be recognized and bound by transcription factors that are related to hormones and stress responses. NtCIPK2 was ubiquitously and robustly expressed in all tested organs, and was induced by salinity, drought, heat, and cold stress. The overexpression of NtCIPK2 in Escherichia coli caused better growth against high salinity, alkalinity, and osmotic conditions, dehydration, and extreme temperatures (i.e., heat and cold) compared to the control. Thus, NtCIPK2 is a candidate gene that might improve the stress tolerance of crops and herbs through genetic manipulation. PMID:25062407

  12. Ectopic expression of wheat TaCIPK14, encoding a calcineurin B-like protein-interacting protein kinase, confers salinity and cold tolerance in tobacco.

    PubMed

    Deng, Xiaomin; Zhou, Shiyi; Hu, Wei; Feng, Jialu; Zhang, Fan; Chen, Lihong; Huang, Chao; Luo, Qingchen; He, Yanzhen; Yang, Guangxiao; He, Guangyuan

    2013-11-01

    Calcineurin B-like protein-interacting protein kinases (CIPKs) are components of Ca(2+) signaling in responses to abiotic stresses. In this work, the full-length cDNA of a novel CIPK gene (TaCIPK14) was isolated from wheat and was found to have significant sequence similarity to OsCIPK14/15. Subcellular localization assay revealed the presence of TaCIPK14 throughout the cell. qRT-PCR analysis showed that TaCIPK14 was upregulated under cold conditions or when treated with salt, PEG or exogenous stresses related signaling molecules including ABA, ethylene and H2 O2 . Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar, higher catalase activity, while decreased amounts of H2 O2 and malondialdehyde, and lesser ion leakage under cold and salt stresses. In addition, overexpression also increased seed germination rate, root elongation and decreased Na(+) content in the transgenic lines under salt stress. Higher expression of stress-related genes was observed in lines overexpressing TaCIPK14 compared to controls under stress conditions. In summary, these results suggested that TaCIPK14 is an abiotic stress-responsive gene in plants. PMID:23534344

  13. Arg Kinase-binding Protein 2 (ArgBP2) Interaction with α-Actinin and Actin Stress Fibers Inhibits Cell Migration*

    PubMed Central

    Anekal, Praju Vikas; Yong, Jeffery; Manser, Ed

    2015-01-01

    Cell migration requires dynamic remodeling of the actomyosin network. We report here that an adapter protein, ArgBP2, is a component of α-actinin containing stress fibers and inhibits migration. ArgBP2 is undetectable in many commonly studied cancer-derived cell lines. COS-7 and HeLa cells express ArgBP2 (by Western analysis), but expression was detectable only in approximately half the cells by immunofluorescence. Short term clonal analysis demonstrated 0.2–0.3% of cells switch ArgBP2 expression (on or off) per cell division. ArgBP2 can have a fundamental impact on the actomyosin network: ArgBP2 positive COS-7 cells, for example, are clearly distinguishable by their denser actomyosin (stress fiber) network. ArgBP2γ binding to α-actinin appears to underlie its ability to localize to stress fibers and decrease cell migration. We map a small α-actinin binding region in ArgBP2 (residues 192–228) that is essential for these effects. Protein kinase A phosphorylation of ArgBP2γ at neighboring Ser-259 and consequent 14-3-3 binding blocks its interaction with α-actinin. ArgBP2 is known to be down-regulated in some aggressively metastatic cancers. Our work provides a biochemical explanation for the anti-migratory effect of ArgBP2. PMID:25429109

  14. The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots1[OPEN

    PubMed Central

    Ragel, Paula; Ródenas, Reyes; García-Martín, Elena; Andrés, Zaida; Villalta, Irene; Nieves-Cordones, Manuel; Martínez, Vicente

    2015-01-01

    Plant growth and development requires efficient acquisition of essential elements. Potassium (K+) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K+ uptake systems in the roots is essential to secure K+ supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K+ concentration is very low (<10 µm), K+ nutrition depends exclusively on the high-affinity K+ transporter5 (HAK5). Low-K+-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca2+ sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K+ transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K+. Experiments of K+ (Rb+) uptake and growth measurements in low-K+ medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta. PMID:26474642

  15. Loss of c-Jun N-terminal kinase-interacting protein-1 does not affect axonal transport of the amyloid precursor protein or Aβ production

    PubMed Central

    Vagnoni, Alessio; Glennon, Elizabeth B.C.; Perkinton, Michael S.; Gray, Emma H.; Noble, Wendy; Miller, Christopher C.J.

    2013-01-01

    Disruption to axonal transport is an early pathological feature in Alzheimer's disease. The amyloid precursor protein (APP) is a key axonal transport cargo in Alzheimer's disease since perturbation of its transport increases APP processing and production of amyloid-β peptide (Aβ) that is deposited in the brains of Alzheimer's disease patients. APP is transported anterogradely through axons on kinesin-1 motors. One favoured route for attachment of APP to kinesin-1 involves the scaffolding protein c-Jun N-terminal kinase-interacting protein-1 (JIP1), which has been shown to bind both APP and kinesin-1 light chain (KLC). However, direct experimental evidence to support a role of JIP1 in APP transport is lacking. Notably, the effect of loss of JIP1 on movement of APP through axons of living neurons, and the impact of such loss on APP processing and Aβ production has not been reported. To address these issues, we monitored how siRNA mediated loss of JIP1 influenced transport of enhanced green fluorescent protein (EGFP)-tagged APP through axons and production of endogenous Aβ in living neurons. Surprisingly, we found that knockdown of JIP1 did not affect either APP transport or Aβ production. These results have important implications for our understanding of APP trafficking in Alzheimer's disease. PMID:23825109

  16. Simultaneous quantification of cardiolipin, bis(monoacylglycero)phosphate and their precursors by hydrophilic interaction LC-MS/MS including correction of isotopic overlap.

    PubMed

    Scherer, Max; Schmitz, Gerd; Liebisch, Gerhard

    2010-11-01

    Cardiolipin (CL) and bis(monoacylglycero)phosphate (BMP) are unique lipid structures with important biological roles for mitochondrial integrity and endolysosomal degradation, respectively. They are synthesized from common precursors, phosphatidylglycerol (PG) and phosphatidic acid (PA). Here we present a rapid method for the simultaneous quantification of BMP, CL, PG, and PA using hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry (HILIC-MS/MS). HILIC provides coelution of lipid species and their internal standards required for accurate quantification. Coelution leads to isotope overlap of lipid species which was successfully corrected. This assay was validated in mouse heart tissue and primary human skin fibroblasts. It shows reproducibility and limits of detection sufficient for biomarker studies contributing to basic research on BMP and CL metabolism. PMID:20945919

  17. Analysis of music-brain interaction with simultaneous measurement of regional cerebral blood flow and electroencephalogram beta rhythm in human subjects.

    PubMed

    Nakamura, S; Sadato, N; Oohashi, T; Nishina, E; Fuwamoto, Y; Yonekura, Y

    1999-11-19

    To elucidate the neural substrates of the receptive aspect of music, we measured regional cerebral blood flow (rCBF) with positron emission tomography (PET) and simultaneously recorded the electroencephalogram (EEG) in eight normal volunteers. Compared with the rest condition, listening to music caused a significant increase in EEG beta power spectrum (13-30 Hz) averaged over the posterior two third of the scalp. The averaged beta power spectrum was positively correlated with rCBF in the premotor cortex and adjacent prefrontal cortices bilaterally, the anterior portion of the precuneus and the anterior cingulate cortex in both the rest and the music conditions. Listening to music newly recruited the posterior portion of the precuneus bilaterally. This may reflect the interaction of the music with the cognitive processes, such as music-evoked memory recall or visual imagery. PMID:10580715

  18. A new “angle” on kinase inhibitor design: Prioritizing amphosteric activity above kinase inhibition

    PubMed Central

    Meyerowitz, Justin G; Weiss, William A; Gustafson, W Clay

    2015-01-01

    The MYCN oncoprotein has remained an elusive target for decades. We recently reported a new class of kinase inhibitors designed to disrupt the conformation of Aurora kinase A enough to block its kinase-independent interaction with MYCN, resulting in potent degradation of MYCN. These studies provide proof-of-principle for a new method of targeting enzyme activity-independent functions of kinases and other enzymes. PMID:27308435

  19. Simultaneous determination of residues of dipyrone metabolites in goat tissues by hydrophilic interaction liquid chromatography tandem mass spectrometry.

    PubMed

    Zhang, Chong; Zhang, Lifang; Cao, Suqing; Jiang, Zhaoling; Wu, Hao; Yan, Ming; Zhang, Xiaoxiao; Jiang, Shanxiang; Xue, Feiqun

    2016-04-01

    A reliable LC-MS/MS method with high sensitivity was developed and validated for the determination of dipyrone (DIP) metabolites in goat muscle, fat, liver, and kidney samples. Analytes were extracted using acetonitrile mixed with ammonia solution. After dehydration and evaporation to dryness, extracts were purified using an Oasis MAX cartridge. Chromatographic separation was performed on a hydrophilic interaction liquid chromatography column. The analytes were then detected using triple-quadrupole mass spectrometry in positive electrospray ionization and multiple reaction monitoring mode. Calibration plots were constructed using matrix-matched standards and showed good linearity. Limits of quantification for 4-methylaminoantipyrine (MAA), 4-formylaminoantipyrine (FAA), and 4-acetylaminoantipyrone (AAA) ranged from 0.4 μg kg(-1) to 6 μg kg(-1), while those for 4-aminoantipyrone (AA) ranged from 10 μg kg(-1) to 125 μg kg(-1) in all tissues. The developed method was successfully applied in the determination of DIP metabolite residues in actual goat tissues. PMID:26593468

  20. Sld3-MCM Interaction Facilitated by Dbf4-Dependent Kinase Defines an Essential Step in Eukaryotic DNA Replication Initiation

    PubMed Central

    Fang, Dingqiang; Cao, Qinhong; Lou, Huiqiang

    2016-01-01

    Sld3/Treslin is an evolutionarily conserved protein essential for activation of DNA helicase Mcm2-7 and replication initiation in all eukaryotes. Nevertheless, it remains elusive how Sld3 is recruited to origins. Here, we have identified the direct physical association of Sld3 with Mcm2 and Mcm6 subunits in vitro, which is significantly enhanced by DDK in vivo. The Sld3-binding domain (SBD) is mapped to the N-termini of Mcm2 and Mcm6, both of them are essential for cell viability and enriched with the DDK phosphorylation sites. Glutamic acid substitution of four conserved positively charged residues of Sld3 (sld3-4E), near the Cdc45-binding region, interrupts its interaction with Mcm2/6 and causes cell death. By using a temperature-inducible degron (td), we show that deletion of Mcm6 SBD (mcm6ΔN122) abolishes not only Sld3 enrichment at early origins in G1 phase, but also subsequent recruitment of GINS and RPA during S phase. These findings elucidate the in vivo molecular details of the DDK-dependent Sld3-MCM association, which plays a crucial role in MCM helicase activation and origin unwinding. PMID:27375603

  1. Discovery of a Selective and Potent Inhibitor of Mitogen-Activated Protein Kinase-Interacting Kinases 1 and 2 (MNK1/2) Utilizing Structure-Based Drug Design.

    PubMed

    Han, Wooseok; Ding, Yu; Xu, Yongjin; Pfister, Keith; Zhu, Shejin; Warne, Bob; Doyle, Mike; Aikawa, Mina; Amiri, Payman; Appleton, Brent; Stuart, Darrin D; Fanidi, Abdallah; Shafer, Cynthia M

    2016-04-14

    The discovery of a highly potent and selective small molecule inhibitor 9 for in vitro target validation of MNK1/2 kinases is described. The aminopyrazine benzimidazole series was derived from an HTS hit and optimized by utilization of a docking model, conformation analysis, and binding pocket comparison against antitargets. PMID:27002243

  2. Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078.

    PubMed

    Tang, Yong; Zhao, Chun-Yan; Tan, Shu-Tang; Xue, Hong-Wei

    2016-08-01

    Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5-1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5-1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5-1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5-1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

  3. Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078

    PubMed Central

    Tan, Shu-Tang; Xue, Hong-Wei

    2016-01-01

    Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5–1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5–1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5–1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5–1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

  4. Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62.

