Science.gov

Sample records for agc kinase family

  1. Evolutionary Adaptations of Plant AGC Kinases: From Light Signaling to Cell Polarity Regulation

    PubMed Central

    Rademacher, Eike H.; Offringa, Remko

    2012-01-01

    Signaling and trafficking over membranes involves a plethora of transmembrane proteins that control the flow of compounds or relay specific signaling events. Next to external cues, internal stimuli can modify the activity or abundance of these proteins at the plasma membrane (PM). One such regulatory mechanism is protein phosphorylation by membrane-associated kinases, several of which are AGC kinases. The AGC kinase family is one of seven kinase families that are conserved in all eukaryotic genomes. In plants evolutionary adaptations introduced specific structural changes within the AGC kinases that most likely allow modulation of kinase activity by external stimuli (e.g., light). Starting from the well-defined structural basis common to all AGC kinases we review the current knowledge on the structure-function relationship in plant AGC kinases. Nine of the 39 Arabidopsis AGC kinases have now been shown to be involved in the regulation of auxin transport. In particular, AGC kinase-mediated phosphorylation of the auxin transporters ABCB1 and ABCB19 has been shown to regulate their activity, while auxin transporters of the PIN family are located to different positions at the PM depending on their phosphorylation status, which is a result of counteracting AGC kinase and PP6 phosphatase activities. We therefore focus on regulation of AGC kinase activity in this context. Identified structural adaptations of the involved AGC kinases may provide new insight into AGC kinase functionality and demonstrate their position as central hubs in the cellular network controlling plant development and growth. PMID:23162562

  2. The wheat AGC kinase TaAGC1 is a positive contributor to host resistance to the necrotrophic pathogen Rhizoctonia cerealis.

    PubMed

    Zhu, Xiuliang; Yang, Kun; Wei, Xuening; Zhang, Qiaofeng; Rong, Wei; Du, Lipu; Ye, Xingguo; Qi, Lin; Zhang, Zengyan

    2015-11-01

    Considerable progress has been made in understanding the roles of AGC kinases in mammalian systems. However, very little is known about the roles of AGC kinases in wheat (Triticum aestivum). The necrotrophic fungus Rhizoctonia cerealis is the major pathogen of the destructive disease sharp eyespot of wheat. In this study, the wheat AGC kinase gene TaAGC1, responding to R. cerealis infection, was isolated, and its properties and role in wheat defence were characterized. R. cerealis-resistant wheat lines expressed TaAGC1 at higher levels than susceptible wheat lines. Sequence and phylogenetic analyses showed that the TaAGC1 protein is a serine/threonine kinase belonging to the NDR (nuclear Dbf2-related) subgroup of AGC kinases. Kinase activity assays proved that TaAGC1 is a functional kinase and the Asp-239 residue located in the conserved serine/threonine kinase domain of TaAGC1 is required for the kinase activity. Subcellular localization assays indicated that TaAGC1 localized in the cytoplasm and nucleus. Virus-induced TaAGC1 silencing revealed that the down-regulation of TaAGC1 transcripts significantly impaired wheat resistance to R. cerealis. The molecular characterization and responses of TaAGC1 overexpressing transgenic wheat plants indicated that TaAGC1 overexpression significantly enhanced resistance to sharp eyespot and reduced the accumulation of reactive oxygen species (ROS) in wheat plants challenged with R. cerealis. Furthermore, ROS-scavenging and certain defence-associated genes were up-regulated in resistant plants overexpressing TaAGC1 but down-regulated in susceptible knock-down plants. These results suggested that the kinase TaAGC1 positively contributes to wheat immunity to R. cerealis through regulating expression of ROS-related and defence-associated genes. PMID:26220083

  3. The wheat AGC kinase TaAGC1 is a positive contributor to host resistance to the necrotrophic pathogen Rhizoctonia cerealis

    PubMed Central

    Zhu, Xiuliang; Yang, Kun; Wei, Xuening; Zhang, Qiaofeng; Rong, Wei; Du, Lipu; Ye, Xingguo; Qi, Lin; Zhang, Zengyan

    2015-01-01

    Considerable progress has been made in understanding the roles of AGC kinases in mammalian systems. However, very little is known about the roles of AGC kinases in wheat (Triticum aestivum). The necrotrophic fungus Rhizoctonia cerealis is the major pathogen of the destructive disease sharp eyespot of wheat. In this study, the wheat AGC kinase gene TaAGC1, responding to R. cerealis infection, was isolated, and its properties and role in wheat defence were characterized. R. cerealis-resistant wheat lines expressed TaAGC1 at higher levels than susceptible wheat lines. Sequence and phylogenetic analyses showed that the TaAGC1 protein is a serine/threonine kinase belonging to the NDR (nuclear Dbf2-related) subgroup of AGC kinases. Kinase activity assays proved that TaAGC1 is a functional kinase and the Asp-239 residue located in the conserved serine/threonine kinase domain of TaAGC1 is required for the kinase activity. Subcellular localization assays indicated that TaAGC1 localized in the cytoplasm and nucleus. Virus-induced TaAGC1 silencing revealed that the down-regulation of TaAGC1 transcripts significantly impaired wheat resistance to R. cerealis. The molecular characterization and responses of TaAGC1 overexpressing transgenic wheat plants indicated that TaAGC1 overexpression significantly enhanced resistance to sharp eyespot and reduced the accumulation of reactive oxygen species (ROS) in wheat plants challenged with R. cerealis. Furthermore, ROS-scavenging and certain defence-associated genes were up-regulated in resistant plants overexpressing TaAGC1 but down-regulated in susceptible knock-down plants. These results suggested that the kinase TaAGC1 positively contributes to wheat immunity to R. cerealis through regulating expression of ROS-related and defence-associated genes. PMID:26220083

  4. Myotonin protein-kinase [AGC]n trinucleotide repeat in seven nonhuman primates

    SciTech Connect

    Novelli, G.; Sineo, L.; Pontieri, E. ||

    1994-09-01

    Myotonic dystrophy (DM) is due to a genomic instability of a trinucleotide [AGC]n motif, located at the 3{prime} UTR region of a protein-kinase gene (myotonin protein kinase, MT-PK). The [AGC] repeat is meiotically and mitotically unstable, and it is directly related to the manifestations of the disorder. Although a gene dosage effect of the MT-PK has been demonstrated n DM muscle, the mechanism(s) by which the intragenic repeat expansion leads to disease is largely unknown. This non-standard mutational event could reflect an evolutionary mechanism widespread among animal genomes. We have isolated and sequenced the complete 3{prime}UTR region of the MT-PK gene in seven primates (macaque, orangutan, gorilla, chimpanzee, gibbon, owl monkey, saimiri), and examined by comparative sequence nucleotide analysis the [AGC]n intragenic repeat and the surrounding nucleotides. The genomic organization, including the [AGC]n repeat structure, was conserved in all examined species, excluding the gibbon (Hylobates agilis), in which the [AGC]n upstream sequence (GGAA) is replaced by a GA dinucleotide. The number of [AGC]n in the examined species ranged between 7 (gorilla) and 13 repeats (owl monkeys), with a polymorphism informative content (PIC) similar to that observed in humans. These results indicate that the 3{prime}UTR [AGC] repeat within the MT-PK gene is evolutionarily conserved, supporting that this region has important regulatory functions.

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

    PubMed

    Xi, Yu; Niu, Jianhua; Shen, Yun; Li, Dongmei; Peng, Xinyu; Wu, Xiangwei

    2016-09-01

    The AGC kinase family is important cell proliferation and survival. Dysregulation of this family contributes to gastric cancer progression. Here, we evaluated the potential activity of AT13148, a first-in-class multi-AGC kinase inhibitor, against gastric cancer cells. Our results showed that AT13148 exerted potent cytotoxic and anti-proliferative activities against a panel human gastric cancer cell lines (HGC-27, AGS, SNU-601, N87 and MKN-28), possibly via inducing cancer cell apoptotic death. Apoptosis inhibition by the Caspase blockers dramatically attenuated AT13148-caused cytotoxicity against gastric cancer cells. Intriguingly, same AT13148 treatment was not cytotoxic/pro-apoptotic to the non-cancerous human gastric epithelial GEC-1 cells. At the signaling level, AT13148 treatment in gastric cancer cells dramatically suppressed activation of multiple AGC kinases, including Akt (at p-Thr-308), p70S6 kinase (p70S6K), glycogen synthase kinase 3β (GSK-3β) and p90 ribosomal S6 kinase (RSK). Our in vivo studies demonstrated that daily oral gavage of AT13148 at well-tolerated doses significantly inhibited HGC27 xenograft tumor growth in nude mice. AGC activity was also dramatically decreased in AT13148-administrated HGC27 tumors. Therefore, targeting AGC kinases by AT13148 demonstrates superior anti-gastric cancer activity both in vitro and in vivo. The preclinical results of this study support the progression of this molecule into future evaluation as a valuable anti-gastric cancer candidate. PMID:26828267

  6. Regulation of planar growth by the Arabidopsis AGC protein kinase UNICORN.

    PubMed

    Enugutti, Balaji; Kirchhelle, Charlotte; Oelschner, Maxi; Torres Ruiz, Ramón Angel; Schliebner, Ivo; Leister, Dario; Schneitz, Kay

    2012-09-11

    The spatial coordination of growth is of central importance for the regulation of plant tissue architecture. Individual layers, such as the epidermis, are clonally propagated and structurally maintained by symmetric cell divisions that are oriented along the plane of the layer. The developmental control of this process is poorly understood. The simple cellular basis and sheet-like structure of Arabidopsis integuments make them an attractive model system to address planar growth. Here we report on the characterization of the Arabidopsis UNICORN (UCN) gene. Analysis of ucn integuments reveals localized distortion of planar growth, eventually resulting in an ectopic multicellular protrusion. In addition, ucn mutants exhibit ectopic growth in filaments and petals, as well as aberrant embryogenesis. We further show that UCN encodes an active AGC VIII kinase. Genetic, biochemical, and cell biological data suggest that UCN suppresses ectopic growth in integuments by directly repressing the KANADI transcription factor ABERRANT TESTA SHAPE. Our findings indicate that UCN represents a unique plant growth regulator that maintains planar growth of integuments by repressing a developmental regulator involved in the control of early integument growth and polarity. PMID:22927420

  7. Regulation of planar growth by the Arabidopsis AGC protein kinase UNICORN

    PubMed Central

    Enugutti, Balaji; Kirchhelle, Charlotte; Oelschner, Maxi; Torres Ruiz, Ramón Angel; Schliebner, Ivo; Leister, Dario; Schneitz, Kay

    2012-01-01

    The spatial coordination of growth is of central importance for the regulation of plant tissue architecture. Individual layers, such as the epidermis, are clonally propagated and structurally maintained by symmetric cell divisions that are oriented along the plane of the layer. The developmental control of this process is poorly understood. The simple cellular basis and sheet-like structure of Arabidopsis integuments make them an attractive model system to address planar growth. Here we report on the characterization of the Arabidopsis UNICORN (UCN) gene. Analysis of ucn integuments reveals localized distortion of planar growth, eventually resulting in an ectopic multicellular protrusion. In addition, ucn mutants exhibit ectopic growth in filaments and petals, as well as aberrant embryogenesis. We further show that UCN encodes an active AGC VIII kinase. Genetic, biochemical, and cell biological data suggest that UCN suppresses ectopic growth in integuments by directly repressing the KANADI transcription factor ABERRANT TESTA SHAPE. Our findings indicate that UCN represents a unique plant growth regulator that maintains planar growth of integuments by repressing a developmental regulator involved in the control of early integument growth and polarity. PMID:22927420

  8. Phosphorylation of the TOR ATP binding domain by AGC kinase constitutes a novel mode of TOR inhibition.

    PubMed

    Hálová, Lenka; Du, Wei; Kirkham, Sara; Smith, Duncan L; Petersen, Janni

    2013-11-25

    TOR (target of rapamycin) signaling coordinates cell growth, metabolism, and cell division through tight control of signaling via two complexes, TORC1 and TORC2. Here, we show that fission yeast TOR kinases and mTOR are phosphorylated on an evolutionarily conserved residue of their ATP-binding domain. The Gad8 kinase (AKT homologue) phosphorylates fission yeast Tor1 at this threonine (T1972) to reduce activity. A T1972A mutation that blocked phosphorylation increased Tor1 activity and stress resistance. Nitrogen starvation of fission yeast inhibited TOR signaling to arrest cell cycle progression in G1 phase and promoted sexual differentiation. Starvation and a Gad8/T1972-dependent decrease in Tor1 (TORC2) activity was essential for efficient cell cycle arrest and differentiation. Experiments in human cell lines recapitulated these yeast observations, as mTOR was phosphorylated on T2173 in an AKT-dependent manner. In addition, a T2173A mutation increased mTOR activity. Thus, TOR kinase activity can be reduced through AGC kinase-controlled phosphorylation to generate physiologically significant changes in TOR signaling. PMID:24247430