    PubMed

    Tsai, Li-Chun Lisa; Xie, Lei; Dore, Kim; Xie, Li; Del Rio, Jason C; King, Charles C; Martinez-Ariza, Guillermo; Hulme, Christopher; Malinow, Roberto; Bourne, Philip E; Newton, Alexandra C

    2015-09-01

    Atypical protein kinase C (aPKC) enzymes signal on protein scaffolds, yet how they are maintained in an active conformation on scaffolds is unclear. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of the atypical PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity; however, we have previously shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu-Zhang, A. X., Schramm, C. L., Nabavi, S., Malinow, R., and Newton, A. C. (2012) J. Biol. Chem. 287, 12879-12885). Here we sought to identify a bona fide target of ZIP and, in so doing, unveiled a novel mechanism by which aPKCs are maintained in an active conformation on a protein scaffold. Specifically, we used protein-protein interaction network analysis, structural modeling, and protein-protein docking to predict that ZIP binds an acidic surface on the Phox and Bem1 (PB1) domain of p62, an interaction validated by peptide array analysis. Using a genetically encoded reporter for PKC activity fused to the p62 scaffold, we show that ZIP inhibits the activity of wild-type aPKC, but not a construct lacking the pseudosubstrate. These data support a model in which the pseudosubstrate of aPKCs is tethered to the acidic surface on p62, locking aPKC in an open, signaling-competent conformation. ZIP competes for binding to the acidic surface, resulting in displacement of the pseudosubstrate of aPKC and re-engagement in the substrate-binding cavity. This study not only identifies a cellular target for ZIP, but also unveils a novel mechanism by which scaffolded aPKC is maintained in an active conformation. PMID:26187466

  5. Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62*

    PubMed Central

    Tsai, Li-Chun Lisa; Xie, Lei; Dore, Kim; Xie, Li; Del Rio, Jason C.; King, Charles C.; Martinez-Ariza, Guillermo; Hulme, Christopher; Malinow, Roberto; Bourne, Philip E.; Newton, Alexandra C.

    2015-01-01

    Atypical protein kinase C (aPKC) enzymes signal on protein scaffolds, yet how they are maintained in an active conformation on scaffolds is unclear. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of the atypical PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity; however, we have previously shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu-Zhang, A. X., Schramm, C. L., Nabavi, S., Malinow, R., and Newton, A. C. (2012) J. Biol. Chem. 287, 12879–12885). Here we sought to identify a bona fide target of ZIP and, in so doing, unveiled a novel mechanism by which aPKCs are maintained in an active conformation on a protein scaffold. Specifically, we used protein-protein interaction network analysis, structural modeling, and protein-protein docking to predict that ZIP binds an acidic surface on the Phox and Bem1 (PB1) domain of p62, an interaction validated by peptide array analysis. Using a genetically encoded reporter for PKC activity fused to the p62 scaffold, we show that ZIP inhibits the activity of wild-type aPKC, but not a construct lacking the pseudosubstrate. These data support a model in which the pseudosubstrate of aPKCs is tethered to the acidic surface on p62, locking aPKC in an open, signaling-competent conformation. ZIP competes for binding to the acidic surface, resulting in displacement of the pseudosubstrate of aPKC and re-engagement in the substrate-binding cavity. This study not only identifies a cellular target for ZIP, but also unveils a novel mechanism by which scaffolded aPKC is maintained in an active conformation. PMID:26187466

  6. Protein Kinase C-δ (PKCδ) Regulates Proinflammatory Chemokine Expression through Cytosolic Interaction with the NF-κB Subunit p65 in Vascular Smooth Muscle Cells*

    PubMed Central

    Ren, Jun; Wang, Qiwei; Morgan, Stephanie; Si, Yi; Ravichander, Aarthi; Dou, Changlin; Kent, K. Craig; Liu, Bo

    2014-01-01

    Proinflammatory chemokines released by vascular smooth muscle cells (VSMCs) play a critical role in vascular inflammation. Protein kinase C-δ (PKCδ) has been shown to be up-regulated in VSMCs of injured arteries. PKCδ knock-out (Prkcd−/−) mice are resistant to inflammation as well as apoptosis in models of abdominal aortic aneurysm. However, the precise mechanism by which PKCδ modulates inflammation remains incompletely understood. In this study, we identified four inflammatory chemokines (Ccl2/Mcp-1, Ccl7, Cxcl16, and Cx3cl1) of over 45 PKCδ-regulated genes associated with inflammatory response by microarray analysis. Using CCL2 as a prototype, we demonstrated that PKCδ stimulated chemokine expression at the transcriptional level. Inhibition of the NF-κB pathway or siRNA knockdown of subunit p65, but not p50, eliminated the effect of PKCδ on Ccl2 expression. Overexpressing PKCδ followed by incubation with phorbol 12-myristate 13-acetate resulted in an increase in p65 Ser-536 phosphorylation and enhanced DNA binding affinity without affecting IκB degradation or p65 nuclear translocation. Prkcd gene deficiency impaired p65 Ser-536 phosphorylation and DNA binding affinity in response to TNFα. Results from in situ proximity ligation analysis and co-immunoprecipitation performed on cultured VSMCs and aneurysmal aorta demonstrated physical interaction between PKCδ and p65 that took place largely outside the nucleus. Promoting nuclear translocation of PKCδ with peptide ψδRACK diminished Ccl2 production, whereas inhibition of PKCδ translocation with peptide δV1-1 enhanced Ccl2 expression. Together, these results suggest that PKCδ modulates inflammation at least in part through the NF-κB-mediated chemokines. Mechanistically, PKCδ activates NF-κB through an IκB-independent cytosolic interaction, which subsequently leads to enhanced p65 phosphorylation and DNA binding affinity. PMID:24519937

  7. Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel).

    PubMed Central

    Hruska-Hageman, Alesia M; Wemmie, John A; Price, Margaret P; Welsh, Michael J

    2002-01-01

    Neuronal members of the degenerin/epithelial Na(+) channel (DEG/ENaC) family of cation channels include the mammalian brain Na(+) channel 1 (BNC1), acid-sensing ion channel (ASIC) and dorsal-root acid-sensing ion channel (DRASIC). Their response to acidic pH, their sequence similarity to nematode proteins involved in mechanotransduction and their modulation by neuropeptides suggest that they may function as receptors for a number of different stimuli. Using the yeast two-hybrid assay, we found that the PDZ domain-containing protein PICK1 (protein interacting with C kinase) interacts specifically with the C-termini of BNC1 and ASIC, but not DRASIC or the related alphaENaC or betaENaC. In both the yeast two-hybrid system and mammalian cells, deletion of the BNC1 and ASIC C-termini abolished the interaction with PICK1. Likewise, mutating the PDZ domain in PICK1 abolished its interaction with BNC1 and ASIC. In addition, in a heterologous expression system PICK1 altered the distribution of BNC1 channels; this effect was dependent on the PDZ domain of PICK1 and the C-terminus of BNC1. We found crude synaptosomal fractions of brain to be enriched in ASIC, suggesting a possible synaptic localization. Moreover, in transfected hippocampal neurons ASIC co-localized with PICK1 in a punctate pattern at synapses. These data suggest that PICK1 binds ASIC and BNC1 via its PDZ domain. This interaction may be important for the localization and/or function of these channels in both the central and peripheral nervous systems. PMID:11802773

  8. OVATE Family Protein 8 Positively Mediates Brassinosteroid Signaling through Interacting with the GSK3-like Kinase in Rice

    PubMed Central

    He, Yong; Tian, Zhihong; Li, Jianxiong

    2016-01-01

    OVATE gene was first identified as a key regulator of fruit shape in tomato. OVATE family proteins (OFPs) are characterized as plant-specific transcription factors and conserved in Arabidopsis, tomato, and rice. Roles of OFPs involved in plant development and growth are largely unknown. Brassinosteroids (BRs) are a class of steroid hormones involved in diverse biological functions. OsGKS2 plays a critical role in BR signaling by phosphorylating downstream components such as OsBZR1 and DLT. Here we report in rice that OsOFP8 plays a positive role in BR signaling pathway. BL treatment induced the expression of OsOFP8 and led to enhanced accumulation of OsOFP8 protein. The gain-of-function mutant Osofp8 and OsOFP8 overexpression lines showed enhanced lamina joint inclination, whereas OsOFP8 RNAi transgenic lines showed more upright leaf phenotype, which suggest that OsOFP8 is involved in BR responses. Further analyses indicated that OsGSK2 interacts with and phosphorylates OsOFP8. BRZ treatment resulted in the cytoplasmic distribution of OsOFP8, and bikinin treatment reduced the cytoplasmic accumulation of OsOFP8. Phosphorylation of OsOFP8 by OsGSK2 is needed for its nuclear export. The phospphorylated OsOFP8 shuttles to the cytoplasm and is targeted for proteasomal degradation. These results indicate that OsOFP8 is a substrate of OsGSK2 and the function of OsOFP8 in plant growth and development is at least partly through the BR signaling pathway. PMID:27332964

  9. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  10. The side-by-side exploratory test: a simple automated protocol for the evaluation of adult zebrafish behavior simultaneously with social interaction.

    PubMed

    Schaefer, Isabel C; Siebel, Anna M; Piato, Angelo L; Bonan, Carla D; Vianna, Mônica R; Lara, Diogo R

    2015-10-01

    The assessment of shoaling in adult zebrafish is technically difficult, but important, given their social nature. The present study aimed to characterize a new protocol using simple automated tracking software to evaluate general behavior and social interaction simultaneously. To this end, we used a single tank with a central transparent glass division and placed one zebrafish on each side for 5 min. This strategy allows fish to interact visually at the same time that individual automated evaluation of behavior can be easily performed. Our results showed that, when two fish are placed side-by-side, there is an increase in their height in the tank compared with isolated fish and they remain close to each other. The pharmacological treatments with benzodiazepines (bromazepam and clonazepam) and the serotonergic drugs buspirone, fluoxetine, and escitalopram did not affect locomotion at the concentrations tested, except for the highest concentration of buspirone. Nevertheless, benzodiazepines increased interfish distance (i.e. reduced shoaling behavior) and serotonergic drugs elevated height in the tank. These results support the use of the side-by-side exploratory test for behavioral studies with the zebrafish, including high-throughput behavioral screening for antidepressants and anxiolytics. PMID:26061352

  11. The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast.

    PubMed

    Silva, Richard C; Dautel, Martina; Di Genova, Bruno M; Amberg, David C; Castilho, Beatriz A; Sattlegger, Evelyn

    2015-01-01

    The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown. Here, we show that Yih1 is involved in the cell cycle. Yeast lacking Yih1 displays morphological patterns and DNA content indicative of a delay in the G2/M phases of the cell cycle, and this phenotype is independent of Gcn1 and Gcn2. Accordingly, the levels of phosphorylated eIF2α, which show a cell cycle-dependent fluctuation, are not altered in cells devoid of Yih1. We present several lines of evidence indicating that Yih1 is in a complex with Cdc28. Yih1 pulls down endogenous Cdc28 in vivo and this interaction is enhanced when Cdc28 is active, suggesting that Yih1 modulates the function of Cdc28 in specific stages of the cell cycle. We also demonstrate, by Bimolecular Fluorescence Complementation, that endogenous Yih1 and Cdc28 interact with each other, confirming Yih1 as a bona fide Cdc28 binding partner. Amino acid substitutions within helix H2 of the RWD domain of Yih1 enhance Yih1-Cdc28 association. Overexpression of this mutant, but not of wild type Yih1, leads to a phenotype similar to that of YIH1 deletion, supporting the view that Yih1 is involved through Cdc28 in the regulation of the cell cycle. We further show that IMPACT, the mammalian homologue of Yih1, interacts with CDK1, the mammalian counterpart of Cdc28, indicating that the involvement with the cell cycle is conserved. Together, these data provide insights into the cellular function of Yih1/IMPACT, and provide the basis for future studies on the role of this protein in the cell cycle. PMID:26176233

  12. The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast

    PubMed Central

    Silva, Richard C.; Dautel, Martina; Di Genova, Bruno M.; Amberg, David C.; Castilho, Beatriz A.; Sattlegger, Evelyn

    2015-01-01

    The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown. Here, we show that Yih1 is involved in the cell cycle. Yeast lacking Yih1 displays morphological patterns and DNA content indicative of a delay in the G2/M phases of the cell cycle, and this phenotype is independent of Gcn1 and Gcn2. Accordingly, the levels of phosphorylated eIF2α, which show a cell cycle-dependent fluctuation, are not altered in cells devoid of Yih1. We present several lines of evidence indicating that Yih1 is in a complex with Cdc28. Yih1 pulls down endogenous Cdc28 in vivo and this interaction is enhanced when Cdc28 is active, suggesting that Yih1 modulates the function of Cdc28 in specific stages of the cell cycle. We also demonstrate, by Bimolecular Fluorescence Complementation, that endogenous Yih1 and Cdc28 interact with each other, confirming Yih1 as a bona fide Cdc28 binding partner. Amino acid substitutions within helix H2 of the RWD domain of Yih1 enhance Yih1-Cdc28 association. Overexpression of this mutant, but not of wild type Yih1, leads to a phenotype similar to that of YIH1 deletion, supporting the view that Yih1 is involved through Cdc28 in the regulation of the cell cycle. We further show that IMPACT, the mammalian homologue of Yih1, interacts with CDK1, the mammalian counterpart of Cdc28, indicating that the involvement with the cell cycle is conserved. Together, these data provide insights into the cellular function of Yih1/IMPACT, and provide the basis for future studies on the role of this protein in the cell cycle. PMID:26176233

  13. Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions*

    PubMed Central

    Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P.; Burgoyne, Robert D.; Mayans, Olga; Derrick, Jeremy P.; Lian, Lu-Yun

    2015-01-01

    Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca2+-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca2+/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca2+/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178–Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca2+/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178–Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. PMID:25979333

  14. G-Protein-Coupled Receptor Kinase 4 Polymorphisms and Blood Pressure Response to Metoprolol Among African Americans: Sex-Specificity and Interactions

    PubMed Central

    Bhatnagar, Vibha; O’Connor, Daniel T.; Brophy, Victoria H.; Schork, Nicholas J.; Richard, Erin; Salem, Rany M.; Nievergelt, Caroline M.; Bakris, George L.; Middleton, John P.; Norris, Keith C.; Wright, Jackson; Hiremath, Leena; Contreras, Gabriel; Appel, Lawrence J.; Lipkowitz, Michael S.

    2009-01-01

    BACKGROUND African Americans have a disproportionate burden of hypertension and comorbid disease. Pharmacogenetic markers of blood pressure response have yet to be defined clearly. This study explores the association between G-protein-coupled receptor kinase type 4 (GRK4) variants and blood pressure response to metoprolol among African Americans with early hypertensive nephrosclerosis. METHODS Participants from the African American Study of Kidney Disease and Hypertension (AASK) trial were genotyped at three GRK4 polymorphisms: R65L, A142V, and A486V. A Cox proportional hazards model, stratified by gender, was used to determine the relationship between GRK4 variants and time to reach a mean arterial pressure (MAP) of 107 mm Hg, adjusted for other predictors of blood pressure response. Potential interactions between the three polymorphisms were explored by analyzing the effects of gene haplotypes and by stratifying the analysis by neighboring sites. RESULTS The hazard ratio with 95% confidence interval by A142V among men randomized to a usual MAP (102–107 mm Hg) was 1.54 (1.11–2.44; P = 0.0009). The hazard ratio by A142V with R65/L65 or L65/L65 was 2.14 (1.35–3.39; P = 0.001). Haplotype analyses were consistent but inconclusive. There was no association between A142V and blood pressure response among women. CONCLUSIONS Results suggest a sex-specific relationship between GRK4 A142V and blood pressure response among African-American men with early hypertensive nephrosclerosis. Men with a GRK4 A142 were less responsive to metoprolol if they had a GRK4 L65 variant. The effect of GRK4 variants and blood pressure response to metoprolol should be studied in larger clinical trials. PMID:19119263

  15. Molecular Interaction of a Kinase Inhibitor Midostaurin with Anticancer Drug Targets, S100A8 and EGFR: Transcriptional Profiling and Molecular Docking Study for Kidney Cancer Therapeutics

    PubMed Central

    Mirza, Zeenat; Schulten, Hans-Juergen; Farsi, Hasan Ma; Al-Maghrabi, Jaudah A.; Gari, Mamdooh A.; Chaudhary, Adeel Ga; Abuzenadah, Adel M.; Al-Qahtani, Mohammed H.; Karim, Sajjad

    2015-01-01

    The S100A8 and epidermal growth factor receptor (EGFR) proteins are proto-oncogenes that are strongly expressed in a number of cancer types. EGFR promotes cellular proliferation, differentiation, migration and survival by activating molecular pathways. Involvement of proinflammatory S100A8 in tumor cell differentiation and progression is largely unclear and not studied in kidney cancer (KC). S100A8 and EGFR are potential therapeutic biomarkers and anticancer drug targets for KC. In this study, we explored molecular mechanisms of interaction profiles of both molecules with potential anticancer drugs. We undertook transcriptional profiling in Saudi KCs using Affymetrix HuGene 1.0 ST arrays. We identified 1478 significantly expressed genes, including S100A8 and EGFR overexpression, using cut-off p value <0.05 and fold change ≥2. Additionally, we compared and confirmed our findings with expression data available at NCBI’s GEO database. A significant number of genes associated with cancer showed involvement in cell cycle progression, DNA repair, tumor morphology, tissue development, and cell survival. Atherosclerosis signaling, leukocyte extravasation signaling, notch signaling, and IL-12 signaling were the most significantly disrupted signaling pathways. The present study provides an initial transcriptional profiling of Saudi KC patients. Our analysis suggests distinct transcriptomic signatures and pathways underlying molecular mechanisms of KC progression. Molecular docking analysis revealed that the kinase inhibitor "midostaurin" has amongst the selected drug targets, the best ligand properties to S100A8 and EGFR, with the implication that its binding inhibits downstream signaling in KC. This is the first structure-based docking study for the selected protein targets and anticancer drug, and the results indicate S100A8 and EGFR as attractive anticancer targets and midostaurin with effective drug properties for therapeutic intervention in KC. PMID:25789858

  16. Genetic ablation of homeodomain-interacting protein kinase 2 selectively induces apoptosis of cerebellar Purkinje cells during adulthood and generates an ataxic-like phenotype

    PubMed Central

    Anzilotti, S; Tornincasa, M; Gerlini, R; Conte, A; Brancaccio, P; Cuomo, O; Bianco, G; Fusco, A; Annunziato, L; Pignataro, G; Pierantoni, G M

    2015-01-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a multitalented coregulator of an increasing number of transcription factors and cofactors involved in cell death and proliferation in several organs and systems. As Hipk2−/− mice show behavioral abnormalities consistent with cerebellar dysfunction, we investigated whether Hipk2 is involved in these neurological symptoms. To this aim, we characterized the postnatal developmental expression profile of Hipk2 in the brain cortex, hippocampus, striatum, and cerebellum of mice by real-time PCR, western blot analysis, and immunohistochemistry. Notably, we found that whereas in the brain cortex, hippocampus, and striatum, HIPK2 expression progressively decreased with age, that is, from postnatal day 1 to adulthood, it increased in the cerebellum. Interestingly, mice lacking Hipk2 displayed atrophic lobules and a visibly smaller cerebellum than did wild-type mice. More important, the cerebellum of Hipk2−/− mice showed a strong reduction in cerebellar Purkinje neurons during adulthood. Such reduction is due to the activation of an apoptotic process associated with a compromised proteasomal function followed by an unpredicted accumulation of ubiquitinated proteins. In particular, Purkinje cell dysfunction was characterized by a strong accumulation of ubiquitinated β-catenin. Moreover, our behavioral tests showed that Hipk2−/− mice displayed muscle and balance impairment, indicative of Hipk2 involvement in cerebellar function. Taken together, these results indicate that Hipk2 exerts a relevant role in the survival of cerebellar Purkinje cells and that Hipk2 genetic ablation generates cerebellar dysfunction compatible with an ataxic-like phenotype. PMID:26633710

  17. Structural insights into the interactions of phorbol ester and bryostatin complexed with protein kinase C: a comparative molecular dynamics simulation study.

    PubMed

    Thangsunan, Patcharapong; Tateing, Suriya; Hannongbua, Supa; Suree, Nuttee

    2016-07-01

    Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer's disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin - the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn(2+)-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators. PMID:26292580

  18. Protein kinase C activation is involved in ultraviolet B irradiation-induced endothelial cell ICAM-1 up-regulation and lymphocyte-endothelium interaction in vitro.

    PubMed

    Funk, J O; Holler, E; Kohlhuber, F; Ueffing, M; Bornkamm, G W; Kind, P; Eissner, G

    1996-10-01

    Lymphocyte-endothelium interactions are pivotal steps in mediating inflammatory responses. The authors have analysed the influence of ultraviolet B (UVB) irradiation on intercellular adhesion molecule (ICAM)-1 expression on cells of the human microvascular endothelial cell line (HMEC)-1 and the intracellular signalling pathways involved. Flow cytometry revealed dose-dependent ICAM-1 up-regulation with maximum induced expression 24h after sublethal UVB irradiation of 10 mJ/cm2. While anti-tumour necrosis factor (TNF)-alpha antibodies or recombinant human interleukin (IL)-10 did not influence this response, anti-interferon (IFN)-gamma antibodies blocked the UVB-induced ICAM-1 up-regulation. Significant induction of intracellular/membrane-bound IFN-gamma was measured as early as 6 h post-UVB. Since previous work has shown a differential role of protein kinase C (PKC) in cytokine induced ICAM-1 expression, the effect of a selective bisindolylmaleimide-derived PKC-inhibitor (GF109203X) was studied. Ultraviolet B-induced ICAM-1 up-regulation was effectively blocked by the PKC-inhibitor, whereas a PKA-inhibitor was ineffective. Moreover, immunofluorescence analysis showed a radiation-induced membrane translocation of PKC-alpha, indicative of enzyme activation, in HMEC-1 cells already 30 min post-UVB. The functional relevance of the UVB-induced ICAM-1 expression and involvement of PKC in this process was demonstrated in an adhesion assay with peripheral blood mononuclear cells. In conclusion, UVB-induced ICAM-1 expression on human endothelial cells involves PKC-dependent pathways and can be prevented by a PKC-inhibitor. The use of PKC-inhibitors as additive modulators in immune reactions may bear clinical potential. The mechanisms of IFN-gamma induction in endothelial cells by UVB deserve further investigation. PMID:8845028

  19. The receptor-like kinase SOBIR1 interacts with Brassica napus LepR3 and is required for Leptosphaeria maculans AvrLm1-triggered immunity.

    PubMed

    Ma, Lisong; Borhan, M Hossein

    2015-01-01

    The fungus Leptosphaeria maculans (L. maculans) is the causal agent of blackleg disease of canola/oilseed rape (Brassica napus) worldwide. We previously reported cloning of the B. napus blackleg resistance gene, LepR3, which encodes a receptor-like protein. LepR3 triggers localized cell death upon recognition of its cognate Avr protein, AvrLm1. Here, we exploited the Nicotiana benthamiana model plant to investigate the recognition mechanism of AvrLm1 by LepR3. Co-expression of the LepR3/AvrLm1 gene pair in N. benthamiana resulted in development of a hypersensitive response (HR). However, a truncated AvrLm1 lacking its indigenous signal peptide was compromised in its ability to induce LepR3-mediated HR, indicating that AvrLm1 is perceived by LepR3 extracellularly. Structure-function analysis of the AvrLm1 protein revealed that the C-terminal region of AvrLm1 was required for LepR3-mediated HR in N. benthamiana and for resistance to L. maculans in B. napus. LepR3 was shown to be physically interacting with the B. napus receptor like kinase, SOBIR1 (BnSOBIR1). Silencing of NbSOBIR1 or NbSERK3 (BAK1) compromised LepR3-AvrLm1-dependent HR in N. benthamiana, suggesting that LepR3-mediated resistance to L. maculans in B. napus requires SOBIR1 and BAK1/SERK3. Using this model system, we determined that BnSOBIR1 and SERK3/BAK1 are essential partners in the LepR3 signaling complex and were able to define the AvrLm1 effector domain. PMID:26579176

  20. KIDFamMap: a database of kinase-inhibitor-disease family maps for kinase inhibitor selectivity and binding mechanisms.