  9. PINOID AGC kinases are necessary for phytochrome-mediated enhancement of hypocotyl phototropism in Arabidopsis.

    PubMed

    Haga, Ken; Hayashi, Ken-ichiro; Sakai, Tatsuya

    2014-11-01

    Several members of the AGCVIII kinase subfamily, which includes PINOID (PID), PID2, and WAVY ROOT GROWTH (WAG) proteins, have previously been shown to phosphorylate PIN-FORMED (PIN) auxin transporters and control the auxin flow in plants. PID has been proposed as a key component of the phototropin signaling pathway that induces phototropic responses, although the responses were not significantly impaired in the pid single and pid wag1 wag2 triple mutants. This raises questions about the functional roles of the PID family in phototropic responses. Here, we investigated hypocotyl phototropism in the pid pid2 wag1 wag2 quadruple mutant in detail to clarify the roles of the PID family in Arabidopsis (Arabidopsis thaliana). The pid quadruple mutants exhibited moderate responses in continuous light-induced phototropism with a decrease in growth rates of hypocotyls and normal responses in pulse-induced phototropism. However, they showed serious defects in enhancements of pulse-induced phototropic curvatures and lateral fluorescent auxin transport by red light pretreatment. Red light pretreatment significantly reduced the expression level of PID, and the constitutive expression of PID prevented pulse-induced phototropism, irrespective of red light pretreatment. This suggests that the PID family plays a significant role in phytochrome-mediated phototropic enhancement but not the phototropin signaling pathway. Red light treatment enhanced the intracellular accumulation of PIN proteins in response to the vesicle-trafficking inhibitor brefeldin A in addition to increasing their expression levels. Taken together, these results suggest that red light preirradiation enhances phototropic curvatures by up-regulation of PIN proteins, which are not being phosphorylated by the PID family. PMID:25281709

  10. 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

  11. 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

  12. S6K2: The Neglected S6 Kinase Family Member.

    PubMed

    Pardo, Olivier E; Seckl, Michael J

    2013-01-01

    S6 kinase 2 (S6K2) is a member of the AGC kinases super-family. Its closest homolog, S6K1, has been extensively studied along the years. However, due to the belief in the community that the high degree of identity between these two isoforms would translate in essentially identical biological functions, S6K2 has been largely neglected. Nevertheless, recent research has clearly highlighted that these two proteins significantly differ in their roles in vitro as well as in vivo. These findings are significant to our understanding of S6 kinase signaling and the development of therapeutic strategies for several diseases including cancer. Here, we will focus on S6K2 and review the protein-protein interactions and specific substrates that determine the selective functions of this kinase. PMID:23898460

  13. Modification of tobacco plant development by sense and antisense expression of the tomato viroid-induced AGC VIIIa protein kinase PKV suggests involvement in gibberellin signaling

    PubMed Central

    2009-01-01

    Background The serine-threonine protein kinase gene, designated pkv (protein kinase- viroid induced) was previously found to be transcriptionally activated in tomato plants infected with the plant pathogen Potato spindle tuber viroid (PSTVd). These plants exhibited symptoms of stunting, and abnormal development of leaf, root, and vascular tissues. The encoded protein, PKV, is a novel member of the AGC VIIIa group of signal-transducing protein kinases; however, the role of PKV in plant development is unknown. In this communication, we report the phenotypic results of over expression and silencing of pkv in transgenic tobacco. Results Over expression of pkv in Nicotiana tabacum cv. Xanthi (tobacco) resulted in stunting, reduced root formation, and delay in flowering, phenotypes similar to symptoms of PSTVd infection of tomato. In addition, homozygous T2 tobacco plants over expressing PKV were male sterile. Antisense expression of pkv, on the other hand, resulted in plants that were taller than non-transformed plants, produced an increased number of flowers, and were fertile. Exogenous application of GA3 stimulated stem elongation in the stunted, sense-expressing plants. PKV sense and antisense expression altered transcript levels of GA biosynthetic genes and genes involved in developmental and signaling pathways, but not genes involved in salicylic acid- or jasmonic acid-dependent pathways. Our data provide evidence suggesting that PKV plays an important role in a GA signaling pathway that controls plant height and fertility. Conclusion We have found that the over expression of the tomato protein kinase PKV resulted in stunting, modified vascular tissue development, reduced root formation, and male sterility in tobacco, and we propose that PKV regulates plant development by functioning in critical signaling pathways involved in gibberellic acid metabolism. PMID:19689802

  14. Tec family kinases in inflammation and disease.

    PubMed

    Horwood, Nicole J; Urbaniak, Ania M; Danks, Lynett

    2012-04-01

    Over the last decade, the Tec family of nonreceptor tyrosine kinases (Btk, Tec, Bmx, Itk, and Rlk) have been shown to play a key role in inflammation and bone destruction. Bruton's tyrosine kinase (Btk) has been the most widely studied due to the critical role of this kinase in B-cell development and recent evidence showing that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. This review will examine the role of TFK in myeloid cell function and the potential of targeting these kinases as a therapeutic intervention in autoimmune disorders such as rheumatoid arthritis. PMID:22449071

  15. AGC-2 Disassembly Report

    SciTech Connect

    William Windes

    2014-05-01

    The Next Generation Nuclear Plant (NGNP) Graphite Research and Development (R&D) Program is currently measuring irradiated material properties for predicting the behavior and operating performance of new nuclear graphite grades available for use within the cores of new very high temperature reactor designs. The Advanced Graphite Creep (AGC) experiment, consisting of six irradiation capsules, will generate irradiated graphite performance data for NGNP reactor operating conditions. The AGC experiment is designed to determine the changes to specific material properties such as thermal diffusivity, thermal expansion, elastic modulus, mechanical strength, irradiation induced dimensional change rate, and irradiation creep for a wide variety of nuclear grade graphite types over a range of high temperature, and moderate doses. A series of six capsules containing graphite test specimens will be used to expose graphite test samples to a dose range from 1 to 7 dpa at three different temperatures (600, 900, and 1200°C) as described in the Graphite Technology Development Plan. Since irradiation induced creep within graphite components is considered critical to determining the operational life of the graphite core, some of the samples will also be exposed to an applied load to determine the creep rate for each graphite type under both temperature and neutron flux. All six AGC capsules in the experiment will be irradiated in the Advanced Test Reactor (ATR). AGC-1 and AGC-2 will be irradiated in the south flux trap and AGC-3–AGC-6 will be irradiated in the east flux trap. The change in flux traps is due to NGNP irradiation priorities requiring the AGC experiment to be moved to accommodate Fuel irradiation experiments. After irradiation, all six AGC capsules will be cooled in the ATR Canal, sized for shipment, and shipped to the Materials and Fuels Complex (MFC) where the capsule will be disassembled in the Hot Fuel Examination Facility (HFEF). During disassembly, the metallic

  16. The Ror receptor tyrosine kinase family.

    PubMed

    Forrester, W C

    2002-01-01

    Receptor tyrosine kinases (RTKs) participate in numerous developmental decisions. Ror RTKs are a family of orphan receptors that are related to muscle specific kinase (MuSK) and Trk neurotrophin receptors. MuSK assembles acetylcholine receptors at the neuromuscular junction, and Trk receptors function in the developing nervous system (reviewed in [3-5]). Rors have been identified in nematodes, insects and mammals. Recent studies have begun to shed light on Ror function during development. In most species, Rors are expressed in many tissue types during development. Analyses of mutants that are defective in the single nematode Ror demonstrate a role in cell migration and in orienting cell polarity. Mice lacking one of the two Ror gene products display defects in bone and heart formation. Similarly, two different human bone development disorders, dominant brachydactyly B and recessive Robinow syndrome, result from mutations in one of the human Ror genes. PMID:11846036

  17. Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction

    PubMed Central

    Liu, Guofa; Beggs, Hilary; Jürgensen, Claudia; Park, Hwan-Tae; Tang, Hao; Gorski, Jessica; Jones, Kevin R; Reichardt, Louis F; Wu, Jane; Rao, Yi

    2008-01-01

    Although netrins are an important family of neuronal guidance proteins, intracellular mechanisms that mediate netrin function are not well understood. Here we show that netrin-1 induces tyrosine phosphorylation of proteins including focal adhesion kinase (FAK) and the Src family kinase Fyn. Blockers of Src family kinases inhibited FAK phosphorylation and axon outgrowth and attraction by netrin. Dominant-negative FAK and Fyn mutants inhibited the attractive turning response to netrin. Axon outgrowth and attraction induced by netrin-1 were significantly reduced in neurons lacking the FAK gene. Our results show the biochemical and functional links between netrin, a prototypical neuronal guidance cue, and FAK, a central player in intracellular signaling that is crucial for cell migration. PMID:15494732

  18. X-Ray Crystal Structure of Bone Marrow Kinase in the X Chromosome: A Tec Family Kinase

    SciTech Connect

    Muckelbauer, Jodi; Sack, John S.; Ahmed, Nazia; Burke, James; Chang, ChiehYing Y.; Gao, Mian; Tino, Joseph; Xie, Dianlin; Tebben, Andrew J.

    2012-06-27

    Bone marrow kinase in the X chromosome, a member of the Tec family of tyrosine kinases, plays a role in both monocyte/macrophage trafficking as well as cytokine secretion. Although the structures of Tec family kinases Bruton's tyrosine kinase and IL-2-inducible T-cell kinase are known, the crystal structures of other Tec family kinases have remained elusive. We report the X-ray crystal structures of bone marrow kinase in the X chromosome in complex with dasatinib at 2.4 {angstrom} resolution and PP2 at 1.9 {angstrom} resolution. The bone marrow kinase in the X chromosome structures reveal a typical kinase protein fold; with well-ordered protein conformation that includes an open/extended activation loop and a stabilized DFG-motif rendering the kinase in an inactive conformation. Dasatinib and PP2 bind to bone marrow kinase in the X chromosome in the ATP binding pocket and display similar binding modes to that observed in other Tec and Src protein kinases. The bone marrow kinase in the X chromosome structures identify conformational elements of the DFG-motif that could potentially be utilized to design potent and/or selective bone marrow kinase in the X chromosome inhibitors.

  19. AGC-3 Graphite Preirradiation Data Analysis Report

    SciTech Connect

    William Windes; David Swank; David Rohrbaugh; Joseph Lord

    2013-09-01

    This report describes the specimen loading order and documents all pre-irradiation examination material property measurement data for the graphite specimens contained within the third Advanced Graphite Capsule (AGC-3) irradiation capsule. The AGC-3 capsule is third in six planned irradiation capsules comprising the Advanced Graphite Creep (AGC) test series. The AGC test series is used to irradiate graphite specimens allowing quantitative data necessary for predicting the irradiation behavior and operating performance of new nuclear graphite grades to be generated which will ascertain the in-service behavior of the graphite for pebble bed and prismatic Very High Temperature Reactor (VHTR) designs. The general design of AGC-3 test capsule is similar to the AGC-2 test capsule, material property tests were conducted on graphite specimens prior to loading into the AGC-3 irradiation assembly. However the 6 major nuclear graphite grades in AGC-2 were modified; two previous graphite grades (IG-430 and H-451) were eliminated and one was added (Mersen’s 2114 was added). Specimen testing from three graphite grades (PCEA, 2114, and NBG-17) was conducted at Idaho National Laboratory (INL) and specimen testing for two grades (IG-110 and NBG-18) were conducted at Oak Ridge National Laboratory (ORNL) from May 2011 to July 2013. This report also details the specimen loading methodology for the graphite specimens inside the AGC-3 irradiation capsule. The AGC-3 capsule design requires "matched pair" creep specimens that have similar dose levels above and below the neutron flux profile mid-plane to provide similar specimens with and without an applied load. This document utilized the neutron flux profile calculated for the AGC-3 capsule design, the capsule dimensions, and the size (length) of the selected graphite and silicon carbide samples to create a stacking order that can produce "matched pairs" of graphite samples above and below the AGC-3 capsule elevation mid-point to

  20. AGC-2 Graphite Preirradiation Data Analysis Report

    SciTech Connect

    William Windes; W. David Swank; David Rohrbaugh; Joseph Lord

    2013-08-01

    This report described the specimen loading order and documents all pre-irradiation examination material property measurement data for the graphite specimens contained within the second Advanced Graphite Capsule (AGC-2) irradiation capsule. The AGC-2 capsule is the second in six planned irradiation capsules comprising the Advanced Graphite Creep (AGC) test series. The AGC test series is used to irradiate graphite specimens allowing quantitative data necessary for predicting the irradiation behavior and operating performance of new nuclear graphite grades to be generated which will ascertain the in-service behavior of the graphite for pebble bed and prismatic Very High Temperature Reactor (VHTR) designs. Similar to the AGC-1 specimen pre-irradiation examination report, material property tests were conducted on specimens from 18 nuclear graphite types but on an increased number of specimens (512) prior to loading into the AGC-2 irradiation assembly. All AGC-2 specimen testing was conducted at Idaho National Laboratory (INL) from October 2009 to August 2010. This report also details the specimen loading methodology for the graphite specimens inside the AGC-2 irradiation capsule. The AGC-2 capsule design requires “matched pair” creep specimens that have similar dose levels above and below the neutron flux profile mid-plane to provide similar specimens with and without an applied load. This document utilized the neutron flux profile calculated for the AGC-2 capsule design, the capsule dimensions, and the size (length) of the selected graphite and silicon carbide samples to create a stacking order that can produce “matched pairs” of graphite samples above and below the AGC-2 capsule elevation mid-point to provide specimens with similar neutron dose levels.

  1. 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

  2. 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

  3. The role of Tec family kinases in the regulation of T-helper-cell differentiation.

    PubMed

    Boucheron, Nicole; Ellmeier, Wilfried

    2012-04-01

    ABSTRACT Members of the Tec kinase family (Tec, Btk, Itk, Rlk, and Bmx) play an important role during innate and adaptive immune responses, and mutations in Tec family kinases are linked with immunodeficiencies in humans and mice. Three members of the Tec kinase family are expressed in T cells (Tec, Itk, and Rlk), and biochemical and genetic studies have revealed important roles for Tec family kinases during T-cell development and in the control of T-cell function. Here the authors review the role of Tec family kinases in the regulation of T-helper-cell differentiation. PMID:22449074

  4. The EGFR Family: Not So Prototypical Receptor Tyrosine Kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph; Ferguson, Kathryn M.