    PubMed

    Chiu, Yi-Yuan; Lin, Chih-Ta; Huang, Jhang-Wei; Hsu, Kai-Cheng; Tseng, Jen-Hu; You, Syuan-Ren; Yang, Jinn-Moon

    2013-01-01

    Kinases play central roles in signaling pathways and are promising therapeutic targets for many diseases. Designing selective kinase inhibitors is an emergent and challenging task, because kinases share an evolutionary conserved ATP-binding site. KIDFamMap (http://gemdock.life.nctu.edu.tw/KIDFamMap/) is the first database to explore kinase-inhibitor families (KIFs) and kinase-inhibitor-disease (KID) relationships for kinase inhibitor selectivity and mechanisms. This database includes 1208 KIFs, 962 KIDs, 55 603 kinase-inhibitor interactions (KIIs), 35 788 kinase inhibitors, 399 human protein kinases, 339 diseases and 638 disease allelic variants. Here, a KIF can be defined as follows: (i) the kinases in the KIF with significant sequence similarity, (ii) the inhibitors in the KIF with significant topology similarity and (iii) the KIIs in the KIF with significant interaction similarity. The KIIs within a KIF are often conserved on some consensus KIDFamMap anchors, which represent conserved interactions between the kinase subsites and consensus moieties of their inhibitors. Our experimental results reveal that the members of a KIF often possess similar inhibition profiles. The KIDFamMap anchors can reflect kinase conformations types, kinase functions and kinase inhibitor selectivity. We believe that KIDFamMap provides biological insights into kinase inhibitor selectivity and binding mechanisms. PMID:23193279

  1. KIDFamMap: a database of kinase-inhibitor-disease family maps for kinase inhibitor selectivity and binding mechanisms

    PubMed Central

    Chiu, Yi-Yuan; Lin, Chih-Ta; Huang, Jhang-Wei; Hsu, Kai-Cheng; Tseng, Jen-Hu; You, Syuan-Ren; Yang, Jinn-Moon

    2013-01-01

    Kinases play central roles in signaling pathways and are promising therapeutic targets for many diseases. Designing selective kinase inhibitors is an emergent and challenging task, because kinases share an evolutionary conserved ATP-binding site. KIDFamMap (http://gemdock.life.nctu.edu.tw/KIDFamMap/) is the first database to explore kinase-inhibitor families (KIFs) and kinase-inhibitor-disease (KID) relationships for kinase inhibitor selectivity and mechanisms. This database includes 1208 KIFs, 962 KIDs, 55 603 kinase-inhibitor interactions (KIIs), 35 788 kinase inhibitors, 399 human protein kinases, 339 diseases and 638 disease allelic variants. Here, a KIF can be defined as follows: (i) the kinases in the KIF with significant sequence similarity, (ii) the inhibitors in the KIF with significant topology similarity and (iii) the KIIs in the KIF with significant interaction similarity. The KIIs within a KIF are often conserved on some consensus KIDFamMap anchors, which represent conserved interactions between the kinase subsites and consensus moieties of their inhibitors. Our experimental results reveal that the members of a KIF often possess similar inhibition profiles. The KIDFamMap anchors can reflect kinase conformations types, kinase functions and kinase inhibitor selectivity. We believe that KIDFamMap provides biological insights into kinase inhibitor selectivity and binding mechanisms. PMID:23193279

  2. The stimulus-dependent co-localization of serum- and glucocorticoid-regulated protein kinase (Sgk) and Erk/MAPK in mammary tumor cells involves the mutual interaction with the importin-alpha nuclear import protein

    SciTech Connect

    Buse, Patricia; Maiyar, Anita C.; Failor, Kim L.; Tran, Susan; Leong, Meredith L.L.; Firestone, Gary L.

    2007-09-10

    In Con8 rat mammary epithelial tumor cells, indirect immunofluorescence revealed that Sgk (serum- and glucocorticoid-regulated kinase) and Erk/MAPK (extracellular signal-regulated protein kinase/mitogen activated protein kinase) co-localized to the nucleus in serum-treated cells and to the cytoplasmic compartment in cells treated with the synthetic glucocorticoid dexamethasone. Moreover, the subcellular distribution of the importin-alpha nuclear transport protein was similarly regulated in a signal-dependent manner. In vitro GST-pull down assays revealed the direct interaction of importin-alpha with either Sgk or Erk/MAPK, while RNA interference knockdown of importin-alpha expression disrupted the localization of both Sgk and Erk into the nucleus of serum-treated cells. Wild type or kinase dead forms of Sgk co-immunoprecipitated with Erk/MAPK from either serum- or dexamethasone-treated mammary tumor cells, suggesting the existence of a protein complex containing both kinases. In serum-treated cells, nucleus residing Sgk and Erk/MAPK were both hyperphosphorylated, indicative of their active states, whereas, in dexamethasone-treated cells Erk/MAPK, but not Sgk, was in its inactive hypophosphorylated state. Treatment with a MEK inhibitor, which inactivates Erk/MAPK, caused the relocalization of both Sgk and ERK to the cytoplasm. We therefore propose that the signal-dependent co-localization of Sgk and Erk/MAPK mediated by importin-alpha represents a new pathway of signal integration between steroid and serum/growth factor-regulated pathways.

  3. Ligand stimulation induces clathrin- and Rab5-dependent downregulation of the kinase-dead EphB6 receptor preceded by the disruption of EphB6-Hsp90 interaction.

    PubMed

    Allonby, Odette; El Zawily, Amr M; Freywald, Tanya; Mousseau, Darrell D; Chlan, Jennifer; Anderson, Deborah; Benmerah, Alexandre; Sidhu, Vishaldeep; Babu, Mohan; DeCoteau, John; Freywald, Andrew

    2014-12-01

    Ligand-induced internalisation and subsequent downregulation of receptor tyrosine kinases (RTKs) serve to determine biological outputs of their signalling. Intrinsically kinase-deficient RTKs control a variety of biological responses, however, the mechanism of their downregulation is not well understood and its analysis is focused exclusively on the ErbB3 receptor. The Eph group of RTKs is represented by the EphA and EphB subclasses. Each bears one kinase-inactive member, EphA10 and EphB6, respectively, suggesting an important role for these molecules in the Eph signalling network. While EphB6 effects on cell behaviour have been assessed, the mechanism of its downregulation remains elusive. Our work reveals that EphB6 and its kinase-active relative, and signalling partner, EphB4, are downregulated in a similar manner in response to their common ligand, ephrin-B2. Following stimulation, both receptors are internalised through clathrin-coated pits and are degraded in lysosomes. Their targeting for lysosomal degradation relies on the activity of an early endosome regulator, the Rab5 GTPase, as this process is inhibited in the presence of a Rab5 dominant-negative mutant. EphB6 also interacts with the Hsp90 chaperone and EphB6 downregulation is preceded by their rapid dissociation. Moreover, the inhibition of Hsp90 results in EphB6 degradation, mimicking its ligand-induced downregulation. These processes appear to rely on overlapping mechanisms, since Hsp90 inhibition does not significantly enhance ligand-induced EphB6 elimination. Taken together, our observations define a novel mechanism for intrinsically kinase-deficient RTK downregulation and support an intriguing model, where Hsp90 dissociation acts as a trigger for ligand-induced receptor removal. PMID:25152371

  4. p21-Activated kinase 2 (PAK2) inhibits TGF-β signaling in Madin-Darby canine kidney (MDCK) epithelial cells by interfering with the receptor-Smad interaction.

    PubMed

    Yan, Xiaohua; Zhang, Junyu; Sun, Qinyu; Tuazon, Polygena T; Wu, Xiaoping; Traugh, Jolinda A; Chen, Ye-Guang

    2012-04-20

    TGF-β (transforming growth factor β) plays a variety of cellular functions mainly through the Smad pathway. Phosphorylation of the carboxyl SXS motif in R-Smads (Smad2 and Smad3) by the type I receptor TβRI is a key step for their activation. It has been reported that the serine/threonine kinase PAK2 (p21-activated kinase 2) can mediate TGF-β signaling in mesenchymal cells. Here, we report that PAK2 restricts TGF-β-induced Smad2/3 activation and transcriptional responsiveness in MDCK epithelial cells. Mechanistically, PAK2 associates with Smad2 and Smad3 in a kinase activity-dependent manner and blocks their activation. PAK2 phosphorylates Smad2 at Ser-417, which is adjacent to the L3 loop that contributes to the TβRI-R-Smad association. Consistently, substitution of Ser-417 with glutamic acid attenuates the interaction of Smad2 with TβRI. Together, our results indicate that PAK2 negatively modulate TGF-β signaling by attenuating the receptor-Smad interaction and thus Smad activation. PMID:22393057

  5. Simultaneous Determination of Metformin, Metoprolol and its Metabolites in Rat Plasma by LC-MS-MS: Application to Pharmacokinetic Interaction Study.

    PubMed

    Ma, Yan-rong; Rao, Zhi; Shi, A-xi; Wang, Ya-feng; Huang, Jing; Han, Miao; Wang, Xin-dong; Jin, Yong-wen; Zhang, Guo-qiang; Zhou, Yan; Zhang, Fan; Qin, Hong-yan; Wu, Xin-an

    2016-01-01

    A simple, rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the simultaneous quantitation of metformin (MTF), metoprolol (MET), α-hydroxymetoprolol (HMT) and O-desmethylmetoprolol (DMT) in rat plasma using paracetamol as an internal standard (IS), respectively. The sample preparation involved a protein-precipitation method with methanol after the addition of IS. The separation was performed on an Agilent HC-C18 column (4.6 × 250 mm, 5 µm) at a flow rate of 1.0 mL/min, using methanol-water containing 0.1% formic acid (39:61, v/v) as mobile phase, and total run time was 8.5 min. MS-MS detection was accomplished in multiple reaction monitoring mode with positive electrospray ionization. The monitored transitions were m/z 130.1 → 60.2 for MTF, m/z 268.2 → 116.1 for MET, m/z 284.2 → 116.1 for HMT, m/z 254.2 → 116.1 for DMT and m/z 152.3 → 110.1 for IS. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 19.53-40,000 ng/mL for MTF, 3.42-7,000 ng/mL for MET, 2.05-4,200 ng/mL for HMT and 1.95-4,000 ng/mL for DMT, respectively. The analytical method was successfully applied to drug interaction study of MTF and MET after oral administration of MTF and MET. Results suggested that the coadministration of MTF and MET results in a significant drug interaction in rat. PMID:26187926

  6. Simultaneous profiling of seed-associated bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum and Brassica seeds

    PubMed Central

    Links, Matthew G; Demeke, Tigst; Gräfenhan, Tom; Hill, Janet E; Hemmingsen, Sean M; Dumonceaux, Tim J

    2014-01-01

    In order to address the hypothesis that seeds from ecologically and geographically diverse plants harbor characteristic epiphytic microbiota, we characterized the bacterial and fungal microbiota associated with Triticum and Brassica seed surfaces. The total microbial complement was determined by amplification and sequencing of a fragment of chaperonin 60 (cpn60). Specific microorganisms were quantified by qPCR. Bacteria and fungi corresponding to operational taxonomic units (OTU) that were identified in the sequencing study were isolated and their interactions examined. A total of 5477 OTU were observed from seed washes. Neither total epiphytic bacterial load nor community richness/evenness was significantly different between the seed types; 578 OTU were shared among all samples at a variety of abundances. Hierarchical clustering revealed that 203 were significantly different in abundance on Triticum seeds compared with Brassica. Microorganisms isolated from seeds showed 99–100% identity between the cpn60 sequences of the isolates and the OTU sequences from this shared microbiome. Bacterial strains identified as Pantoea agglomerans had antagonistic properties toward one of the fungal isolates (Alternaria sp.), providing a possible explanation for their reciprocal abundances on both Triticum and Brassica seeds. cpn60 enabled the simultaneous profiling of bacterial and fungal microbiota and revealed a core seed-associated microbiota shared between diverse plant genera. PMID:24444052

  7. Simultaneous profiling of seed-associated bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum and Brassica seeds.

    PubMed

    Links, Matthew G; Demeke, Tigst; Gräfenhan, Tom; Hill, Janet E; Hemmingsen, Sean M; Dumonceaux, Tim J

    2014-04-01

    In order to address the hypothesis that seeds from ecologically and geographically diverse plants harbor characteristic epiphytic microbiota, we characterized the bacterial and fungal microbiota associated with Triticum and Brassica seed surfaces. The total microbial complement was determined by amplification and sequencing of a fragment of chaperonin 60 (cpn60). Specific microorganisms were quantified by qPCR. Bacteria and fungi corresponding to operational taxonomic units (OTU) that were identified in the sequencing study were isolated and their interactions examined. A total of 5477 OTU were observed from seed washes. Neither total epiphytic bacterial load nor community richness/evenness was significantly different between the seed types; 578 OTU were shared among all samples at a variety of abundances. Hierarchical clustering revealed that 203 were significantly different in abundance on Triticum seeds compared with Brassica. Microorganisms isolated from seeds showed 99-100% identity between the cpn60 sequences of the isolates and the OTU sequences from this shared microbiome. Bacterial strains identified as Pantoea agglomerans had antagonistic properties toward one of the fungal isolates (Alternaria sp.), providing a possible explanation for their reciprocal abundances on both Triticum and Brassica seeds. cpn60 enabled the simultaneous profiling of bacterial and fungal microbiota and revealed a core seed-associated microbiota shared between diverse plant genera. PMID:24444052

  8. Simultaneous analysis of acetaminophen, p-aminophenol and aspirin metabolites by hydrophilic interaction and strong anion exchange capillary liquid chromatography coupled to amperometric detection.

    PubMed

    Zheng, Minmin; Wu, Yimin; Lu, Lanxiang; Ding, Kang; Tang, Fengxiang; Lin, Zian; Wu, Xiaoping

    2011-08-01

    A simple and sensitive method has been developed for the simultaneous determination of polar nonsteroidal pharmaceuticals and metabolites, including acetaminophen, p-aminophenol and several aspirin metabolites (salicylic acid, gentisic acid, salicyluric acid and 2,3-dihydroxybenzoic acid), by capillary liquid chromatography with amperometric detection. Using a capillary monolithic column with mixed mode stationary phases and a mobile phase composed of acetonitrile and Tris buffer, rapid separation of six polar analytes was achieved within 8 min, and a hydrophilic interaction and strong anion exchange separation mechanism were exhibited. Method detection limits of six analytes ranged from 10 to 50 ng/mL. In terms of precision, the intra- and interday relative standard deviation values in all analytes never exceeded 3.1% for migration time and 8.9% for peak areas, respectively. This method provided a simple, rapid and cost-effective approach for the analysis of polar pharmaceuticals. The applicability of the method in pharmacokinetics was verified by spiking human serum samples with the compounds and analyzing the recoveries. PMID:21644253

  9. A new hydrophilic interaction liquid chromatographic (HILIC) procedure for the simultaneous determination of pseudoephedrine hydrochloride (PSH), diphenhydramine hydrochloride (DPH) and dextromethorphan hydrobromide (DXH) in cough-cold formulations.

    PubMed

    Ali, Mohammed Shahid; Ghori, Mohsin; Rafiuddin, Syed; Khatri, Aamer Roshanali

    2007-01-01

    A new HILIC method has been developed for the simultaneous determination of pseudoephedrine hydrochloride (PSH), diphenhydramine hydrochloride (DPH) and dextromethorphan hydrobromide (DXH) in cough-cold syrup. Mobile phase consists of methanol:water (containing 6.0 g of ammonium acetate and 10 mL of triethylamine per liter, pH adjusted to 5.2 with orthophosphoric acid), 95:5 (v/v). Column containing porous silica particles (Supelcosil LC-Si, 25 cm x 4.6 mm, 5 microm) is used as stationary phase. Detection is carried out using a variable wavelength UV-vis detector at 254 nm for PSH and DPH, and at 280 nm for DXH. Solutions are injected into the chromatograph under isocratic condition at constant flow rate of 1.2 mL/min. Linearity range and percent recoveries for PSH, DPH and DXH were 150-600, 62.5-250, 75-300 microg/mL and 100.7%, 100.1% and 100.8%, respectively. Method is stability indicating and excipients like saccharin sodium, sodium citrate, flavour and sodium benzoate did not interfere in the analysis. Compounds elute in order of increasing ionization degree caused by cation-exchange mechanism in a run time of less than 15 min. Mobile phase pH is manipulated to regulate ionization and ion-exchange interaction and thereby retention of compounds. PMID:16887317

  10. Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell.

    PubMed

    Nagano, Takashi; Lubling, Yaniv; Yaffe, Eitan; Wingett, Steven W; Dean, Wendy; Tanay, Amos; Fraser, Peter

    2015-12-01

    Hi-C is a powerful method that provides pairwise information on genomic regions in spatial proximity in the nucleus. Hi-C requires millions of cells as input and, as genome organization varies from cell to cell, a limitation of Hi-C is that it only provides a population average of genome conformations. We developed single-cell Hi-C to create snapshots of thousands of chromatin interactions that occur simultaneously in a single cell. To adapt Hi-C to single-cell analysis, we modified the protocol to include in-nucleus ligation. This enables the isolation of single nuclei carrying Hi-C-ligated DNA into separate tubes, followed by reversal of cross-links, capture of biotinylated ligation junctions on streptavidin-coated magnetic beads and PCR amplification of single-cell Hi-C libraries. The entire laboratory protocol can be carried out in 1 week, and although we have demonstrated its use in mouse T helper (TH1) cells, it should be applicable to any cell type or species for which standard Hi-C has been successful. We also developed an analysis pipeline to filter noise and assess the quality of data sets in a few hours. Although the interactome maps produced by single-cell Hi-C are sparse, the data provide useful information to understand cellular variability in nuclear genome organization and chromosome structure. Standard wet and dry laboratory skills in molecular biology and computational analysis are required. PMID:26540590

  11. The split Renilla luciferase complementation assay is useful for identifying the interaction of Epstein-Barr virus protein kinase BGLF4 and a heat shock protein Hsp90.

    PubMed

    Wang, J; Guo, W; Long, C; Zhou, H; Wang, H; Sun, X

    2016-03-01

    Protein-protein interactions can regulate different cellular processes, such as transcription, translation, and oncogenic transformation. The split Renilla luciferase complementation assay (SRLCA) is one of the techniques that detect protein-protein interactions. The SRLCA is based on the complementation of the LN and LC non-functional halves of Renilla luciferase fused to possibly interacting proteins which after interaction form a functional enzyme and emit luminescence. The BGLF4 of Epstein-Barr virus (EBV) is a viral protein kinase that is expressed during the early and late stages of lytic cycles, which can regulate multiple cellular and viral substrates to optimize the DNA replication environment. The heat shock protein Hsp90 is a molecular chaperone that maintains the integrity of structure and function of various interacting proteins, which can form a complex with BGLF4 and stabilize its expression in cells. The interaction between BGLF4 and Hsp90 could be specifically detected through the SRLCA. The region of aa 250-295 of BGLF4 is essential for the BGLF4/Hsp90 interaction and the mutation of Phe-254, Leu-266, and Leu-267 can disrupt this interaction. These results suggest that the SRLCA can specifically detect the BGLF4/Hsp90 interaction and provide a reference to develop inhibitors that disrupt the BGLF4/Hsp90 interaction. PMID:26982469

  12. DNA-dependent protein kinase interacts functionally with the RNA polymerase II complex recruited at the human immunodeficiency virus (HIV) long terminal repeat and plays an important role in HIV gene expression.

    PubMed

    Tyagi, Shilpi; Ochem, Alex; Tyagi, Mudit

    2011-07-01

    DNA-dependent protein kinase (DNA-PK), a nuclear protein kinase that specifically requires association with DNA for its kinase activity, plays important roles in the regulation of different DNA transactions, including transcription, replication and DNA repair, as well as in the maintenance of telomeres. Due to its large size, DNA-PK is also known to facilitate the activities of other factors by providing the docking platform at their site of action. In this study, by running several chromatin immunoprecipitation assays, we demonstrate the parallel distribution of DNA-PK with RNA polymerase II (RNAP II) along the human immunodeficiency virus (HIV) provirus before and after activation with tumour necrosis factor alpha. The association between DNA-PK and RNAP II is also long-lasting, at least for up to 4 h (the duration analysed in this study). Knockdown of endogenous DNA-PK using specific small hairpin RNAs expressed from lentiviral vectors resulted in significant reduction in HIV gene expression and replication, demonstrating the importance of DNA-PK for HIV gene expression. Sequence analysis of the HIV-1 Tat protein revealed three potential target sites for phosphorylation by DNA-PK and, by using kinase assays, we confirmed that Tat is an effective substrate of DNA-PK. Through peptide mapping, we found that two of these three potential phosphorylation sites are recognized and phosphorylated by DNA-PK. Mutational studies on the DNA-PK target sites of Tat further demonstrated the functional significance of the Tat-DNA-PK interaction. Thus, overall our results clearly demonstrate the functional interaction between DNA-PK and RNAP II during HIV transcription. PMID:21450944

  13. Estrogen Regulates MAPK-Related Genes through Genomic and Nongenomic Interactions between IGF-I Receptor Tyrosine Kinase and Estrogen Receptor-Alpha Signaling Pathways in Human Uterine Leiomyoma Cells.

    PubMed

    Yu, Linda; Moore, Alicia B; Castro, Lysandra; Gao, Xiaohua; Huynh, Hoang-Long C; Klippel, Michelle; Flagler, Norris D; Lu, Yi; Kissling, Grace E; Dixon, Darlene

    2012-01-01

    Estrogen and growth factors play a major role in uterine leiomyoma (UtLM) growth possibly through interactions of receptor tyrosine kinases (RTKs) and estrogen receptor-alpha (ERα) signaling. We determined the genomic and nongenomic effects of 17β-estradiol (E(2)) on IGF-IR/MAPKp44/42 signaling and gene expression in human UtLM cells with intact or silenced IGF-IR. Analysis by RT(2) Profiler PCR-array showed genes involved in IGF-IR/MAPK signaling were upregulated in UtLM cells by E(2) including cyclin D kinases, MAPKs, and MAPK kinases; RTK signaling mediator, GRB2; transcriptional factors ELK1 and E2F1; CCNB2 involved in cell cycle progression, proliferation, and survival; and COL1A1 associated with collagen synthesis. Silencing (si)IGF-IR attenuated the above effects and resulted in upregulation of different genes, such as transcriptional factor ETS2; the tyrosine kinase receptor, EGFR; and DLK1 involved in fibrosis. E(2) rapidly activated IGF-IR/MAPKp44/42 signaling nongenomically and induced phosphorylation of ERα at ser118 in cells with a functional IGF-IR versus those without. E(2) also upregulated IGF-I gene and protein expression through a prolonged genomic event. These results suggest a pivotal role of IGF-IR and possibly other RTKs in mediating genomic and nongenomic hormone receptor interactions and signaling in fibroids and provide novel genes and targets for future intervention and prevention strategies. PMID:23094148

  14. Simultaneous active intracellular delivery of doxorubicin and C6-ceramide shifts the additive/antagonistic drug interaction of non-encapsulated combination.

    PubMed

    Fonseca, Nuno A; Gomes-da-Silva, Lígia C; Moura, Vera; Simões, Sérgio; Moreira, João Nuno

    2014-12-28

    Drug resistance remains the Achilles tendon undermining the success of chemotherapy. It has been recognized that success requires the identification of compounds that, when combined, lead to synergistic tumor inhibition while simultaneously minimizing systemic toxicity. However, in vivo application of such protocols is dependent on the ability to deliver the appropriate drug ratio at the tumor level. In this respect, nanotechnology-based delivery platforms, like liposomes, offer an elegant solution for the in vivo translation of such strategy. In this work, we propose the active intracellular delivery of combinations of doxorubicin and the pro-apoptotic sphingolipid, C6-ceramide, using our previously described cytosolic triggered release-enabling liposomes, targeting nucleolin with the F3 peptide. Combination of doxorubicin (DXR):C6-ceramide (C6-Cer) at 1:2 molar ratio interacted synergistically against drug resistant/triple negative MDA-MB-231 breast cancer cells, as well as drug sensitive MDA-MB-435S melanoma cells. Cell viability studies indicated that F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molar ratio (p[F3]DC12) performed similarly as targeted liposomal DXR (p[F3]SL), encapsulating twice the amount of DXR, at the IC50, for an incubation time of 24 h. Importantly, F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molar ratio (p[F3]DC12) enabled a cell death above 90% at 24 h of treatment against both DXR-resistant and sensitive cells, unattainable by the F3-targeted liposomal doxorubicin. Furthermore, a F3-targeted formulation encapsulating a mildly additive/antagonistic DXR:C6-Cer 1:1 molar ratio (p[F3]DC11) enabled an effect above 90% for an incubation period as short as 4 h, suggesting that the delivery route at the cell level may shift the nature of drug interaction. Such activity, including the one for p[F3]DC12, induced a marked cell and nucleus swelling at similar extent, consistent with necrotic cell death. Overall, these results

  15. Interactions between protein kinase C and arachidonic acid in the gonadotropin response to salmon and chicken gonadotropin-releasing hormone-II in goldfish.

    PubMed

    Chang, J P; Van Goor, F; Neumann, C M

    1994-03-01

    Previous studies have shown that, in goldfish, the gonadotropin (GTH) response to salmon GTH-releasing hormone (sGnRH) is partly mediated by arachidonic acid (AA) metabolism via the lipoxygenase enzyme system, whereas protein kinase C (PKC) participates in both sGnRH- and chicken (c)GnRH-II-induced GTH secretion. In this study, the interactions between AA- and PKC-dependent pathways in mediating the long-term GnRH stimulation of GTH release were further investigated using dispersed goldfish pituitary cell cultures in static incubation. Treatments with AA or the PKC activator tetradecanoylphorbol acetate (TPA) increased GTH release. The GTH responses to AA and TPA were additive. The lipoxygenase inhibitor nordihydroguairetic acid (NDGA) and the PKC inhibitor H7 selectively reduced AA- and TPA-stimulated GTH release, respectively. These findings suggest that the GTH responses to stimulation by AA- and PKC-dependent signaling pathways are independent of one another. In other experiments, the GTH response to cGnRH-II was unaffected by NDGA but was abolished by H7. In contrast, sGnRH-induced GTH release was attenuated by NDGA and H7. Furthermore, in the presence of both NDGA and H7, the GTH response to sGnRH was abolished. These data suggest that sGnRH stimulation of GTH secretion involves both AA- and PKC-dependent mechanisms; in contrast, cGnRH-II action is not dependent on AA metabolism. The pathway by which AA might be mobilized in response to a GnRH challenge was also investigated by pharmacological manipulations. The diacylglcerol (DG) lipase inhibitor, U-57908, did not decrease sGnRH- and cGnRH-II-induced GTH secretion. On the other hand, the phospholipase A2 (PLA2) inhibitors, bromophenacyl bromide (BPB), chloroquine, and quinacrine, reduced sGnRH-elicited, but not cGnRH-II-stimulated GTH release. The addition of AA reversed the inhibitory action of BPB on sGnRH-elicited GTH release. In addition, the GTH response to AA was additive to the cGnRH-II-induced, but

  16. THI1, a Thiamine Thiazole Synthase, Interacts with Ca2+-Dependent Protein Kinase CPK33 and Modulates the S-Type Anion Channels and Stomatal Closure in Arabidopsis.