    2014-01-01

    The epidermal growth factor receptor (EGFR) was among the first receptor tyrosine kinases (RTKs) for which ligand binding was studied and for which the importance of ligand-induced dimerization was established. As a result, EGFR and its relatives have frequently been termed “prototypical” RTKs. Many years of mechanistic studies, however, have revealed that—far from being prototypical—the EGFR family is quite unique. As we discuss in this review, the EGFR family uses a distinctive “receptor-mediated” dimerization mechanism, with ligand binding inducing a dramatic conformational change that exposes a dimerization arm. Intracellular kinase domain regulation in this family is also unique, being driven by allosteric changes induced by asymmetric dimer formation rather than the more typical activation-loop phosphorylation. EGFR family members also distinguish themselves from other RTKs in having an intracellular juxtamembrane (JM) domain that activates (rather than autoinhibits) the receptor and a very large carboxy-terminal tail that contains autophosphorylation sites and serves an autoregulatory function. We discuss recent advances in mechanistic aspects of all of these components of EGFR family members, attempting to integrate them into a view of how RTKs in this important class are regulated at the cell surface. PMID:24691965

  5. The venus kinase receptor (VKR) family: structure and evolution

    PubMed Central

    2013-01-01

    Background Receptor tyrosine kinases (RTK) form a family of transmembrane proteins widely conserved in Metazoa, with key functions in cell-to-cell communication and control of multiple cellular processes. A new family of RTK named Venus Kinase Receptor (VKR) has been described in invertebrates. The VKR receptor possesses a Venus Fly Trap (VFT) extracellular module, a bilobate structure that binds small ligands to induce receptor kinase activity. VKR was shown to be highly expressed in the larval stages and gonads of several invertebrates, suggesting that it could have functions in development and/or reproduction. Results Analysis of recent genomic data has allowed us to extend the presence of VKR to five bilaterian phyla (Platyhelminthes, Arthropoda, Annelida, Mollusca, Echinodermata) as well as to the Cnidaria phylum. The presence of NveVKR in the early-branching metazoan Nematostella vectensis suggested that VKR arose before the bilaterian radiation. Phylogenetic and gene structure analyses showed that the 40 receptors identified in 36 animal species grouped monophyletically, and likely evolved from a common ancestor. Multiple alignments of tyrosine kinase (TK) and VFT domains indicated their important level of conservation in all VKRs identified up to date. We showed that VKRs had inducible activity upon binding of extracellular amino-acids and molecular modeling of the VFT domain confirmed the structure of the conserved amino-acid binding site. Conclusions This study highlights the presence of VKR in a large number of invertebrates, including primitive metazoans like cnidarians, but also its absence from nematodes and chordates. This little-known RTK family deserves to be further explored in order to determine its evolutionary origin, its possible interest for the emergence and specialization of Metazoa, and to understand its function in invertebrate development and/or reproductive biology. PMID:23721482

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

    SciTech Connect

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

    2010-11-15

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

  7. MAP4K Family Kinases in Immunity and Inflammation.

    PubMed

    Chuang, Huai-Chia; Wang, Xiaohong; Tan, Tse-Hua

    2016-01-01

    MAP kinase kinase kinase kinases (MAP4Ks) belong to the mammalian Ste20-like family of serine/threonine kinases. MAP4Ks including MAP4K1/HPK1, MAP4K2/GCK, MAP4K3/GLK, MAP4K4/HGK, MAP4K5/KHS, and MAP4K6/MINK have been reported to induce JNK activation through activating the MAP3K-MAP2K cascade. The physiological roles of MAP4Ks in immunity and inflammation are largely unknown until recent studies using biochemical approaches and knockout mice. Surprisingly, JNK is not the major target of MAP4Ks in immune cells; MAP4Ks regulate immune responses through novel targets. HPK1 inhibits T-cell receptor (TCR) signaling and B-cell receptor signaling via inducing phosphorylation/ubiquitination of SLP-76 and BLNK, respectively. GLK activates TCR signaling through phosphorylating/activating PKCθ. T-cell-mediated immune responses and Th17-mediated experimental autoimmune diseases are enhanced in HPK1 knockout mice but ameliorated in GLK knockout mice. Consistently, HPK1 levels are decreased in peripheral blood mononuclear cells and T cells from patients with psoriatic arthritis and systemic lupus erythematosus (SLE), respectively. Moreover, GLK levels are increased in T cells from patients with SLE, rheumatoid arthritis, or adult-onset Still's disease; the percentages of GLK-overexpression T cells are correlated with the disease activity. In addition, HGK phosphorylates and induces TRAF2 protein degradation, leading to negative regulation of IL-6 production in resting T cells. Loss of HGK in T cells results in spontaneous systemic inflammation and type 2 diabetes in mice. HGK is also involved in cancer cell migration. To date, the phenotypes of knockout mice for GCK, KHS, and MINK have not been reported; the roles of these three MAP4Ks in immune cell signaling are discussed in this review. Taken together, MAP4K family kinases play diverse roles in immune cell signaling, immune responses, and inflammation. PMID:26791862

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

    SciTech Connect

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

    2012-12-05

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

  9. RF front end interface and AGC modification

    NASA Technical Reports Server (NTRS)

    Yost, S. R.

    1983-01-01

    Preliminary results indicate that the new front end automatic gain control (AGC) combination perform satisfactorily. Side by side bench tests with Trimble 10A and Texas Instruments 9900 LORAN-C receivers have proven that the proper time differences are being obtained. Further optimization of the AGC circuit will occur as software to track all of the stations in a LORAN-C chain is developed. The AGC circuit was designed to sample up to six separate LORAN-C stations. Along with expanded station tracking software, a more sophisticated search routine is also under development. A printed circuit board for the AGC is planned and will be enclosed with the RF front end in a sealed enclosure to reduce interference from the other digital circuits of the microcomputer.

  10. AGC-2 Graphite Preirradiation Data Package

    SciTech Connect

    David Swank; Joseph Lord; David Rohrbaugh; William Windes

    2012-10-01

    The NGNP Graphite R&D program is currently establishing the safe operating envelope of graphite core components for a Very High Temperature Reactor (VHTR) design. The program is generating quantitative data necessary for predicting the behavior and operating performance of the new nuclear graphite grades. To determine the in-service behavior of the graphite for pebble bed and prismatic designs, the Advanced Graphite Creep (AGC) experiment is underway. This experiment is examining the properties and behavior of nuclear grade graphite over a large spectrum of temperatures, neutron fluences and compressive loads. Each experiment consists of over 400 graphite specimens that are characterized prior to irradiation and following irradiation. Six experiments are planned with the first, AGC-1, currently being irradiated in the Advanced Test Reactor (ATR) and pre-irradiation characterization of the second, AGC-2, completed. This data package establishes the readiness of 512 specimens for assembly into the AGC-2 capsule.

  11. Cell cycle regulation by the NEK family of protein kinases.

    PubMed

    Fry, Andrew M; O'Regan, Laura; Sabir, Sarah R; Bayliss, Richard

    2012-10-01

    Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer. PMID:23132929

  12. RF Front End Interface and AGC Modification

    NASA Technical Reports Server (NTRS)

    Yost, S. R.

    1982-01-01

    The latest RF front end built by Burhans (Mini-L-82) was successfully interfaced to the Ohio University LORAN-C receiver. In order for the front end to operate optimally, modifications were made to existing automatic gain control (AGC) circuitry already developed for the Ohio University LORAN-C receiver. The hardware modifications to the AGC and other interface circuitry, as well as some preliminary results are discussed.

  13. AGC-1 Post Irradiation Examination Status

    SciTech Connect

    David Swank

    2011-09-01

    The Next Generation Nuclear Plant (NGNP) Graphite R&D program is currently measuring irradiated material property changes in several grades of nuclear graphite for predicting their behavior and operating performance within the core of new Very High Temperature Reactor (VHTR) designs. The Advanced Graphite Creep (AGC) experiment consisting of six irradiation capsules will generate this irradiated graphite performance data for NGNP reactor operating conditions. All six AGC capsules in the experiment will be irradiated in the Advanced Test Reactor (ATR), disassembled in the Hot Fuel Examination Facility (HFEF), and examined at the INL Research Center (IRC) or Oak Ridge National Laboratory (ORNL). This is the first in a series of status reports on the progress of the AGC experiment. As the first capsule, AGC1 was irradiated from September 2009 to January 2011 to a maximum dose level of 6-7 dpa. The capsule was removed from ATR and transferred to the HFEF in April 2011 where the capsule was disassembled and test specimens extracted from the capsules. The first irradiated samples from AGC1 were shipped to the IRC in July 2011and initial post irradiation examination (PIE) activities were begun on the first 37 samples received. PIE activities continue for the remainder of the AGC1 specimen as they are received at the IRC.

  14. rlk/TXK Encodes Two Forms of a Novel Cysteine String Tyrosine Kinase Activated by Src Family Kinases

    PubMed Central

    Debnath, Jayantha; Chamorro, Mario; Czar, Michael J.; Schaeffer, Edward M.; Lenardo, Michael J.; Varmus, Harold E.; Schwartzberg, Pamela L.

    1999-01-01

    Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR. PMID:9891083

  15. Family-wide Structural Analysis of Human Numb-Associated Protein Kinases

    PubMed Central

    Sorrell, Fiona J.; Szklarz, Marta; Abdul Azeez, Kamal R.; Elkins, Jon M.; Knapp, Stefan

    2016-01-01

    Summary The highly diverse Numb-associated kinase (NAK) family has been linked to broad cellular functions including receptor-mediated endocytosis, Notch pathway modulation, osteoblast differentiation, and dendrite morphogenesis. Consequently, NAK kinases play a key role in a diverse range of diseases from Parkinson's and prostate cancer to HIV. Due to the plasticity of this kinase family, NAK kinases are often inhibited by approved or investigational drugs and have been associated with side effects, but they are also potential drug targets. The presence of cysteine residues in some NAK family members provides the possibility for selective targeting via covalent inhibition. Here we report the first high-resolution structures of kinases AAK1 and BIKE in complex with two drug candidates. The presented data allow a comprehensive structural characterization of the NAK kinase family and provide the basis for rational design of selective NAK inhibitors. PMID:26853940

  16. Family-wide Structural Analysis of Human Numb-Associated Protein Kinases.

    PubMed

    Sorrell, Fiona J; Szklarz, Marta; Abdul Azeez, Kamal R; Elkins, Jon M; Knapp, Stefan

    2016-03-01

    The highly diverse Numb-associated kinase (NAK) family has been linked to broad cellular functions including receptor-mediated endocytosis, Notch pathway modulation, osteoblast differentiation, and dendrite morphogenesis. Consequently, NAK kinases play a key role in a diverse range of diseases from Parkinson's and prostate cancer to HIV. Due to the plasticity of this kinase family, NAK kinases are often inhibited by approved or investigational drugs and have been associated with side effects, but they are also potential drug targets. The presence of cysteine residues in some NAK family members provides the possibility for selective targeting via covalent inhibition. Here we report the first high-resolution structures of kinases AAK1 and BIKE in complex with two drug candidates. The presented data allow a comprehensive structural characterization of the NAK kinase family and provide the basis for rational design of selective NAK inhibitors. PMID:26853940

  17. Robust AGC : Traditional Structure Versus Restructured Scheme

    NASA Astrophysics Data System (ADS)

    Bevrani, Hassan; Mitani, Yasunori; Tsuji, Kiichiro

    In this paper, a decentralized robust approach is proposed for the Automatic Generation Control (AGC) system based on a modified traditional AGC structure. This work addresses the new strategy to adapt well-tested classical AGC scheme to the changing environment of power system operation under deregulation. The effect of bilateral contracts is considered as a set of new input signals in each control area dynamical model. In practice, AGC systems use simple proportional-integral (PI) controllers. However, since the PI controller parameters are usually tuned based on classical or trial-and-error approaches, they are incapable of obtaining good dynamical performance for a wide range of operating conditions and various scenarios in deregulated environment. In this paper with regard to this problem, the AGC synthesis is formulated as an H∞ static output control problem and is solved using a developed iterative linear matrix inequalities (ILMI) algorithm to design of robust PI controllers in the restructured power system control areas. A three area power system example with possible contract scenarios and wide range of load changes is given to illustrate the proposed approach. The resulting controllers are shown to minimize the effect of disturbances and maintain the robust performance.

  18. Fertilization triggers localized activation of Src-family protein kinases in the zebrafish egg.

    PubMed

    Sharma, Dipika; Kinsey, William H

    2006-07-15

    Fertilization triggers activation of Src-family kinases in eggs of various species including marine invertebrates and lower vertebrates. While immunofluorescence studies have localized Src-family kinases to the plasma membrane or cortical cytoplasm, no information is available regarding the extent to which these kinases are activated in different regions of the zygote. The objective of the present study was to detect the subcellular distribution of activated Src-family kinases in the fertilized zebrafish egg. An antibody specific for the active, non-phosphorylated form of Src-family PTKs was used to detect these activated kinases by immunofluorescence. The results demonstrate that Fyn, and possibly other Src family members are activated by dephosphorylation of the C-terminal tyrosine at fertilization. The activated Src-family kinases are asymmetrically distributed around the egg cortex with an area of higher kinase activity localized adjacent to the micropyle near the presumptive animal pole. Fertilization initially caused elevation of kinase activity in the cytoplasm underlying the micropyle, but this quickly spread to involve the entire zygote cortex. Later, during egg activation, formation of the blastodisc involved concentration of active Src-family kinase in the blastodisc cortex. As cytokinesis began, activated Src-family kinases were no longer limited to the cortex, but became more evenly distributed in the clear apical cytoplasm of the blastomeres. The results demonstrate that the cortex of the zebrafish egg is functionally differentiated and that fertilization triggers localized activation of Src-family kinases at the point of sperm entry, which subsequently progresses through the entire egg cortex. PMID:16698010

  19. FY-2010 AGC-1 Disassembly Preparation

    SciTech Connect

    Philip L. Winston

    2010-09-01

    The Next Generation Nuclear Plant Project Graphite Research and Development program is currently establishing the safe operating envelope of graphite core components for a very high temperature reactor design. The program is generating quantitative data necessary for predicting the behavior and operating performance of the new nuclear graphite grades. To determine the in-service behavior of the graphite for pebble bed and prismatic designs, the Advanced Graphite Creep (AGC) experiment is underway. This experiment is examining the properties and behavior of nuclear grade graphite over a large spectrum of temperatures, neutron fluencies, and compressive loads. Each experiment consists of over 400 graphite specimens that are characterized prior to irradiation and following irradiation. Six experiments are planned with the first, AGC-1, currently being irradiated in the Advanced Test Reactor and preirradiation characterization of the second, AGC-2, completed.

  20. AGC 226067: A possible interacting low-mass system

    NASA Astrophysics Data System (ADS)

    Adams, E. A. K.; Cannon, J. M.; Rhode, K. L.; Janesh, W. F.; Janowiecki, S.; Leisman, L.; Giovanelli, R.; Haynes, M. P.; Oosterloo, T. A.; Salzer, J. J.; Zaidi, T.

    2015-08-01

    We present Arecibo, GBT, VLA, and WIYN/pODI observations of the ALFALFA source AGC 226067. Originally identified as an ultra-compact high velocity cloud and candidate Local Group galaxy, AGC 226067 is spatially and kinematically coincident with the Virgo cluster, and the identification by multiple groups of an optical counterpart with no resolved stars supports the interpretation that this systems lies at the Virgo distance (D = 17 Mpc). The combined observations reveal that the system consists of multiple components: a central H i source associated with the optical counterpart (AGC 226067), a smaller H i-only component (AGC 229490), a second optical component (AGC 229491), and extended low-surface brightness H i. Only ~1/4 of the single-dish H i emission is associated with AGC 226067; as a result, we find MHI/Lg ~ 6M⊙/L⊙ which is lower than previous work. At D = 17 Mpc, AGC 226067 has an H i mass of 1.5 × 107M⊙ and Lg = 2.4 × 106L⊙, AGC 229490 (the H i-only component) has MHI = 3.6 × 106M⊙, and AGC 229491 (the second optical component) has Lg = 3.6 × 105L⊙. The nature of this system of three sources is uncertain: AGC 226067 and AGC 229490 may be connected by an H i bridge, and AGC 229490 and AGC 229491 are separated by only 0.5'. The current data do not resolve the H i in AGC 229490 and its origin is unclear. We discuss possible scenarios for this system of objects: an interacting system of dwarf galaxies, accretion of material onto AGC 226067, or stripping of material from AGC 226067.

  1. Pulse-width-modulated attenuator for AGC

    NASA Technical Reports Server (NTRS)

    Macconnell, J. W.

    1979-01-01

    Automatic-gain-control (AGC) circuit regulates gain of intermediate-frequency and radio-frequency signals over wide dynamic range with high linearity and very low phase shift. Device has potential uses in radio and television receivers, signal distribution systems, and test and measurement instruments.

  2. Src family kinases differentially influence glioma growth and motility.

    PubMed

    Lewis-Tuffin, Laura J; Feathers, Ryan; Hari, Priya; Durand, Nisha; Li, Zhimin; Rodriguez, Fausto J; Bakken, Katie; Carlson, Brett; Schroeder, Mark; Sarkaria, Jann N; Anastasiadis, Panos Z

    2015-11-01

    Src-family kinase (SFK) signaling impacts multiple tumor-related properties, particularly in the context of the brain tumor glioblastoma. Consequently, the pan-SFK inhibitor dasatinib has emerged as a therapeutic strategy, despite physiologic limitations to its effectiveness in the brain. We investigated the importance of individual SFKs (Src, Fyn, Yes, and Lyn) to glioma tumor biology by knocking down individual SFK expression both in culture (LN229, SF767, GBM8) and orthotopic xenograft (GBM8) contexts. We evaluated the effects of these knockdowns on tumor cell proliferation, migration, and motility-related signaling in culture, as well as overall survival in the orthotopic xenograft model. The four SFKs differed significantly in their importance to these properties. In culture, Src, Fyn, and Yes knockdown generally reduced growth and migration and altered motility-related phosphorylation patterns while Lyn knockdown did so to a lesser extent. However the details of these effects varied significantly depending on the cell line: in no case were conclusions about the role of a particular SFK applicable to all of the measures or all of the cell types examined. In the orthotopic xenograft model, mice implanted with non-target or Src or Fyn knockdown cells showed no differences in survival. In contrast, mice implanted with Yes knockdown cells had longer survival, associated with reduced tumor cell proliferation. Those implanted with Lyn knockdown cells had shorter survival, associated with higher overall tumor burden. Together, our results suggest that Yes signaling directly affects tumor cell biology in a pro-tumorigenic manner, while Lyn signaling affects interactions between tumor cells and the microenvironment in an anti-tumor manner. In the context of therapeutic targeting of SFKs, these results suggest that pan-SFK inhibitors may not produce the intended therapeutic benefit when Lyn is present. PMID:26105207

  3. Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

    PubMed

    Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

    2016-04-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

  4. Benzobisthiazoles Represent a Novel Scaffold for Kinase Inhibitors of CLK Family Members

    PubMed Central

    2015-01-01

    Protein kinases are essential regulators of most cellular processes and are involved in the etiology and progression of multiple diseases. The cdc2-like kinases (CLKs) have been linked to various neurodegenerative disorders, metabolic regulation, and virus infection, and the kinases have been recognized as potential drug targets. Here, we have developed a screening workflow for the identification of potent CLK2 inhibitors and identified compounds with a novel chemical scaffold structure, the benzobisthiazoles, that has not been previously reported for kinase inhibitors. We propose models for binding of these compounds to CLK family proteins and key residues in CLK2 that are important for the compound interactions and the kinase activity. We identified structural elements within the benzobisthiazole that determine CLK2 and CLK3 inhibition, thus providing a rationale for selectivity assays. In summary, our results will inform structure-based design of CLK family inhibitors based on the novel benzobisthiazole scaffold. PMID:26701387

  5. AGC-1 Irradiation Experiment Test Plan

    SciTech Connect

    R. L. Bratton

    2006-05-01

    The Advanced Graphite Capsule (AGC) irradiation test program supports the acquisition of irradiated graphite performance data to assist in the selection of the technology to be used for the VHTR. Six irradiations are planned to investigate compressive creep in graphite subjected to a neutron field and obtain irradiated mechanical properties of vibrationally molded, extruded, and iso-molded graphites for comparison. The experiments will be conducted at three temperatures: 600, 900, and 1200°C. At each temperature, two different capsules will be irradiated to different fluence levels, the first from 0.5 to 4 dpa and the second from 4 to 7 dpa. AGC-1 is the first of the six capsules designed for ATR and will focus on the prismatic fluence range.

  6. AGC1/2, the mitochondrial aspartate-glutamate carriers.

    PubMed

    Amoedo, N D; Punzi, G; Obre, E; Lacombe, D; De Grassi, A; Pierri, C L; Rossignol, R

    2016-10-01

    In this review we discuss the structure and functions of the aspartate/glutamate carriers (AGC1-aralar and AGC2-citrin). Those proteins supply the aspartate synthesized within mitochondrial matrix to the cytosol in exchange for glutamate and a proton. A structure of an AGC carrier is not available yet but comparative 3D models were proposed. Moreover, transport assays performed by using the recombinant AGC1 and AGC2, reconstituted into liposome vesicles, allowed to explore the kinetics of those carriers and to reveal their specific transport properties. AGCs participate to a wide range of cellular functions, as the control of mitochondrial respiration, calcium signaling and antioxydant defenses. AGC1 might also play peculiar tissue-specific functions, as it was found to participate to cell-to-cell metabolic symbiosis in the retina. On the other hand, AGC1 is involved in the glutamate-mediated excitotoxicity in neurons and AGC gene or protein alterations were discovered in rare human diseases. Accordingly, a mice model of AGC1 gene knock-out presented with growth delay and generalized tremor, with myelinisation defects. More recently, AGC was proposed to play a crucial role in tumor metabolism as observed from metabolomic studies showing that the asparate exported from the mitochondrion by AGC1 is employed in the regeneration of cytosolic glutathione. Therefore, given the central role of AGCs in cell metabolism and human pathology, drug screening are now being developed to identify pharmacological modulators of those carriers. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou. PMID:27132995

  7. The Tec family kinase Itk differentially controls mast cell responses*

    PubMed Central

    Iyer, Archana S.; August, Avery

    2008-01-01

    The Tec family tyrosine kinase Itk is expressed in T cells and mast cells. Mice lacking Itk exhibit impaired TH2 cytokine secretion; however, they have increased circulating serum IgE but exhibit few immunological symptoms of allergic airway responses. We have examined the role of Itk in mast cell function and FcεRI signaling. We report here that Itk null mice have reduced allergen/IgE induced histamine release as well as early airway hyperresponsiveness in vivo. This is due to the increased levels of IgE in the serum of these mice, since the transfer of Itk null BMMC into mast cell deficient W/Wv animals is able to fully rescue histamine release in the W/Wv mice. Further analysis of Itk null BMMC in vitro revealed that while they have normal degranulation responses, they secrete elevated levels of cytokines, including IL-13 and TNF-α, particularly in response to unliganded IgE. Analysis of biochemical events downstream of the FcεRI revealed little difference in overall tyrosine phosphorylation of specific substrates or calcium responses, however, these cells express elevated levels of NFAT which was largely nuclear. Our results suggest that the reduced mast cell response in vivo in Itk null mice is due to elevated levels of IgE in these mice. Our results also suggest that Itk differentially modulates mast cell degranulation and cytokine production in part by regulating expression and activation of NFAT proteins in these cells. “This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any

  8. The Src-family tyrosine kinase inhibitor PP1 interferes with the activation of ribosomal protein S6 kinases.

    PubMed Central

    Shah, O Jameel; Kimball, Scot R; Jefferson, Leonard S

    2002-01-01

    Considerable biochemical and pharmacological evidence suggests that the activation of ribosomal protein S6 kinases (S6Ks) by activated receptor tyrosine kinases involves multiple co-ordinated input signals. However, the identities of many of these inputs remain poorly described, and their precise involvement in S6K activation has been the subject of great investigative effort. In the present study, we have shown that 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), a selective inhibitor of the Src family of non-receptor tyrosine kinases, interferes with the activation of 70 and 85 kDa S6K gene products (p70S6K1 and p85S6K1) by insulin, insulin-like growth factor 1, sodium orthovanadate and activated alleles of phosphoinositide 3-kinase and H-Ras. PP1 also impedes the activation of AKT/protein kinase B and the extracellular signal-regulated protein kinases 1 and 2 by these various stimuli. Insulin-like growth factor 1 was observed to induce a sustained increase in c-Src autophosphorylation as revealed using anti-phospho-Y416 antisera, but this effect was absent from the cells treated with PP1. To conclude, an activated allele of p70S6K1 is compared with the wild-type allele, resistant to inhibition by PP1 when co-expressed with phosphoinositide-dependent kinase 1 (PDK1), suggesting that PP1 affects p70S6K1 via a PDK1-independent pathway. Thus activation of Src may supply a necessary signal for the activation of p70S6K1 and possibly other S6Ks. PMID:12014987

  9. AGC-2 Irradiation Data Qualification Final Report

    SciTech Connect

    Laurence C. Hull

    2012-07-01

    The Graphite Technology Development Program will run a series of six experiments to quantify the effects of irradiation on nuclear grade graphite. The second Advanced Graphite Creep (AGC) experiment (AGC-2) began with Advanced Test Reactor (ATR) Cycle 149A on April 12, 2011, and ended with ATR Cycle 151B on May 5, 2012. The purpose of this report is to qualify AGC-2 irradiation monitoring data following INL Management and Control Procedure 2691, Data Qualification. Data that are Qualified meet the requirements for data collection and use as described in the experiment planning and quality assurance documents. Data that do not meet the requirements are Failed. Some data may not quite meet the requirements, but may still provide some useable information. These data are labeled as Trend. No Trend data were identified for the AGC-2 experiment. All thermocouples functioned throughout the AGC-2 experiment. There was one instance where spurious signals or instrument power interruption resulted in a recorded temperature value being well outside physical reality. This value was identified and labeled as Failed data. All other temperature data are Qualified. All helium and argon gas flow data are within expected ranges. Total gas flow was approximately 50 sccm through the capsule. Helium gas flow was briefly increased to 100 sccm during reactor shutdown. All gas flow data are Qualified. At the start of the experiment, moisture in the outflow gas line increased to 200 ppmv then declined to less than 10 ppmv over a period of 5 days. This increase in moisture coincides with the initial heating of the experiment and drying of the system. Moisture slightly exceeded 10 ppmv three other times during the experiment. While these moisture values exceed the 10 ppmv threshold value, the reported measurements are considered accurate and to reflect moisture conditions in the capsule. All moisture data are Qualified. Graphite creep specimens are subjected to one of three loads, 393 lbf

  10. Nek8, a NIMA family kinase member, is overexpressed in primary human breast tumors.

    PubMed

    Bowers, Alex J; Boylan, John F

    2004-03-17

    The family of human Nek (NIMA Related Kinase) kinases currently contains 11 members. We have identified Nek8 as a new member of the Nek kinase family. For many of the Nek family members, primary tumor expression data and function have been limited. However, all of the Nek family proteins share considerable homology with the Never In Mitosis, gene A (NIMA) kinase from the filamentous fungus Aspergillus nidulans. NIMA, as well as its most closely related human ortholog, Nek2, are required for G(2)/M progression and promote centrosome maturation during mitosis. We isolated Nek8 from a primary human colon cDNA library, and found it to be highly homologous to murine Nek8. Recently, a previously named Nek8 sequence was renamed Nek9/Nercc1 in Genbank due to its lack of homology to murine Nek8 and its high homology to murine Nek9. Interestingly, in our study, phylogenetic analysis suggests that human Nek8 and Nek9 form a subfamily within the Nek family. Nek8 has high homology to the Nek family kinase domain as well as to a regulator of chromosome condensation domain (RCC1), which is also present in Nek9. The open reading frame of human Nek8 encodes a 692 amino-acid protein with a calculated molecular weight of 75 kDa. Nek8 is differently expressed between normal human breast tissue and breast tumors. Overexpression of a mutated kinase domain Nek8 in U2-0S cells led to a decrease in actin protein, and a small increase in the level of cdk1/cyclinB1. Our data demonstrate for the first time that Nek8 is a novel tumor associated gene, and shares considerable sequence homology with the Nek family of protein kinases and may be involved in G(2)/M progression. PMID:15019993

  11. Phosphorylation of spore coat proteins by a family of atypical protein kinases.

    PubMed

    Nguyen, Kim B; Sreelatha, Anju; Durrant, Eric S; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R; Pawłowski, Krzysztof; Dixon, Jack E; Tagliabracci, Vincent S

    2016-06-21

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology. PMID:27185916

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

    SciTech Connect

    Nesic, D.; Miller, M; Quinkert, Z; Stein, M; Chait, B; Stebbins, C

    2010-01-01

    The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.