    PubMed

    Li, Chun-Long; Wang, Mei; Wu, Xiao-Meng; Chen, Dong-Hua; Lv, Hong-Jun; Shen, Jian-Lin; Qiao, Zhu; Zhang, Wei

    2016-02-01

    Thiamine is required for both plant growth and development. Here, the involvement of a thiamine thiazole synthase, THI1, has been demonstrated in both guard cell abscisic acid (ABA) signaling and the drought response in Arabidopsis (Arabidopsis thaliana). THI1 overexpressors proved to be more sensitive to ABA than the wild type with respect to both the activation of guard cell slow type anion channels and stomatal closure; this effectively reduced the rate of water loss from the plant and thereby enhanced its level of drought tolerance. A yeast two-hybrid strategy was used to screen a cDNA library from epidermal strips of leaves for THI1 regulatory factors, and identified CPK33, a Ca(2+)-dependent protein kinase, as interactor with THI1 in a plasma membrane-delimited manner. Loss-of-function cpk33 mutants were hypersensitive to ABA activation of slow type anion channels and ABA-induced stomatal closure, while the CPK33 overexpression lines showed opposite phenotypes. CPK33 kinase activity was essential for ABA-induced stomatal closure. Consistent with their contrasting regulatory role over stomatal closure, THI1 suppressed CPK33 kinase activity in vitro. Together, our data reveal a novel regulatory role of thiamine thiazole synthase to kinase activity in guard cell signaling. PMID:26662273

  17. High-throughput wide dynamic range procedure for the simultaneous quantification of nicotine and cotinine in multiple biological matrices using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    PubMed

    Pérez-Ortuño, Raúl; Martínez-Sánchez, Jose M; Fernández, Esteve; Pascual, José A

    2015-11-01

    A straightforward, high-throughput method was developed and fully validated for the simultaneous determination of the specific tobacco biomarkers nicotine and its main metabolite cotinine in a wide dynamic range and supporting the most common human biological matrices (urine, oral fluid and hair). Sample preparation was performed inside the very HPLC injection vials by pipetting 0.5 mL of the liquid samples, deuterated internal standards in alkaline solution and dichloromethane as extraction solvent. Solid samples (i.e. around 10 mg hair) were first submitted to alkaline digestion in the HPLC vials and processed accordingly. The organic phase (reached through the upper aqueous layer) was directly injected without further treatment. Instrumental analysis was performed using hydrophilic interaction (HILIC) ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Total chromatographic time was 2 min. The method covers a wide dynamic range making it fit-for-purpose for the analysis of samples covering entire populations, irrespective of the level of exposure or tobacco use. Calibration curves (r (2) > 0.995) covered the range 1-2000 ng/mL (or 0.05-100 ng/mg hair) for nicotine and 0.1-2000 ng/mL (or 0.005-100 ng/mg hair) for cotinine. Within-run and between-run precision and accuracy were typically below 10 %, and always below 20 % at the lower limit of quantification. The method was successfully applied to the analysis of samples from different projects involving multiple matrices. PMID:26342312

  18. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    NASA Technical Reports Server (NTRS)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  19. Simultaneous determination of the novel tyrosine kinase inhibitor meditinib and its active metabolite demethylation meditinib in monkey plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.

    PubMed

    Liang, Feng; Kong, Qi; Guo, Yongqi; Wang, Yu; Sun, Dejie; Liu, Shi; Cai, Jinling; Guan, Yongbiao; Ding, Rigao

    2015-08-01

    Meditinib (ME) is a novel tyrosine kinase inhibitor used as an antichronic myeloid leukemia drug. A simple, sensitive and specific LC/MS/MS method was developed and validated for the analysis of ME and its metabolite demethylation meditinib (PI) in monkey plasma using naltrexone as the internal standard. Sample preparation involved protein precipitation with methanol. The analysis was carried out on an Agilent C8 column (3.5 µm, 2.1 × 50 mm). Elution was achieved with a mobile phase gradient varying the proportion of a water solution containing 0.1% formic acid (solvent A) and a 0.1% formic acid in methanol solution (solvent B) at a flow rate of 300 μL/min. The method had a linear calibration curve over the concentration range of 2-1000 ng/mL for ME and 2-1000 ng/mL for PI. The lower limits of quantification of ME and PI were 2 and 2 ng/mL, respectively. The intra- and inter-day precision values were <15% and accuracy values were within ±10.0%. The mean recoveries of ME and PI from plasma were >85%. The assay has been successfully used for pharmacokinetic evaluation of ME and PI using the monkey as an animal model, and those data are reported for the first time. PMID:25616210

  20. Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase)

    PubMed Central

    Neerathilingam, Muniasamy; Bairy, Sneha G.; Mysore, Sumukh

    2016-01-01

    Intrinsically disordered proteins (IDPs) play a major role in various cellular functions ranging from transcription to cell migration. Mutations/modifications in such IDPs are shown to be associated with various diseases. Current strategies to study the mode of action and regulatory mechanisms of disordered proteins at the structural level are time consuming and challenging. Therefore, using simple and swift strategies for identifying functionally important regions in unstructured segments and understanding their underlying mechanisms is critical for many applications. Here we propose a simple strategy that employs dissection of human paxillin (residues 1–313) that comprises intrinsically disordered regions, followed by its interaction study using FAT (Focal adhesion targeting domain of focal adhesion kinase) as its binding partner to retrace structural behavior. Our findings show that the paxillin interaction with FAT exhibits a masking and unmasking effect by a putative intra-molecular regulatory region. This phenomenon suggests how cancer associated mutations in paxillin affect its interactions with Focal Adhesion Kinase (FAK). The strategy could be used to decipher the mode of regulations and identify functionally relevant constructs for other studies. PMID:26928467

  1. Chronic Lead Exposure and Mixed Factors of Gender×Age×Brain Regions Interactions on Dendrite Growth, Spine Maturity and NDR Kinase

    PubMed Central

    Xue, Weizhen; Yang, Qian-Qian; Wang, Shuang; Xu, Yi; Wang, Hui-Li

    2015-01-01

    NDR1/2 kinase is essential in dendrite morphology and spine formation, which is regulated by cellular Ca2+. Lead (Pb) is a potent blocker of L-type calcium channel and our recent work showed Pb exposure impairs dendritic spine outgrowth in hippocampal neurons in rats. But the sensitivity of Pb-induced spine maturity with mixed factors (gender×age×brain regions) remains unknown. This study aimed to systematically investigate the effect of Pb exposure on spine maturity in rat brain with three factors (gender×age×brain regions), as well as the NDR1/2 kinase expression. Sprague–Dawley rats were exposed to Pb from parturition to postnatal day 30, 60, 90, respectively. Golgi-Cox staining was used to examine spine maturity. Western blot assay was applied to measure protein expression and real-time fluorescence quantitative PCR assay was used to examine mRNA levels. The results showed chronic Pb exposure significantly decreased dendritic length and impaired spine maturity in both rat hippocampus and medial prefrontal cortex. The impairment of dendritic length induced by Pb exposure tended to adolescence > adulthood, hippocampus > medial prefrontal cortex and female > male. Pb exposure induced significant damage in spine maturity during adolescence and early adult while little damage during adult in male rat brain and female medial prefrontal cortex. Besides, there was sustained impairment from adolescence to adulthood in female hippocampus. Interestingly, impairment of spine maturity followed by Pb exposure was correlated with NDR1/2 kinase. The reduction of NDR1/2 kinase protein expression after Pb exposure was similar to the result of spine maturity. In addition, NDR2 and their substrate Rabin3 mRNA levels were significantly decreased by Pb exposure in developmental rat brain. Taken together, Pb exposure impaired dendrite growth and maturity which was subject to gender×age×brain regions effects and related to NDR1/2 signal expression. PMID:26368815

  2. Augmentor α and β (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand–receptor interactions

    PubMed Central

    Reshetnyak, Andrey V.; Murray, Phillip B.; Shi, Xiarong; Mo, Elizabeth S.; Mohanty, Jyotidarsini; Tome, Francisco; Bai, Hanwen; Gunel, Murat; Lax, Irit; Schlessinger, Joseph

    2015-01-01

    Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. The orphan receptor anaplastic lymphoma kinase (ALK) is one of very few RTKs that remain without a firmly established protein ligand. Here we present a novel cytokine, FAM150B, which we propose naming augmentor-α (AUG-α), as a ligand for ALK. AUG-α binds ALK with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK or the related receptor leukocyte tyrosine kinase (LTK) demonstrate that AUG-α binds and robustly activates both ALK and LTK. We show that the previously established LTK ligand FAM150A (AUG-β) is specific for LTK and only weakly binds to ALK. Furthermore, expression of AUG-α stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer partly driven by ALK. These experiments reveal the hierarchy and specificity of two cytokines as ligands for ALK and LTK and set the stage for elucidating their roles in development and disease states. PMID:26630010

  3. Phosphorylation of Calcineurin B-like (CBL) Calcium Sensor Proteins by Their CBL-interacting Protein Kinases (CIPKs) Is Required for Full Activity of CBL-CIPK Complexes toward Their Target Proteins*

    PubMed Central

    Hashimoto, Kenji; Eckert, Christian; Anschütz, Uta; Scholz, Martin; Held, Katrin; Waadt, Rainer; Reyer, Antonella; Hippler, Michael; Becker, Dirk; Kudla, Jörg

    2012-01-01

    Calcineurin B-like proteins (CBLs) represent a family of calcium sensor proteins that interact with a group of serine/threonine kinases designated as CBL-interacting protein kinases (CIPKs). CBL-CIPK complexes are crucially involved in relaying plant responses to many environmental signals and in regulating ion fluxes. However, the biochemical characterization of CBL-CIPK complexes has so far been hampered by low activities of recombinant CIPKs. Here, we report on an efficient wheat germ extract-based in vitro transcription/translation protocol that yields active full-length wild-type CIPK proteins. We identified a conserved serine residue within the C terminus of CBLs as being phosphorylated by their interacting CIPKs. Remarkably, our studies revealed that CIPK-dependent CBL phosphorylation is strictly dependent on CBL-CIPK interaction via the CIPK NAF domain. The phosphorylation status of CBLs does not appear to influence the stability, localization, or CIPK interaction of these calcium sensor proteins in general. However, proper phosphorylation of CBL1 is absolutely required for the in vivo activation of the AKT1 K+ channel by CBL1-CIPK23 and CBL9-CIPK23 complexes in oocytes. Moreover, we show that by combining CBL1, CIPK23, and AKT1, we can faithfully reconstitute CBL-dependent enhancement of phosphorylation of target proteins by CIPKs in vitro. In addition, we report that phosphorylation of CBL1 by CIPK23 is also required for the CBL1-dependent enhancement of CIPK23 activity toward its substrate. Together, these data identify a novel general regulatory mechanism of CBL-CIPK complexes in that CBL phosphorylation at their flexible C terminus likely provokes conformational changes that enhance specificity and activity of CBL-CIPK complexes toward their target proteins. PMID:22253446

  4. Hydrophilic interaction chromatography combined with dispersive liquid-liquid microextraction as a preconcentration tool for the simultaneous determination of the panel of underivatized neurotransmitters in human urine samples.

    PubMed

    Konieczna, Lucyna; Roszkowska, Anna; Niedźwiecki, Maciej; Bączek, Tomasz

    2016-01-29

    A simple and sensitive method using dispersive liquid-liquid microextraction (DLLME) followed by liquid chromatography coupled to mass spectrometry (LC-MS) with a hydrophilic interaction chromatography (HILIC) column was developed for the simultaneous determination of 13 compounds of different polarities, comprising monoamine neurotransmitters (dopamine, norepinephrine, epinephrine and serotonin) along with their respective precursors and metabolites, in human urine samples. The microextraction procedure was based on the fast injection of a mixture of ethanol (disperser solvent) and dichloromethane (extraction solvent) into a human urine sample, forming a cloudy solution in the Eppendorf tube. After centrifugation, the sedimented phase was collected and subsequently analyzed by LC-HILIC-MS in about 12min without a derivatization step. The separation was performed on an XBridge Amide™ BEH column 3.0×100mm, 3.5mm and the mobile phase consisted of phase A: 10mM ammonium formate buffer in water pH 3.0 and phase B: 10 mM ammonium formate buffer in acetonitrile, under gradient program elution. Tyrosine, tryptophan, 5-hydroxytryptophan, dopamine, epinephrine, norepinephrine, serotonin, 3-methoxytyramine, 5-hydroxyindole-3-acetic acid, 3,4-dihydroxy-l-phenylalanine and norvaline (internal standard) were detected in the positive ionization mode. While vanillylmandelic acid, homovanillic acid, 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxybenzylamine (internal standard) were detected in the negative ionization mode. Parameters influencing DLLME and LC-HILIC-MS were investigated. Under the optimum conditions, the proposed method exhibited a low detection limit (5-10ngmL(-1)), and good linearity with R between 0.9991 and 0.9998. The recoveries in human urine samples were 99.0%±3.6%. for the 13 studied biogenic amines with intra- and inter-day RSDs of 0.24-9.55% and 0.31-10.0%, respectively. The developed DLLME-LC-MS method could be successfully applied for the

  5. A novel method for the simultaneous analysis of seven biothiols in rice (Oryza sativa L.) using hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry.