  13. Assessing the range of kinase autoinhibition mechanisms in the insulin receptor family

    PubMed Central

    Artim, Stephen C.; Mendrola, Jeannine M.; Lemmon, Mark A.

    2012-01-01

    To investigate the range of autoinhibitory mechanisms used by TKDs (tyrosine kinase domains) from the insulin receptor family of RTKs (receptor tyrosine kinases), we determined crystal structures of TKDs from TrkA (tropomyosin receptor kinase A, a nerve growth factor receptor) and Ror2 (receptor tyrosine kinase-like orphan receptor 2, an unconventional Wnt receptor). TrkA autoinhibition closely resembles that seen for the insulin receptor, relying on projection of an activation loop tyrosine residue into the substrate-binding site and occlusion of the ATP-binding site by the activation loop. Ror2 employs similar mechanisms, but the unusual replacement of the phenylalanine residue in its Asp-Phe-Gly motif with leucine necessitates occlusion of the ATP-binding site by other means. The unusual Asp-Leu-Gly motif in Ror2 is displaced compared with other inactive kinases, allowing the activation loop to interact directly with the TKD's αC helix, in another mode of autoinhibition that is characteristic of the other extreme of this receptor family: ALK (anaplastic lymphoma kinase) and Met. These findings provide insight into the expected range of activating mutations in these TKDs in cancer. We also describe symmetrical dimers of the inactive TrkA TKD resembling those found in other RTKs, possibly reflecting an arrangement of kinase domains in a pre-formed TrkA dimer. PMID:22992069

  14. Genome-wide identification and expression analysis of WNK kinase gene family in rice.

    PubMed

    Manuka, Rakesh; Saddhe, Ankush Ashok; Kumar, Kundan

    2015-12-01

    Eukaryotic protein kinases represent one of the largest gene families involved in diverse regulatory functions. WNK (With No Lysine) kinases are members of ser/thr protein kinase family, which lack conserved catalytic lysine (K) residue at protein kinase subdomain II and is replaced by either asparagine, serine or glycine residues. They are involved in regulation of flowering time, circadian rhythms and abiotic stresses in Arabidopsis thaliana. In the present study, we have identified 9 members of WNK in rice, showed resemblance to Arabidopsis and human WNK and clustered into five main clades phylogenetically. The predicted genes structure, bonafide conserved signature motif and domains strongly support their identity, as members of WNK kinase family. We have analyzed their chromosomal distribution, physio-chemical properties, subcellular localizations and cis-elements in the promoter regions in silico. Further, transcript analysis of OsWNK by qRT-PCR revealed their differential regulation in tissue specific and abiotic stresses libraries. In conclusion, the identification of nine OsWNK and transcript level expression pattern under abiotic stress using qRT-PCR in rice will significantly contribute towards the understanding of WNK genes in monocots and thus provide a set up for functional genomics studies of WNK protein kinases. PMID:26414948

  15. Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition.

    PubMed Central

    Komander, David; Kular, Gursant S; Bain, Jennifer; Elliott, Matthew; Alessi, Dario R; Van Aalten, Daan M F

    2003-01-01

    PDK1 (3-phosphoinositide-dependent protein kinase-1) is a member of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases, and has a key role in insulin and growth-factor signalling through phosphorylation and subsequent activation of a number of other AGC kinase family members, such as protein kinase B. The staurosporine derivative UCN-01 (7-hydroxystaurosporine) has been reported to be a potent inhibitor for PDK1, and is currently undergoing clinical trials for the treatment of cancer. Here, we report the crystal structures of staurosporine and UCN-01 in complex with the kinase domain of PDK1. We show that, although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group, which is not present in staurosporine, generates direct and water-mediated hydrogen bonds with active-site residues. Inhibition data from UCN-01 tested against a panel of 29 different kinases show a different pattern of inhibition compared with staurosporine. We discuss how these differences in inhibition could be attributed to specific interactions with the additional 7-hydroxy group, as well as the size of the 7-hydroxy-group-binding pocket. This information could lead to opportunities for structure-based optimization of PDK1 inhibitors. PMID:12892559

  16. Divergent modulation of Rho‐kinase and Ca2+ influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle

    PubMed Central

    Shaifta, Yasin; Irechukwu, Nneka; Prieto‐Lloret, Jesus; MacKay, Charles E; Marchon, Keisha A; Ward, Jeremy P T

    2015-01-01

    Background and Purpose The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src‐family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR‐mediated ASM contraction and associated signalling events. Experimental Approach Contraction was recorded in intact or α‐toxin permeabilized rat bronchioles. Phosphorylation of SrcFK, FAK, myosin light‐chain‐20 (MLC20) and myosin phosphatase targeting subunit‐1 (MYPT‐1) was evaluated in cultured human ASM cells (hASMC). [Ca2+]i was evaluated in Fura‐2 loaded hASMC. Responses to carbachol (CCh) and bradykinin (BK) and the contribution of SrcFK and FAK to these responses were determined. Key Results Contractile responses in intact bronchioles were inhibited by antagonists of SrcFK, FAK and Rho‐kinase, while after α‐toxin permeabilization, they were sensitive to inhibition of SrcFK and Rho‐kinase, but not FAK. CCh and BK increased phosphorylation of MYPT‐1 and MLC20 and auto‐phosphorylation of SrcFK and FAK. MYPT‐1 phosphorylation was sensitive to inhibition of Rho‐kinase and SrcFK, but not FAK. Contraction induced by SR Ca2+ depletion and equivalent [Ca2+]i responses in hASMC were sensitive to inhibition of both SrcFK and FAK, while depolarization‐induced contraction was sensitive to FAK inhibition only. SrcFK auto‐phosphorylation was partially FAK‐dependent, while FAK auto‐phosphorylation was SrcFK‐independent. Conclusions and Implications SrcFK mediates Ca2+‐sensitization in ASM, while SrcFK and FAK together and individually influence multiple Ca2+ influx pathways. Tyrosine phosphorylation is therefore a key upstream signalling event in ASM contraction and may be a viable target for modulating ASM tone in respiratory disease. PMID:26294392

  17. Coupled AGC-Costas loops with AM/PM conversion

    NASA Astrophysics Data System (ADS)

    Gagliardi, R. M.

    1980-01-01

    Costas loops are invariably designed in conjunction with an automatic gain control (AGC) loop for stabilizing performance. In such systems an inherent coupling between the AGC and Costas loops develops, complicating the standard phase referencing analysis. This coupling is further emphasized if the gain control amplifier introduces an AM/PM conversion, which causes power variations to enter the Costas loop as phase variations. In this paper the coupling effect between AGC and Costas loops is developed, leading to a pair of joint, interconnected dynamical tracking loops. Some degree of solution is attainable by assuming a first order AGC loop, and resorting to quasi-stationary analysis for evaluating the phase referencing generation. Results with and without AM/PM are presented, and illustrate how an improper AGC may in fact degrade the phase referencing from the expected performance.

  18. New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression.

    PubMed

    Galinier, A; Kravanja, M; Engelmann, R; Hengstenberg, W; Kilhoffer, M C; Deutscher, J; Haiech, J

    1998-02-17

    Carbon catabolite repression (CCR) is the prototype of a signal transduction mechanism. In enteric bacteria, cAMP was considered to be the second messenger in CCR by playing a role reminiscent of its actions in eukaryotic cells. However, recent results suggest that CCR in Escherichia coli is mediated mainly by an inducer exclusion mechanism. In many Gram-positive bacteria, CCR is triggered by fructose-1,6-bisphosphate, which activates HPr kinase, presumed to be one of the most ancient serine protein kinases. We here report cloning of the Bacillus subtilis hprK and hprP genes and characterization of the encoded HPr kinase and P-Ser-HPr phosphatase. P-Ser-HPr phosphatase forms a new family of phosphatases together with bacterial phosphoglycolate phosphatase, yeast glycerol-3-phosphatase, and 2-deoxyglucose-6-phosphate phosphatase whereas HPr kinase represents a new family of protein kinases on its own. It does not contain the domain structure typical for eukaryotic protein kinases. Although up to now the HPr modifying/demodifying enzymes were thought to exist only in Gram-positive bacteria, a sequence comparison revealed that they also are present in several Gram-negative pathogenic bacteria. PMID:9465101

  19. The FGGY carbohydrate kinase family: insights into the evolution of functional specificities.

    PubMed

    Zhang, Ying; Zagnitko, Olga; Rodionova, Irina; Osterman, Andrei; Godzik, Adam

    2011-12-01

    Function diversification in large protein families is a major mechanism driving expansion of cellular networks, providing organisms with new metabolic capabilities and thus adding to their evolutionary success. However, our understanding of the evolutionary mechanisms of functional diversity in such families is very limited, which, among many other reasons, is due to the lack of functionally well-characterized sets of proteins. Here, using the FGGY carbohydrate kinase family as an example, we built a confidently annotated reference set (CARS) of proteins by propagating experimentally verified functional assignments to a limited number of homologous proteins that are supported by their genomic and functional contexts. Then, we analyzed, on both the phylogenetic and the molecular levels, the evolution of different functional specificities in this family. The results show that the different functions (substrate specificities) encoded by FGGY kinases have emerged only once in the evolutionary history following an apparently simple divergent evolutionary model. At the same time, on the molecular level, one isofunctional group (L-ribulokinase, AraB) evolved at least two independent solutions that employed distinct specificity-determining residues for the recognition of a same substrate (L-ribulose). Our analysis provides a detailed model of the evolution of the FGGY kinase family. It also shows that only combined molecular and phylogenetic approaches can help reconstruct a full picture of functional diversifications in such diverse families. PMID:22215998

  20. Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

    PubMed

    Chopra, Nikita; Wales, Thomas E; Joseph, Raji E; Boyken, Scott E; Engen, John R; Jernigan, Robert L; Andreotti, Amy H

    2016-03-01

    Bruton's tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site. PMID:27010561

  1. Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases

    PubMed Central

    Chopra, Nikita; Wales, Thomas E.; Joseph, Raji E.; Boyken, Scott E.; Engen, John R.; Jernigan, Robert L.; Andreotti, Amy H.