    PubMed

    Cao, Zhao-Yun; Sun, Li-Hua; Mou, Ren-Xiang; Zhou, Rong; Zhu, Zhi-Wei; Chen, Ming-Xue

    2015-01-22

    Analysis of biothiols is still problematic, due to their high polarity, oxidation sensitivity and time-consuming sample preparation. In this paper, a direct, rapid and sensitive method was developed for simultaneous quantification of unbound cysteine (Cys), glutathione (GSH) and phytochelatins (PCs) in rice leaf, stem and root samples by hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry (HILIC-MS/MS). Homogenized samples were extracted with water containing 50mM dithiothreitol, without derivatization and further clean-up, and the extracts were injected directly onto an Xbridge Amide-HILIC column (3.5μm, 150mm×2.1mm i.d.). The best chromatographic separation and MS sensitivity was achieved using a linear gradient elution with 10mM aqueous ammonium formate and acetonitrile as the mobile phase. In MS/MS mode the detection limit (S/N≥3) of seven biothiols was 3-105nM. Good linearities were observed (r>0.995) with linear dynamic range at least over three orders of magnitude. Recoveries for most analytes were within the range of 77-128%, with relative standard deviations less than 18.2%. The intra-day precision (n=7) was 6.1-11.7%, and the inter-day precision over 15 d (n=15) was 8.5-16.3% for all biothiols. The optimized HILIC-MS/MS method was applied to study the influence of different cadmium (Cd) concentrations (0, 1 and 50μM) on contents of Cys, GSH and PC2-6 in rice tissue. With increasing Cd concentrations in nutrient solutions, contents of PC2-4 in rice roots increased but contents of Cys and GSH decreased. Contents of PC2-4 in both rice leafs and stems increased markedly at high dose Cd (50μM) treatment compared with controls, compared with low Cd concentrations (1μM). However, both PC5 and PC6 were not detected throughout the stress experiment. PMID:25436484

  6. Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

    PubMed

    Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J; Quinlan, Kate G R; Cordwell, Stuart J; Funnell, Alister P W; Pearson, Richard C M; Crossley, Merlin

    2015-03-27

    Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. PMID:25659434

  7. Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains♦

    PubMed Central

    Tanaka, Hiroaki; Akagi, Ken-ichi; Oneyama, Chitose; Tanaka, Masakazu; Sasaki, Yuichi; Kanou, Takashi; Lee, Young-Ho; Yokogawa, Daisuke; Dobenecker, Marc-Werner; Nakagawa, Atsushi; Okada, Masato; Ikegami, Takahisa

    2013-01-01

    Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain. PMID:23548896

  8. The HT1 protein kinase is essential for red light-induced stomatal opening and genetically interacts with OST1 in red light and CO2 -induced stomatal movement responses.

    PubMed

    Matrosova, Anastasia; Bogireddi, Hanumakumar; Mateo-Peñas, Alfonso; Hashimoto-Sugimoto, Mimi; Iba, Koh; Schroeder, Julian I; Israelsson-Nordström, Maria

    2015-12-01

    The question of whether red light-induced stomatal opening is mediated by a photosynthesis-derived reduction in intercellular [CO2 ] (Ci ) remains controversial and genetic analyses are needed. The Arabidopsis thaliana protein kinase HIGH TEMPERATURE 1 (HT1) is a negative regulator of [CO2 ]-induced stomatal closing and ht1-2 mutant plants do not show stomatal opening to low [CO2 ]. The protein kinase mutant ost1-3 exhibits slowed stomatal responses to CO2 . The functions of HT1 and OPEN STOMATA 1 (OST1) to changes in red, blue light or [CO2 ] were analyzed. For comparison we assayed recessive ca1ca4 carbonic anhydrase double mutant plants, based on their slowed stomatal response to CO2 . Here, we report a strong impairment in ht1 in red light-induced stomatal opening whereas blue light was able to induce stomatal opening. The effects on photosynthetic performance in ht1 were restored when stomatal limitation of CO2 uptake, by control of [Ci ], was eliminated. HT1 was found to interact genetically with OST1 both during red light- and low [CO2 ]-induced stomatal opening. Analyses of ca1ca4 plants suggest that more than a low [Ci ]-dependent pathway may function in red light-induced stomatal opening. These results demonstrate that HT1 is essential for red light-induced stomatal opening and interacts genetically with OST1 during stomatal responses to red light and altered [CO2 ]. PMID:26192339

  9. Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

    PubMed Central

    Plocinska, Renata; Martinez, Luis; Gorla, Purushotham; Pandeeti, Emmanuel; Sarva, Krishna; Blaszczyk, Ewelina; Dziadek, Jaroslaw

    2014-01-01

    The septal association of Mycobacterium tuberculosis MtrB, the kinase partner of the MtrAB two-component signal transduction system, is necessary for the optimal expression of the MtrA regulon targets, including ripA, fbpB, and ftsI, which are involved in cell division and cell wall synthesis. Here, we show that MtrB, irrespective of its phosphorylation status, interacts with Wag31, whereas only phosphorylation-competent MtrB interacts with FtsI. We provide evidence that FtsI depletion compromises the MtrB septal assembly and MtrA regulon expression; likewise, the absence of MtrB compromises FtsI localization and, possibly, FtsI activity. We conclude from these results that FtsI and MtrB are codependent for their activities and that FtsI functions as a positive modulator of MtrB activation and MtrA regulon expression. In contrast to FtsI, Wag31 depletion does not affect MtrB septal assembly and MtrA regulon expression, whereas the loss of MtrB increased Wag31 localization and the levels of PknA/PknB (PknA/B) serine-threonine protein kinase-mediated Wag31 phosphorylation. Interestingly, we found that FtsI decreased levels of phosphorylated Wag31 (Wag31∼P) and that MtrB interacted with PknA/B. Overall, our results indicate that MtrB interactions with FtsI, Wag31, and PknA/B are required for its optimal localization, MtrA regulon expression, and phosphorylation of Wag31. Our results emphasize a new role for MtrB in cell division and cell wall synthesis distinct from that regulating the MtrA phosphorylation activities. PMID:25225272

  10. Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates.

    PubMed

    Praseeda, Mullasseril; Pradeep, Kurup K; Krupa, Ananth; Krishna, S Sri; Leena, Suseela; Kumar, R Rajeev; Cheriyan, John; Mayadevi, Madhavan; Srinivasan, Narayanaswamy; Omkumar, Ramakrishnapillai V

    2004-03-01

    CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation. PMID:14558884

  11. Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

    SciTech Connect

    Carrasco-Garcia, Estefania; Saceda, Miguel; Grasso, Silvina; Rocamora-Reverte, Lourdes; Conde, Mariano; Gomez-Martinez, Angeles; Garcia-Morales, Pilar; Ferragut, Jose A.; Martinez-Lacaci, Isabel

    2011-06-10

    Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G{sub 1} arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G{sub 1} arrest. This G{sub 1} arrest was associated with up-regulation of p27{sup kip1}, whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G{sub 1} arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 {Delta}EGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.

  12. Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

    PubMed Central

    Valentine, Cathleen D.; Haggie, Peter M.

    2011-01-01

    The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β1- and β2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β1- and β2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β2AR confinement. For both β1- and β2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β1- or β2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes. PMID:21680711

  13. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    NASA Astrophysics Data System (ADS)

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  14. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

    PubMed Central

    Glenewinkel, Florian; Cohen, Michael J.; King, Cason R.; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S.; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  15. Homeodomain-interacting protein kinase-2 stabilizes p27(kip1) by its phosphorylation at serine 10 and contributes to cell motility.

    PubMed

    Pierantoni, Giovanna Maria; Esposito, Francesco; Tornincasa, Mara; Rinaldo, Cinzia; Viglietto, Giuseppe; Soddu, Silvia; Fusco, Alfredo

    2011-08-19

    HIPK2 is a serine/threonine kinase that acts as a coregulator of an increasing number of factors involved in cell survival and proliferation during development and in response to different types of stress. Here we report on a novel target of HIPK2, the cyclin-dependent kinase inhibitor p27(kip1). HIPK2 phosphorylates p27(kip1) in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27(kip1) phosphorylation and plays a crucial role in the stability of the protein. Indeed, HIPK2 depletion by transient or stable RNA interference in tumor cells of different origin was consistently associated with strong reduction of p27(kip1) phosphorylation at serine 10 and of p27(kip1) stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27(kip1) revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53. PMID:21715331

  16. Diphosphothreonine-specific interaction between an SQ/TQ cluster and an FHA domain in the Rad53-Dun1 kinase cascade.

    PubMed

    Lee, Hyun; Yuan, Chunhua; Hammet, Andrew; Mahajan, Anjali; Chen, Eric S-W; Wu, Ming-Ru; Su, Mei-I; Heierhorst, Jörg; Tsai, Ming-Daw

    2008-06-20

    Forkhead-associated (FHA) domains recognize phosphothreonines, and SQ/TQ cluster domains (SCDs) contain concentrated phosphorylation sites for ATM/ATR-like DNA-damage-response kinases. The Rad53-SCD1 has dual functions in regulating the activation of the Rad53-Dun1 checkpoint kinase cascade but with unknown molecular mechanisms. Here we present structural, biochemical, and genetic evidence that Dun1-FHA possesses an unprecedented diphosphothreonine-binding specificity. The Dun1-FHA has >100-fold increased affinity for diphosphorylated relative to monophosphorylated Rad53-SCD1 due to the presence of two separate phosphothreonine-binding pockets. In vivo, any single threonine of Rad53-SCD1 is sufficient for Rad53 activation and RAD53-dependent survival of DNA damage, but two adjacent phosphothreonines in the Rad53-SCD1 and two phosphothreonine-binding sites in the Dun1-FHA are necessary for Dun1 activation and DUN1-dependent transcriptional responses to DNA damage. The results uncover a phospho-counting mechanism that regulates the specificity of SCD, and provide mechanistic insight into a role of multisite phosphorylation in DNA-damage signaling. PMID:18570878

  17. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2.

    PubMed

    Glenewinkel, Florian; Cohen, Michael J; King, Cason R; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  18. Receptor Tyrosine Kinases with Intracellular Pseudokinase Domains

    PubMed Central

    Mendrola, Jeannine M.; Shi, Fumin; Park, Jin H.; Lemmon, Mark A.

    2013-01-01

    As with other groups of protein kinases, approximately 10% of the receptor tyrosine kinases (RTKs) in the human proteome contain intracellular pseudokinases that lack one or more conserved catalytically important residues. These include ErbB3, a member of the epidermal growth factor receptor (EGFR) family, and a series of unconventional Wnt receptors. We recently showed that, despite its reputation as a pseudokinase, the ErbB3 tyrosine kinase domain (TKD) does retain significant – albeit weak – kinase activity. This led us to suggest that a subgroup of RTKs may be able to signal even with very inefficient kinases. Recent work suggests that this is not the case, however. Other pseudokinase RTKs have not revealed significant kinase activity, and mutations that impair ErbB3’s weak kinase activity have not so far been found to exhibit signaling defects. These findings therefore point to models in which the TKDs of pseudokinase RTKs participate in receptor signaling by allosterically regulating associated kinases (such as ErbB3 regulation of ErbB2) and/or function as regulated ‘scaffolds’ for other intermolecular interactions central to signal propagation. Further structural and functional studies – particularly of the pseudokinase RTKs involved in Wnt signaling – are required to shed new light on these intriguing signaling mechanisms. PMID:23863174

  19. Enzyme kinetics and interaction studies for human JNK1β1 and substrates activating transcription factor 2 (ATF2) and c-Jun N-terminal kinase (c-Jun).