    2016-01-01

    Bruton’s tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site. PMID:27010561

  2. Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

    PubMed

    Rinaldi, Jimena; Arrar, Mehrnoosh; Sycz, Gabriela; Cerutti, María Laura; Berguer, Paula M; Paris, Gastón; Estrín, Darío Ariel; Martí, Marcelo Adrián; Klinke, Sebastián; Goldbaum, Fernando Alberto

    2016-03-27

    In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK. PMID:26851072

  3. A Novel Predicted Calcium-Regulated Kinase Family Implicated in Neurological Disorders

    PubMed Central

    Dudkiewicz, Małgorzata; Lenart, Anna; Pawłowski, Krzysztof

    2013-01-01

    The catalogues of protein kinases, the essential effectors of cellular signaling, have been charted in Metazoan genomes for a decade now. Yet, surprisingly, using bioinformatics tools, we predicted protein kinase structure for proteins coded by five related human genes and their Metazoan homologues, the FAM69 family. Analysis of three-dimensional structure models and conservation of the classic catalytic motifs of protein kinases present in four out of five human FAM69 proteins suggests they might have retained catalytic phosphotransferase activity. An EF-hand Ca2+-binding domain in FAM69A and FAM69B proteins, inserted within the structure of the kinase domain, suggests they may function as Ca2+-dependent kinases. The FAM69 genes, FAM69A, FAM69B, FAM69C, C3ORF58 (DIA1) and CXORF36 (DIA1R), are by large uncharacterised molecularly, yet linked to several neurological disorders in genetics studies. The C3ORF58 gene is found deleted in autism, and resides in the Golgi. Unusually high cysteine content and presence of signal peptides in some of the family members suggest that FAM69 proteins may be involved in phosphorylation of proteins in the secretory pathway and/or of extracellular proteins. PMID:23840464

  4. Src family kinases maintain the balance between replication stress and the replication checkpoint.

    PubMed

    Miura, Takahito; Fukumoto, Yasunori; Morii, Mariko; Honda, Takuya; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2016-01-01

    Progression of DNA replication is tightly controlled by replication checkpoints to ensure the accurate and rapid duplication of genetic information. Upon replication stress, the replication checkpoint slows global DNA replication by inhibiting the late-firing origins and by slowing replication fork progression. Activation of the replication checkpoint has been studied in depth; however, little is known about the termination of the replication checkpoint. Here, we show that Src family kinases promote the recovery from replication checkpoints. shRNA knockdown of a Src family kinase, Lyn, and acute chemical inhibition of Src kinases prevented inactivation of Chk1 after removal of replication stress. Consistently, Src inhibition slowed resumption of DNA replication, after the removal of replication blocks. The effect of Src inhibition was not observed in the presence of an ATM/ATR inhibitor caffeine. These data indicate that Src kinases promote the resumption of DNA replication by suppressing ATR-dependent replication checkpoints. Surprisingly, the resumption of replication was delayed by caffeine. In addition, Src inhibition delayed recovery from replication fork collapse. We propose that Src kinases maintain the balance between replication stress and the activity of the replication checkpoint. PMID:26194897

  5. A biosensor of SRC family kinase conformation by exposable tetracysteine useful for cell-based screening.

    PubMed

    Irtegun, Sevgi; Wood, Rebecca; Lackovic, Kurt; Schweiggert, Jörg; Ramdzan, Yasmin M; Huang, David C S; Mulhern, Terrence D; Hatters, Danny M

    2014-07-18

    We developed a new approach to distinguish distinct protein conformations in live cells. The method, exposable tetracysteine (XTC), involved placing an engineered tetracysteine motif into a target protein that has conditional access to biarsenical dye binding by conformational state. XTC was used to distinguish open and closed regulatory conformations of Src family kinases. Substituting just four residues with cysteines in the conserved SH2 domain of three Src-family kinases (c-Src, Lck, Lyn) enabled open and closed conformations to be monitored on the basis of binding differences to biarsenical dyes FlAsH or ReAsH. Fusion of the kinases with a fluorescent protein tracked the kinase presence, and the XTC approach enabled simultaneous assessment of regulatory state. The c-Src XTC biosensor was applied in a boutique screen of kinase inhibitors, which revealed six compounds to induce conformational closure. The XTC approach demonstrates new potential for assays targeting conformational changes in key proteins in disease and biology. PMID:24828008

  6. Completing the structural family portrait of the human EphB tyrosine kinase domains

    PubMed Central

    Overman, Ross C; Debreczeni, Judit E; Truman, Caroline M; McAlister, Mark S; Attwood, Teresa K

    2014-01-01

    The EphB receptors have key roles in cell morphology, adhesion, migration and invasion, and their aberrant action has been linked with the development and progression of many different tumor types. Their conflicting expression patterns in cancer tissues, combined with their high sequence and structural identity, present interesting challenges to those seeking to develop selective therapeutic molecules targeting this large receptor family. Here, we present the first structure of the EphB1 tyrosine kinase domain determined by X-ray crystallography to 2.5Å. Our comparative crystalisation analysis of the human EphB family kinases has also yielded new crystal forms of the human EphB2 and EphB4 catalytic domains. Unable to crystallize the wild-type EphB3 kinase domain, we used rational engineering (based on our new structures of EphB1, EphB2, and EphB4) to identify a single point mutation which facilitated its crystallization and structure determination to 2.2 Å. This mutation also improved the soluble recombinant yield of this kinase within Escherichia coli, and increased both its intrinsic stability and catalytic turnover, without affecting its ligand-binding profile. The partial ordering of the activation loop in the EphB3 structure alludes to a potential cis-phosphorylation mechanism for the EphB kinases. With the kinase domain structures of all four catalytically competent human EphB receptors now determined, a picture begins to emerge of possible opportunities to produce EphB isozyme-selective kinase inhibitors for mechanistic studies and therapeutic applications. PMID:24677421

  7. Influences to System and Superiority of Model Parameters in Dynamic Setting AGC

    NASA Astrophysics Data System (ADS)

    Xu, LI; Hao-yu, Zhang; Jin, Zhang; Jian-fei, Guo; Cui-hong, Liu; Ping-wen, Cheng

    Dynamic setting AGC is based on the improvement of the BISRA-AGC model. Based on the control algorithms and control models of dynamic setting AGC, the influences of the model parameters to system performance were analyzed using the GUI in MATLAB. At the same time, the superiority and limitation of the dynamic setting AGC were analyzed.

  8. The oncogenic PIM kinase family regulates drug resistance through multiple mechanisms.

    PubMed

    Isaac, Methvin; Siu, Allan; Jongstra, Jan

    2011-01-01

    Resistance to chemotherapeutic drugs is a significant clinical problem for the treatment of cancer patients and has been linked to the activation of survival pathways and expression of multidrug efflux transporters. Thus inhibition of these survival pathways or efflux transporter expression may increase the efficacy of drug treatment. Here we review the role of the oncogenic PIM kinase family in regulating important proliferation and survival pathways in cancer cells and the involvement of PIM kinases in the expression and activity of MDR-1 and BCRP, two of the most important drug efflux transporters. PIM kinases are over expressed in various types of tumors and regulate the activation of signaling pathways that are important for tumor cell proliferation, survival and expression of drug efflux proteins. This makes PIM kinases attractive targets for the development of anti-cancer chemotherapeutic drugs. Focussing mainly on solid tumors, we provide an update on the literature describing the tumorigenic functions of PIM kinases. Also we provide an overview of the development of selective small molecule PIM kinase inhibitors. Because of the intense effort by pharmaceutical companies and academia it is reasonable to expect that PIM kinase inhibitors will enter the clinic in the foreseeable future. We therefore finish this review with a discussion on the most efficient application of these PIM inhibitors. This includes a consideration of which tumor type is the most appropriate target for treatment, how to select the patient population that stands to gain the most from treatment with PIM inhibitors, which molecular markers are suitable to follow the course of treatment and whether PIM kinase inhibitors should be used as monotherapy or in combination with other cytotoxic agents. PMID:21601509

  9. Membrane-bound Dictyostelium myosin heavy chain kinase: a developmentally regulated substrate-specific member of the protein kinase C family.

    PubMed Central

    Ravid, S; Spudich, J A

    1992-01-01

    A cDNA clone corresponding to the Dictyostelium myosin heavy chain kinase (MHCK) gene was isolated using antibodies specific to the purified enzyme. Sequence analysis of the cDNA revealed that the Dictyostelium MHCK possesses all of the domains characteristic of members of the protein kinase C family. The amino-terminal region of the MHCK contains the cysteine-rich motif with an internal duplication that is present in all known protein kinase C species. This domain precedes sequences that are highly homologous to protein kinase catalytic domains. The carboxyl-terminal region contains a cluster of 23 serine and threonine residues that may represent the autophosphorylation domain of the Dictyostelium MHCK. These results, along with previous studies that indicate that this enzyme has very restrictive substrate specificity, incorporates approximately 20 mol of phosphate per mol of kinase through an autophosphorylation reaction, and is expressed only during development, suggest that the Dictyostelium MHCK is a distinct member of the protein kinase C family and imply that this kinase family, which may include members with very specific cellular functions, may be even more heterogeneous than previously thought. Images PMID:1321427

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

    PubMed

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

    2016-08-01

    Understanding the conformational propensities of proteins is key to solving many problems in structural biology and biophysics. The co-variation of pairs of mutations contained in multiple sequence alignments of protein families can be used to build a Potts Hamiltonian model of the sequence patterns which accurately predicts structural contacts. This observation paves the way to develop deeper connections between evolutionary fitness landscapes of entire protein families and the corresponding free energy landscapes which determine the conformational propensities of individual proteins. Using statistical energies determined from the Potts model and an alignment of 2896 PDB structures, we predict the propensity for particular kinase family proteins to assume a "DFG-out" conformation implicated in the susceptibility of some kinases to type-II inhibitors, and validate the predictions by comparison with the observed structural propensities of the corresponding proteins and experimental binding affinity data. We decompose the statistical energies to investigate which interactions contribute the most to the conformational preference for particular sequences and the corresponding proteins. We find that interactions involving the activation loop and the C-helix and HRD motif are primarily responsible for stabilizing the DFG-in state. This work illustrates how structural free energy landscapes and fitness landscapes of proteins can be used in an integrated way, and in the context of kinase family proteins, can potentially impact therapeutic design strategies. PMID:27241634

  11. An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity*

    PubMed Central

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

    2013-01-01

    Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate. PMID:23737529

  12. AGC-2 Graphite Pre-irradiation Data Package

    SciTech Connect

    David Swank; Joseph Lord; David Rohrbaugh; William Windes

    2010-08-01

    The NGNP Graphite R&D program is currently establishing the safe operating envelope of graphite core components for a Very High Temperature Reactor (VHTR) design. The program is generating quantitative data necessary for predicting the behavior and operating performance of the new nuclear graphite grades. To determine the in-service behavior of the graphite for pebble bed and prismatic designs, the Advanced Graphite Creep (AGC) experiment is underway. This experiment is examining the properties and behavior of nuclear grade graphite over a large spectrum of temperatures, neutron fluences and compressive loads. Each experiment consists of over 400 graphite specimens that are characterized prior to irradiation and following irradiation. Six experiments are planned with the first, AGC-1, currently being irradiated in the Advanced Test Reactor (ATR) and pre-irradiation characterization of the second, AGC-2, completed. This data package establishes the readiness of 512 specimens for assembly into the AGC-2 capsule.

  13. Regulation of osteoclast structure and function by FAK family kinases

    PubMed Central

    Ray, Brianne J.; Thomas, Keena; Huang, Cynthia S.; Gutknecht, Michael F.; Botchwey, Edward A.; Bouton, Amy H.

    2012-01-01

    Osteoclasts are highly specialized cells that resorb bone and contribute to bone remodeling. Diseases such as osteoporosis and osteolytic bone metastasis occur when osteoclast-mediated bone resorption takes place in the absence of concurrent bone synthesis. Considerable effort has been placed on identifying molecules that regulate the bone resorption activity of osteoclasts. To this end, we investigated unique and overlapping functions of members of the FAK family (FAK and Pyk2) in osteoclast functions. With the use of a conditional knockout mouse model, in which FAK is selectively targeted for deletion in osteoclast precursors (FAKΔmyeloid), we found that loss of FAK resulted in reduced bone resorption by osteoclasts in vitro, coincident with impaired signaling through the CSF-1R. However, bone architecture appeared normal in FAKΔmyeloid mice, suggesting that Pyk2 might functionally compensate for reduced FAK levels in vivo. This was supported by data showing that podosome adhesion structures, which are essential for bone degradation, were significantly more impaired in osteoclasts when FAK and Pyk2 were reduced than when either molecule was depleted individually. We conclude that FAK contributes to cytokine signaling and bone resorption in osteoclasts and partially compensates for the absence of Pyk2 to maintain proper adhesion structures in these cells. PMID:22941736

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

    PubMed

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

    2014-12-01

    The TYRO3, AXL (also known as UFO) and MERTK (TAM) family of receptor tyrosine kinases (RTKs) are aberrantly expressed in multiple haematological and epithelial malignancies. Rather than functioning as oncogenic drivers, their induction in tumour cells predominately promotes survival, chemoresistance and motility. The unique mode of maximal activation of this RTK family requires an extracellular lipid–protein complex. For example, the protein ligand, growth arrest-specific protein 6 (GAS6), binds to phosphatidylserine (PtdSer) that is externalized on apoptotic cell membranes, which activates MERTK on macrophages. This triggers engulfment of apoptotic material and subsequent anti-inflammatory macrophage polarization. In tumours, autocrine and paracrine ligands and apoptotic cells are abundant, which provide a survival signal to the tumour cell and favour an anti-inflammatory, immunosuppressive microenvironment. Thus, TAM kinase inhibition could stimulate antitumour immunity, reduce tumour cell survival, enhance chemosensitivity and diminish metastatic potential. PMID:25568918

  15. Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors.

    PubMed

    Daulhac, L; Kowalski-Chauvel, A; Pradayrol, L; Vaysse, N; Seva, C

    1999-07-16

    We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc.Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src. IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin. PMID:10400698

  16. Structural Characterization of the Predominant Family of Histidine Kinase Sensor Domains

    SciTech Connect

    Zhang, Z.; Hendrickson, W

    2010-01-01

    Histidine kinase (HK) receptors are used ubiquitously by bacteria to monitor environmental changes, and they are also prevalent in plants, fungi, and other protists. Typical HK receptors have an extracellular sensor portion that detects a signal, usually a chemical ligand, and an intracellular transmitter portion that includes both the kinase domain itself and the site for histidine phosphorylation. While kinase domains are highly conserved, sensor domains are diverse. HK receptors function as dimers, but the molecular mechanism for signal transduction across cell membranes remains obscure. In this study, eight crystal structures were determined from five sensor domains representative of the most populated family, family HK1, found in a bioinformatic analysis of predicted sensor domains from transmembrane HKs. Each structure contains an inserted repeat of PhoQ/DcuS/CitA (PDC) domains, and similarity between sequence and structure is correlated across these and other double-PDC sensor proteins. Three of the five sensors crystallize as dimers that appear to be physiologically relevant, and comparisons between ligated structures and apo-state structures provide insights into signal transmission. Some HK1 family proteins prove to be sensors for chemotaxis proteins or diguanylate cyclase receptors, implying a combinatorial molecular evolution.

  17. Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification

    SciTech Connect

    Kamb, A.; Weir, M.; Rudy, B.; Varmus, H.; Kenyon, C. )

    1989-06-01

    The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. The authors have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, they have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, they have used this method to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, they have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.

  18. Differential Function of Arabidopsis SERK Family Receptor-like Kinases in Stomatal Patterning.

    PubMed

    Meng, Xiangzong; Chen, Xin; Mang, Hyunggon; Liu, Chenglong; Yu, Xiao; Gao, Xiquan; Torii, Keiko U; He, Ping; Shan, Libo

    2015-09-21

    Plants use cell-surface-resident receptor-like kinases (RLKs) to sense diverse extrinsic and intrinsic cues and elicit distinct biological responses. In Arabidopsis, ERECTA family RLKs recognize EPIDERMAL PATTERNING FACTORS (EPFs) to specify stomatal patterning. However, little is known about the molecular link between ERECTA activation and intracellular signaling. We report here that the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family RLKs regulate stomatal patterning downstream of EPF ligands and upstream of a MAP kinase cascade. EPF ligands induce the heteromerization of ERECTA and SERK family RLKs. SERK and ERECTA family RLKs transphosphorylate each other. In addition, SERKs associate with the receptor-like protein (RLP) TMM, a signal modulator of stomata development, in a ligand-independent manner, suggesting that ERECTA, SERKs, and TMM form a multiprotein receptorsome consisting of different RLKs and RLP perceiving peptide ligands to regulate stomatal patterning. In contrast to the differential requirement of individual SERK members in plant immunity, cell-death control, and brassinosteroid (BR) signaling, all four functional SERKs are essential but have unequal genetic contributions to stomatal patterning, with descending order of importance from SERK3/BAK1 to SERK2 to SERK1 to SERK4. Although BR signaling connects stomatal development via multiple components, the function of SERKs in stomatal patterning is uncoupled from their involvement in BR signaling. Our results reveal that the SERK family is a shared key module in diverse Arabidopsis signaling receptorsomes and that different combinatorial codes of individual SERK members regulate distinct functions. PMID:26320950

  19. HIPK family kinases bind and regulate the function of the CCR4-NOT complex.

    PubMed

    Rodriguez-Gil, Alfonso; Ritter, Olesja; Hornung, Juliane; Stekman, Hilda; Krüger, Marcus; Braun, Thomas; Kremmer, Elisabeth; Kracht, Michael; Schmitz, M Lienhard

    2016-06-15

    The serine/threonine kinase HIPK2 functions as a regulator of developmental processes and as a signal integrator of a wide variety of stress signals, such as DNA damage, hypoxia, and reactive oxygen intermediates. Because the kinase is generated in a constitutively active form, its expression levels are restricted by a variety of different mechanisms. Here we identify the CCR4-NOT complex as a new regulator of HIPK2 abundance. Down-regulation or knockout of the CCR4-NOT complex member CNOT2 leads to reduced HIPK2 protein levels without affecting the expression level of HIPK1 or HIPK3. A fraction of all HIPK family members associates with the CCR4-NOT components CNOT2 and CNOT3. HIPKs also phosphorylate the CCR4-NOT complex, a feature that is shared with their yeast progenitor kinase, YAK1. Functional assays reveal that HIPK2 and HIPK1 restrict CNOT2-dependent mRNA decay. HIPKs are well known regulators of transcription, but the mutual regulation between CCR4-NOT and HIPKs extends the regulatory potential of these kinases by enabling posttranscriptional gene regulation. PMID:27122605

  20. Actin cytoskeleton organization regulated by the PAK family of protein kinases.

    PubMed

    Eby, J J; Holly, S P; van Drogen, F; Grishin, A V; Peter, M; Drubin, D G; Blumer, K J

    1998-08-27

    Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton. PMID:9742399

  1. Structural Recognition of an Optimized Substrate for the Ephrin family of Receptor Tyrosine Kinases

    PubMed Central

    Davis, Tara L.; Walker, John R.; Allali-Hassani, Abdellah; Parker, Sirlester A.; Turk, Benjamin E.; Dhe-Paganon, Sirano

    2010-01-01

    Summary Ephrin receptor tyrosine kinase A3 (EphA3, EC 2.7.10.1) is a member of a unique branch of the kinome in which downstream signaling occurs in both ligand- and receptor- expressing cells. Consequently the ephrins and ephrin RTKs often mediate processes involving cell:cell contact, including cellular adhesion or repulsion, developmental remodeling, and neuronal mapping. The receptor is also frequently overexpressed in invasive cancers, including breast, small-cell lung and gastrointestinal cancers. However, little is known about direct substrates of EphA3 kinase and no chemical probes are available. Using a library approach, we found a short peptide sequence that is a good substrate for EphA3 and that is suitable for cocrystallization studies. Complex structures show multiple contacts between kinase and substrates, and in particular two residues undergo conformational changes and by mutation are found to be important for substrate binding and turnover. In addition, a difference in catalytic efficiency between EPH kinase family members is observed. These results provide insight into the mechanism of substrate binding to these developmentally integral enzymes. PMID:19678838

  2. HIPK family kinases bind and regulate the function of the CCR4-NOT complex

    PubMed Central

    Rodriguez-Gil, Alfonso; Ritter, Olesja; Hornung, Juliane; Stekman, Hilda; Krüger, Marcus; Braun, Thomas; Kremmer, Elisabeth; Kracht, Michael; Schmitz, M. Lienhard

    2016-01-01

    The serine/threonine kinase HIPK2 functions as a regulator of developmental processes and as a signal integrator of a wide variety of stress signals, such as DNA damage, hypoxia, and reactive oxygen intermediates. Because the kinase is generated in a constitutively active form, its expression levels are restricted by a variety of different mechanisms. Here we identify the CCR4-NOT complex as a new regulator of HIPK2 abundance. Down-regulation or knockout of the CCR4-NOT complex member CNOT2 leads to reduced HIPK2 protein levels without affecting the expression level of HIPK1 or HIPK3. A fraction of all HIPK family members associates with the CCR4-NOT components CNOT2 and CNOT3. HIPKs also phosphorylate the CCR4-NOT complex, a feature that is shared with their yeast progenitor kinase, YAK1. Functional assays reveal that HIPK2 and HIPK1 restrict CNOT2-dependent mRNA decay. HIPKs are well known regulators of transcription, but the mutual regulation between CCR4-NOT and HIPKs extends the regulatory potential of these kinases by enabling posttranscriptional gene regulation. PMID:27122605

  3. Human immunodeficiency virus type 1 Nef associates with a member of the p21-activated kinase family.

    PubMed Central

    Nunn, M F; Marsh, J W

    1996-01-01

    Although human immunodeficiency virus (HIV) Nef is essential for the induction of AIDS, its biochemical function has remained an enigma. In this study, HIV Nef protein is shown to associate with a serine-threonine kinase that recognizes histone H4 as a substrate, is serologically related to rat p21-activated kinase (PAK), and is specifically activated by Rac and Cdc42. These characteristics define the Nef-associated kinase as belonging to the PAK family. PAKs initiate kinase cascades in response to environmental stimuli, and their identification as a target of Nef implicates these signaling molecules in HIV pathogenesis and provides a novel target for clinical intervention. PMID:8709241

  4. Endocytosis and the Src family of non-receptor tyrosine kinases

    PubMed Central

    Reinecke, James; Caplan, Steve

    2014-01-01

    The regulated intracellular transport of nutrient, adhesion, and growth factor receptors is crucial for maintaining cell and tissue homeostasis. Endocytosis, or endocytic membrane trafficking, involves the steps of intracellular transport that include, but are not limited to: internalization from the plasma membrane, sorting in early endosomes, transport to late endosomes/lysosomes followed by degradation, and/or recycling back to the plasma membrane via tubular recycling endosomes. In addition to regulating the localization of transmembrane receptor proteins, the endocytic pathway also controls the localization of non-receptor molecules. The non-receptor tyrosine kinase c-Src (Src), and its closely related family members Yes and Fyn, represent three proteins whose localization and signaling activities are tightly regulated by endocytic trafficking. Here, we provide a brief overview of endocytosis, Src function and its biochemical regulation. We will then concentrate on recent advances in understanding how Src intracellular localization is regulated and how its subcellular localization ultimately dictates downstream functioning. Since Src kinases are hyperactive in many cancers, it is essential to decipher the spatiotemporal regulation of this important family of tyrosine kinases. PMID:25372749

  5. Structure of acetylglutamate kinase, a key enzyme for arginine biosynthesis and a prototype for the amino acid kinase enzyme family, during catalysis.

    PubMed

    Ramón-Maiques, Santiago; Marina, Alberto; Gil-Ortiz, Fernando; Fita, Ignacio; Rubio, Vicente

    2002-03-01

    N-Acetyl-L-glutamate kinase (NAGK), a member of the amino acid kinase family, catalyzes the second and frequently controlling step of arginine synthesis. The Escherichia coli NAGK crystal structure to 1.5 A resolution reveals a 258-residue subunit homodimer nucleated by a central 16-stranded molecular open beta sheet sandwiched between alpha helices. In each subunit, AMPPNP, as an alphabetagamma-phosphate-Mg2+ complex, binds along the sheet C edge, and N-acetyl-L-glutamate binds near the dyadic axis with its gamma-COO- aligned at short distance from the gamma-phosphoryl, indicating associative phosphoryl transfer assisted by: (1) Mg2+ complexation; (2) the positive charges on Lys8, Lys217, and on two helix dipoles; and (3) by hydrogen bonding with the y-phosphate. The structural resemblance with carbamate kinase and the alignment of the sequences suggest that NAGK is a structural and functional prototype for the amino acid kinase family, which differs from other acylphosphate-making devices represented by phosphoglycerate kinase, acetate kinase, and biotin carboxylase. PMID:12005432

  6. AGC198606: A gas-bearing dark matter minihalo?

    NASA Astrophysics Data System (ADS)

    Adams, E. A. K.; Faerman, Y.; Janesh, W. F.; Janowiecki, S.; Oosterloo, T. A.; Rhode, K. L.; Giovanelli, R.; Haynes, M. P.; Salzer, J. J.; Sternberg, A.; Cannon, J. M.; Muñoz, R. R.

    2015-01-01

    We present neutral hydrogen (H i) imaging observations with the Westerbork Synthesis Radio Telescope of AGC198606, an H i cloud discovered in the ALFALFA 21cm survey. This object is of particular note as it is located 16 km s-1 and 1.̊2 from the gas-bearing ultra-faint dwarf galaxy Leo T while having a similar H i linewidth and approximately twice the flux density. The H i imaging observations reveal a smooth, undisturbed H i morphology with a full extent of 23' × 16' at the 5 × 1018 atoms cm-2 level. The velocity field of AGC198606 shows ordered motion with a gradient of ~25 km s-1 across ~20'. The global velocity dispersion is 9.3 km s-1 with no evidence for a narrow spectral component. No optical counterpart to AGC198606 is detected. The distance to AGC198606 is unknown, and we consider several different scenarios: physical association with Leo T, a minihalo at a distance of ~150 kpc based on the models of Faerman et al. (2013, ApJ, 777, 119), and a cloud in the Galactic halo. At a distance of 420 kpc, AGC198606 would have an H i mass of 6.2 × 105 M⊙, an H i radius of 1.4 kpc, and a dynamical mass within the H i extent of 1.5 × 108 M⊙.