    PubMed

    Figuera-Losada, Mariana; LoGrasso, Philip V

    2012-04-13

    c-Jun N-terminal kinase (JNK) is a stress signal transducer linked to cell death, and survival. JNK1 has been implicated in obesity, glucose intolerance, and insulin resistance. In this study we report the kinetic mechanism for JNK1β1 with transcription factors ATF2 and c-Jun along with interaction kinetics for these substrates. JNK1β1 followed a random sequential mechanism forming a ternary complex between JNK-substrate-ATP. K(m) for ATF2 and c-Jun was 1.1 and 2.8 μM, respectively. Inhibition studies using adenosine 5'-(β,γ-methylenetriphosphate) and a peptide derived from JNK interacting protein 1 (JIP1) supported the proposed kinetic mechanism. Biolayer interferometry studies showed that unphosphorylated JNK1β1 bound to ATF2 with similar affinity as it did to c-Jun (K(D) = 2.60 ± 0.34 versus 1.00 ± 0.35 μM, respectively). The presence of ATP increased the affinity of unphosphorylated JNK1β1 for ATF2 and c-Jun, to 0.80 ± 0.04 versus 0.65 ± 0.07 μM, respectively. Phosphorylation of JNK1β1 decreased the affinity of the kinase for ATF2 to 11.0 ± 1.1 μM and for c-Jun to 17.0 ± 7.5 μM in the absence of ATP. The presence of ATP caused a shift in the K(D) of the active kinase for ATF2 to 1.70 ± 0.25 μM and for c-Jun of 3.50 ± 0.95 μM. These results are the first kinetic and biochemical characterization of JNK1β1 and uncover some of the differences in the enzymatic activity of JNK1β1 compared with other variants and suggest that ATP binding or JNK phosphorylation could induce changes in the interactions with substrates, activators, and regulatory proteins. PMID:22351776

  20. Rapamycin interacts synergistically with idarubicin to induce T-leukemia cell apoptosis in vitro and in a mesenchymal stem cell simulated drug-resistant microenvironment via Akt/mammalian target of rapamycin and extracellular signal-related kinase signaling pathways.

    PubMed

    Wu, Kang-Ni; Zhao, Yan-Min; He, Ying; Wang, Bin-Sheng; Du, Kai-Li; Fu, Shan; Hu, Kai-Min; Zhang, Li-Fei; Liu, Li-Zhen; Hu, Yong-Xian; Wang, Ying-Jia; Huang, He

    2014-03-01

    T-cell acute lymphoblastic leukemias (T-ALLs) are clonal lymphoid malignancies with a poor prognosis, and still a lack of effective treatment. Here we examined the interactions between the mammalian target of rapamycin (mTOR) inhibitor rapamycin and idarubicin (IDA) in a series of human T-ALL cell lines Molt-4, Jurkat, CCRF-CEM and CEM/C1. Co-exposure of cells to rapamycin and IDA synergistically induced T-ALL cell growth inhibition and apoptosis mediated by caspase activation via the intrinsic mitochondrial pathway and extrinsic pathway. Combined treatment with rapamycin and IDA down-regulated Bcl-2 and Mcl-1, and inhibited the activation of phosphoinositide 3-kinase (PI3K)/mTOR and extracellular signal-related kinase (ERK). They also played synergistic pro-apoptotic roles in the drug-resistant microenvironment simulated by mesenchymal stem cells (MSCs) as a feeder layer. In addition, MSCs protected T-ALL cells from IDA cytotoxicity by up-regulating ERK phosphorylation, while rapamycin efficiently reversed this protective effect. Taken together, we confirm the synergistic antitumor effects of rapamycin and IDA, and provide an insight into the potential future clinical applications of combined rapamycin-IDA regimens for treating T-cell malignancies. PMID:23741975

  1. Zipper-interacting protein kinase promotes epithelial-mesenchymal transition, invasion and metastasis through AKT and NF-kB signaling and is associated with metastasis and poor prognosis in gastric cancer patients.

    PubMed

    Li, Jian; Deng, Zhijuan; Wang, Zhu; Wang, Dong; Zhang, Longjuan; Su, Qiao; Lai, Yingrong; Li, Bin; Luo, Zexing; Chen, Xu; Chen, Yu; Huang, Xiaohui; Ma, Jieyi; Wang, Wenjian; Bi, Jiong; Guan, Xinyuan

    2015-04-10

    Zipper-interacting Protein Kinase (ZIPK) belongs to the death-associated protein kinase family. ZIPK has been characterized as a tumor suppressor in various tumors, including gastric cancer. On the other hand, ZIPK also promotes cell survival. In this study, both in vitro and in vivo assays indicated that ZIPK promoted cell growth, proliferation, migration, invasion, tumor formation and metastasis in nude mice. ZIPK induced epithelial-mesenchymal transition (EMT) with increasing expression of β-catenin, mesenchymal markers, Snail and Slug, and with decreasing expression of E-cadherin. Furthermore, ZIPK activated the AKT/IκB/NF-κB pathway, which can promote EMT and metastasis. Additionally, ZIPK expression was detected in human primary gastric cancer and their matched metastatic lymph node samples by immunohistochemistry. Increased expression of ZIPK in lymph node metastases was significantly associated with stage VI and abdominal organ invasion. Survival analysis revealed that patients with increased ZIPK expression in metastatic lymph nodes had poor disease-specific survival. Taken together, our study reveals that ZIPK is a pro-oncogenic factor, which promotes cancer metastasis. PMID:25831050

  2. Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism.

    PubMed

    Patching, Simon G; Edara, Shalini; Ma, Pikyee; Nakayama, Jiro; Hussain, Rohanah; Siligardi, Giuliano; Phillips-Jones, Mary K

    2012-07-01

    FsrC is the membrane-bound histidine kinase component of the Fsr two-component signal transduction system involved in quorum sensing in the hospital-acquired infection agent Enterococcus faecalis. Synchrotron radiation circular dichroism spectroscopy was used here to study the intact purified protein solubilised in detergent micelles. Conditions required for FsrC stability in detergent were firstly determined and tested by prolonged exposure of stabilised protein to far-ultraviolet radiation. Using stabilised purified protein, far-ultraviolet synchrotron radiation circular dichroism revealed that FsrC is 61% alpha-helical and that it is relatively thermostable, retaining at least 57% secondary structural integrity at 90 degrees C in the presence or absence of gelatinase biosynthesis-activating pheromone (GBAP). Whilst binding of the quorum pheromone ligand GBAP did not significantly affect FsrC secondary structure, near-ultraviolet spectra revealed that the tertiary structure in the regions of the Tyr and Trp residues was significantly affected. Titration experiments revealed a calculated kd value of 2 microM indicative of relatively loose binding ofgelatinase biosynthesis-activating pheromone to FsrC. Although use of synchrotron radiation circular dichroism has been applied to membrane proteins previously, to our knowledge this is the first report of its use to determine a kd value for an intact membrane protein. Based on our findings, we suggest that synchrotron radiation circular dichroism will be a valuable technique for characterising ligand binding by other membrane sensor kinases and indeed other membrane proteins in general. It further provides a valuable screening tool for membrane protein stability under a range of detergent conditions prior to downstream structural methods such as crystallisation and NMR experiments particularly when lower detergent concentrations are used. PMID:22366202

  3. Cyclin-dependent kinase 5, a node protein in diminished tauopathy: a systems biology approach

    PubMed Central

    Castro-Alvarez, John F.; Uribe-Arias, S. Alejandro; Mejía-Raigosa, Daniel; Cardona-Gómez, Gloria P.

    2014-01-01

    Alzheimer's disease (AD) is the most common cause of dementia worldwide. One of the main pathological changes that occurs in AD is the intracellular accumulation of hyperphosphorylated Tau protein in neurons. Cyclin-dependent kinase 5 (CDK5) is one of the major kinases involved in Tau phosphorylation, directly phosphorylating various residues and simultaneously regulating various substrates such as kinases and phosphatases that influence Tau phosphorylation in a synergistic and antagonistic way. It remains unknown how the interaction between CDK5 and its substrates promotes Tau phosphorylation, and systemic approaches are needed that allow an analysis of all the proteins involved. In this review, the role of the CDK5 signaling pathway in Tau hyperphosphorylation is described, an in silico model of the CDK5 signaling pathway is presented. The relationship among these theoretical and computational models shows that the regulation of Tau phosphorylation by PP2A and glycogen synthase kinase 3β (GSK3β) is essential under basal conditions and also describes the leading role of CDK5 under excitotoxic conditions, where silencing of CDK5 can generate changes in these enzymes to reverse a pathological condition that simulates AD. PMID:25225483

  4. Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

    PubMed Central

    Van Horn, Wade D.; Sanders, Charles R.

    2013-01-01

    Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble kinases, and that exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a byproduct of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in Gram-positive bacteria, where its importance is evident by the fact that UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic diacylglycerol kinase family, which is based on over 40 years of studies. PMID:22224599

  5. A Glycine soja methionine sulfoxide reductase B5a interacts with the Ca(2+) /CAM-binding kinase GsCBRLK and activates ROS signaling under carbonate alkaline stress.

    PubMed

    Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Qin, Zhiwei; Yang, Kejun; Chen, Chao; Yu, Qingyue; Zhu, Yanming

    2016-06-01

    Although research has extensively illustrated the molecular basis of plant responses to salt and high-pH stresses, knowledge on carbonate alkaline stress is poor and the specific responsive mechanism remains elusive. We have previously characterized a Glycine soja Ca(2+) /CAM-dependent kinase GsCBRLK that could increase salt tolerance. Here, we characterize a methionine sulfoxide reductase (MSR) B protein GsMSRB5a as a GsCBRLK interactor by using Y2H and BiFc assays. Further analyses showed that the N-terminal variable domain of GsCBRLK contributed to the GsMSRB5a interaction. Y2H assays also revealed the interaction specificity of GsCBRLK with the wild soybean MSRB subfamily proteins, and determined that the BoxI/BoxII-containing regions within GsMSRBs were responsible for their interaction. Furthermore, we also illustrated that the N-terminal basic regions in GsMSRBs functioned as transit peptides, which targeted themselves into chloroplasts and thereby prevented their interaction with GsCBRLK. Nevertheless, deletion of these regions allowed them to localize on the plasma membrane (PM) and interact with GsCBRLK. In addition, we also showed that GsMSRB5a and GsCBRLK displayed overlapping tissue expression specificity and coincident expression patterns under carbonate alkaline stress. Phenotypic experiments demonstrated that GsMSRB5a and GsCBRLK overexpression in Arabidopsis enhanced carbonate alkaline stress tolerance. Further investigations elucidated that GsMSRB5a and GsCBRLK inhibited reactive oxygen species (ROS) accumulation by modifying the expression of ROS signaling, biosynthesis and scavenging genes. Summarily, our results demonstrated that GsCBRLK and GsMSRB5a interacted with each other, and activated ROS signaling under carbonate alkaline stress. PMID:27121031

  6. Mnk kinase pathway: Cellular functions and biological outcomes.

    PubMed

    Joshi, Sonali; Platanias, Leonidas C

    2014-08-26

    The mitogen-activated protein kinase (MAPK) interacting protein kinases 1 and 2 (Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs (p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E (eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4E. The role of Mnk kinases in inflammation and inflammation-induced malignancies is also discussed. PMID:25225600

  7. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  8. Suppressor of K+ transport growth defect 1 (SKD1) interacts with RING-type ubiquitin ligase and sucrose non-fermenting 1-related protein kinase (SnRK1) in the halophyte ice plant.

    PubMed

    Chiang, Chih-Pin; Li, Chang-Hua; Jou, Yingtzy; Chen, Yu-Chan; Lin, Ya-Chung; Yang, Fang-Yu; Huang, Nu-Chuan; Yen, Hungchen Emilie

    2013-05-01

    SKD1 (suppressor of K+ transport growth defect 1) is an AAA-type ATPase that functions as a molecular motor. It was previously shown that SKD1 accumulates in epidermal bladder cells of the halophyte Mesembryanthemum crystallinum. SKD1 knock-down Arabidopsis mutants showed an imbalanced Na+/K+ ratio under salt stress. Two enzymes involved in protein post-translational modifications that physically interacted with McSKD1 were identified. McCPN1 (copine 1), a RING-type ubiquitin ligase, has an N-terminal myristoylation site that links to the plasma membrane, a central copine domain that interacts with McSKD1, and a C-terminal RING domain that catalyses protein ubiquitination. In vitro ubiquitination assay demonstrated that McCPN1 was capable of mediating ubiquitination of McSKD1. McSnRK1 (sucrose non-fermenting 1-related protein kinase) is a Ser/Thr protein kinase that contains an N-terminal STKc catalytic domain to phosphorylate McSKD1, and C-terminal UBA and KA1 domains to interact with McSKD1. The transcript and protein levels of McSnRK1 increased as NaCl concentrations increased. The formation of an SKD1-SnRK1-CPN1 ternary complex was demonstrated by yeast three-hybrid and bimolecular fluorescence complementation. It was found that McSKD1 preferentially interacts with McSnRK1 in the cytosol, and salt induced the re-distribution of McSKD1 and McSnRK1 towards the plasma membrane via the microtubule cytoskeleton and subsequently interacted with RING-type E3 McCPN1. The potential effects of ubiquitination a