  7. 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

  8. Deletion of Src family kinase Lyn aggravates endotoxin-induced lung inflammation.

    PubMed

    Gao, Rong; Ma, Zhongsen; Ma, Mengshi; Yu, Jinyan; Chen, Jiao; Li, Zhenyu; Shetty, Sreerama; Fu, Jian

    2015-12-01

    Overwhelming acute inflammation often leads to tissue damage during endotoxemia. In the present study, we investigated the role of Lyn, a member of the Src family tyrosine kinases, in modulating inflammatory responses in a murine model of endotoxemia. We examined lung inflammatory signaling in Lyn knockout (Lyn(-/-)) mice and wild-type littermates (Lyn(+/+)) during endotoxemia. Our data indicate that Lyn deletion aggravates endotoxin-induced pulmonary inflammation and proinflammatory signaling. We found increased activation of proinflammatory transcription factor NF-κB in the lung tissues of Lyn(-/-) mice after endotoxin challenge. Furthermore, during endotoxemia, the lung tissues of Lyn(-/-) mice showed increased inflammasome activation indicated by augmented caspase-1 and IL-1β cleavage and activation. The aggravated lung inflammatory signaling in Lyn(-/-) mice was associated with increased production of proinflammatory mediators and elevated matrix metallopeptidase 9 and reduced VE-cadherin levels. Our results suggest that Lyn kinase modulates inhibitory signaling to suppress endotoxin-induced lung inflammation. PMID:26453518

  9. Expression and Purification of Src-family Kinases for Solution NMR Studies

    PubMed Central

    Piserchio, Andrea; Cowburn, David; Ghose, Ranajeet

    2012-01-01

    Summary NMR analyses of the structure, dynamics and interactions of the Src family kinases (SFKs) have been hindered by the limited ability to obtain sufficient amounts of properly folded, soluble protein from bacterial expression systems, to allow these studies to be performed in an economically viable manner. In this chapter we detail our attempts to overcome these difficulties using the catalytic domain (SrcCD) of c-Src, the prototypical SFK, as an illustrative example. We describe in detail two general methods to express and purify SrcCD from E. coli expression systems in both fully active wild-type and kinase-deficient mutant forms, allowing the efficient and cost-effective labeling by NMR-active isotopes for solution NMR studies. PMID:22167671

  10. AGC-3 IRRADIATION DATA QUALIFICATION FINAL REPORT

    SciTech Connect

    Hull, Laurence

    2014-08-01

    All thermocouples functioned throughout the AGC-3 experiment. There was one interval between Dec 18, 2012 and Dec 20, 2012 where 10 NULL values were reported for various thermocouples. These NULL values were deleted from the database. All temperature data are Qualified. During Oct and Nov 2013, 135,849 helium and argon gas flow values were below 0. These negative gas flow values are not correct and are Failed. The remaining argon, helium, and total gas flow data are within expected ranges and are Qualified. Total gas flow was approximately 50 sccm through the capsule. Helium gas flow was briefly increased to 100 sccm during reactor shutdowns. At the start of the experiment, moisture in the outflow gas line was stuck at a constant value of 335.6174 ppmv for the first cycle. When the capsule was reinstalled in the reactor for Cycle 154B, a new moisture filter was installed. Moisture data from Cycle 152B are Failed. All moisture data from the final three cycles are Qualified. Graphite creep specimens were subjected to one of two loads, 393 lbf or 589 lbf. The experiment plan included three loads, but problems with gas leaks in the rams applying the load to the stacks resulted in lower loads being applied to some of the stacks. While the loads applied are not the loads in the plan, the loads were consistently applied throughout the experiment. Therefore the reported loads are accurate and can be used in analysis of graphite creep. Loads were consistently within 5% of the specified values throughout the experiment. Stack displacement increased consistently throughout the experiment with total displacement ranging from 1 to 1.25 inches. No anomalous values were identified. During reactor outages, a set of pneumatic rams was used to raise the stacks of graphite creep specimens to ensure the specimens had not become stuck within the test train. This stack raising was performed three times; all stacks were raised successfully each time. The load and displacement data are

  11. AGC-3 Experiment Irradiation Monitoring Data Qualification Final Report

    SciTech Connect

    Laurence Hull

    2014-10-01

    The Graphite Technology Development Program will run a series of six experiments to quantify the effects of irradiation on nuclear grade graphite. The third experiment, Advanced Graphite Creep 3 (AGC 3), began with Advanced Test Reactor (ATR) Cycle 152B on November 27, 2012, and ended with ATR Cycle 155B on April 23, 2014. This report documents qualification of AGC 3 experiment irradiation monitoring data for use by the Very High Temperature Reactor (VHTR) Technology Development Office (TDO) Program for research and development activities required to design and license the first VHTR nuclear plant. Qualified data meet the requirements for data collection and use as described in the experiment planning and quality assurance documents. Failed data do not meet the requirements. Trend data may not meet the requirements, but may still provide some useable information. All thermocouples (TCs) functioned throughout the AGC 3 experiment. There was one interval between December 18, 2012, and December 20, 2012, where 10 NULL values were reported for various TCs. These NULL values were deleted from the Nuclear Data Management and Analysis System database. All temperature data are Qualified for use by the VHTR TDO Program. Argon, helium, and total gas flow data were within expected ranges and are Qualified for use by the VHTR TDO Program. Total gas flow was approximately 50 sccm through the AGC 3 experiment capsule. Helium gas flow was briefly increased to 100 sccm during ATR shutdowns. At the start of the AGC 3 experiment, moisture in the outflow gas line was stuck at a constant value of 335.6174 ppmv for the first cycle (Cycle 152B). When the AGC 3 experiment capsule was reinstalled in ATR for Cycle 154B, a new moisture filter was installed. Moisture data from Cycle 152B are Failed. All moisture data from the final three cycles (Cycles 154B, 155A, and 155B) are Qualified for use by the VHTR TDO Program.

  12. Simulation and Analyzing on Model Parameters Effect of BISRA-AGC

    NASA Astrophysics Data System (ADS)

    Xu, Li; Shu-zong, Chen; De-shun, Du; Hua-xin, Chen; Dian-hua, Zhang

    According to the control principle of pressure AGC, the control thought and control model of BISRA-AGC were analyzed. By changing the external disturbance of rolling force and sampling time, the BISRA-AGC and Dynamic Set AGC were simulated under the conditions that the position control system was ideal by using GUI. Then, the advantages and disadvantages were analyzed combining the results of simulation, and the intrinsic factors were quantitatively analyzed.

  13. Changes in Dynamics upon Oligomerization Regulate Substrate Binding and Allostery in Amino Acid Kinase Family Members

    PubMed Central

    Marcos, Enrique; Crehuet, Ramon; Bahar, Ivet

    2011-01-01

    Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions) favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities. PMID:21980279

  14. Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab.

    PubMed

    Wheeler, Deric L; Iida, Mari; Kruser, Tim J; Nechrebecki, Meghan M; Dunn, Emily F; Armstrong, Eric A; Huang, Shyhmin; Harari, Paul M

    2009-04-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a major role in oncogenesis. Cetuximab is an EGFR-blocking antibody that is FDA approved for use in patients with metastatic colorectal cancer (mCRC) and head and neck squamous cell carcinoma (HNSCC). Although cetuximab has shown strong clinical benefit for a subset of cancer patients, most become refractory to cetuximab therapy. We reported that cetuximab-resistant NSCLC line NCI-H226 cells have increased steady-state expression and activity of EGFR secondary to altered trafficking/degradation and this increase in EGFR expression and activity lead to hyper-activation of HER3 and down stream signals to survival. We now present data that Src family kinases (SFKs) are highly activated in cetuximab-resistant cells and enhance EGFR activation of HER3 and PI(3)K/Akt. Studies using the Src kinase inhibitor dasatinib decreased HER3 and PI(3)K/Akt activity. In addition, cetuximab-resistant cells were resensitized to cetuximab when treated with dasatinib. These results indicate that SFKs and EGFR cooperate in acquired resistance to cetuximab and suggest a rationale for clinical strategies that investigate combinatorial therapy directed at both the EGFR and SFKs in patients with acquired resistance to cetuximab. PMID:19276677

  15. Force Sensing by Extension of the Src Family Kinase Substrate, p130Cas

    PubMed Central

    Sawada, Yasuhiro; Tamada, Masako; Dubin-Thaler, Benjamin J.; Cherniavskaya, Oksana; Sakai, Ryuichi; Tanaka, Sakae; Sheetz, Michael P.

    2009-01-01

    SUMMARY Physical force is implicated in many cell functions. However, the molecular mechanisms of force sensing are poorly understood. Here, we show that mechanical extension of p130Cas (Cas) in vitro and in vivo causes phosphorylation by Src family kinases with no apparent change in Src kinase activity and that Cas phosphorylation is involved in stretch-dependent activation of the small GTPase, Rap1. In vitro, we mechanically extended bacterially expressed Cas substrate domain (CasSD) and found a remarkable enhancement of phosphorylation by specific kinases. Using an antibody that recognized extended CasSD in vitro, Cas extension in intact cells was observed in the peripheral regions of spreading cells where higher traction forces are expected and phosphorylated Cas was detected, suggesting that the in vitro extension and phosphorylation of CasSD is relevant to physiological force transduction. Thus, Cas acts as a primary force-sensor through extension of the substrate domain, which primes it for phosphorylation. PMID:17129785

  16. Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

    SciTech Connect

    Takahashi, Akinori; Obata, Yuuki; Fukumoto, Yasunori; Nakayama, Yuji; Kasahara, Kousuke; Kuga, Takahisa; Higashiyama, Yukihiro; Saito, Takashi; Yokoyama, Kazunari K.; Yamaguchi, Naoto

    2009-04-15

    Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G{sub 1} phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

  17. The PIM family of oncoproteins: Small kinases with huge implications in myeloid leukemogenesis and as therapeutic targets

    PubMed Central

    Shah, Parag P.; Mims, Alice S.; Lockwood, William W.; Kraft, Andrew S.; Beverly, Levi J.

    2014-01-01

    PIM kinases are a family of serine/threonine kinases involved in cell survival and proliferation. There is significant structural similarity between the three PIM kinases (PIM1, PIM2 and PIM3) and few amino acid differences. Although, several studies have specifically monitored the role of PIM1 in tumorigenesis, much less is known about PIM2 and PIM3. Therefore, in this study we have used in vitro cell culture models and in vivo bone marrow infection/transplantation to assess the comparative signaling and oncogenic potential of each of the three PIM kinases. All three PIM kinases were able to protect FL5.12 cells from IL-3 withdrawal induced death. Interestingly, the downstream signaling cascades were indistinguishable between the three kinases. Transplantation of murine bone marrow co-expressing MYC and PIM1, PIM2 or PIM3 caused rapid and uniformly lethal myeloid leukemia. De-induction of MYC 18 days following transplantation significantly increased the survival of mice, even with continual expression of PIM kinases. Alternatively, mice treated at the pre-leukemic stage with a PIM kinase inhibitor increased the lifespan of the mice, even with continual expression of the MYC transgene. These data demonstrate the role of PIM kinases in driving myeloid leukemia, and as candidate molecules for therapy against human malignancies. PMID:25238262

  18. Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.

    PubMed

    Dwyer, Amy R; Mouchemore, Kellie A; Steer, James H; Sunderland, Andrew J; Sampaio, Natalia G; Greenland, Eloise L; Joyce, David A; Pixley, Fiona J

    2016-07-01

    A major role of colony-stimulating factor-1 is to stimulate the differentiation of mononuclear phagocytic lineage cells into adherent, motile, mature macrophages. The colony-stimulating factor-1 receptor transduces colony-stimulating factor-1 signaling, and we have shown previously that phosphatidylinositol 3-kinase p110δ is a critical mediator of colony-stimulating factor-1-stimulated motility through the colony-stimulating factor-1 receptor pY721 motif. Src family kinases are also implicated in the regulation of macrophage motility and in colony-stimulating factor-1 receptor signaling, although functional redundancy of the multiple SFKs expressed in macrophages makes it challenging to delineate their specific functions. We report a comprehensive analysis of individual Src family kinase expression in macrophage cell lines and primary macrophages and demonstrate colony-stimulating factor-1-induced changes in Src family kinase subcellular localization, which provides clues to their distinct and redundant functions in macrophages. Moreover, expression of individual Src family kinases is both species specific and dependent on colony-stimulating factor-1-induced macrophage differentiation. Hck associated with the activated colony-stimulating factor-1 receptor, whereas Lyn associated with the receptor in a constitutive manner. Consistent with this, inhibitor studies revealed that Src family kinases were important for both colony-stimulating factor-1 receptor activation and colony-stimulating factor-1-induced macrophage spreading, motility, and invasion. Distinct colony-stimulating factor-1-induced changes in the subcellular localization of individual SFKs suggest specific roles for these Src family kinases in the macrophage response to colony-stimulating factor-1. PMID:26747837

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

    PubMed

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

    2016-04-01

    The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra- and intermolecular binding assays of proteins containing the domains. PMID:26808198

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

    PubMed Central

    Emaduddin, Muhammad; Edelmann, Mariola J; Kessler, Benedikt M; Feller, Stephan M

    2008-01-01

    Background Src family kinases (SFK) are implicated in the development of some colorectal cancers (CRC). One SFK member, Lck, is not detectable in normal colonic epithelium, but becomes aberrantly expressed in a subset of CRCs. Although SFK have been extensively studied in fibroblasts and different types of immune cells, their physical and functional targets in many epithelial cancers remain poorly characterised. Results 64 CRC cell lines were tested for expression of Lck. SW620 CRC cells, which express high levels of Lck and also contain high basal levels of tyrosine phosphorylated (pY) proteins, were then analysed to identify novel SFK targets. Since SH2 domains of SFK are known to often bind substrates after phosphorylation by the kinase domain, the LckSH2 was compared with 14 other SH2s for suitability as affinity chromatography reagent. Mass spectrometric analyses of LckSH2-purified pY proteins subsequently identified several proteins readily known as SFK kinase substrates, including cortactin, Tom1L1 (SRCASM), GIT1, vimentin and AFAP1L2 (XB130). Additional proteins previously reported as substrates of other tyrosine kinase were also detected, including the EGF and PDGF receptor target Odin. Odin was further analysed and found to contain substantially less pY upon inhibition of SFK activity in SW620 cells, indicating that it is a formerly unknown SFK target in CRC cells. Conclusion Rapid identification of known and novel SFK targets in CRC cells is feasible with SH2 domain affinity chromatography. The elucidation of new SFK targets like Odin in epithelial cancer cells is expected to lead to novel insight into cancer cell signalling mechanisms and may also serve to indicate new biomarkers for monitoring tumor cell responses to drug treatments. PMID:18844995

  1. Identification of Aminoimidazole and Aminothiazole Derivatives as Src Family Kinase Inhibitors.

    PubMed

    Francini, Cinzia Maria; Fallacara, Anna Lucia; Artusi, Roberto; Mennuni, Laura; Calgani, Alessia; Angelucci, Adriano; Schenone, Silvia; Botta, Maurizio

    2015-12-01

    Src family kinases (SFKs) are a family of non-receptor tyrosine kinases (TKs) implicated in the regulation of many cellular processes. The aberrant activity of these TKs has been associated with the growth and progression of cancer. In particular, c-Src is overexpressed or hyperactivated in a variety of solid tumors and is most likely a strong promoting factor for the development of metastasis. Herein, the synthesis of new 4-aminoimidazole and 2-aminothiazole derivatives and their in vitro biological evaluation are described for their potential use as SFK inhibitors. Initially, 2-aminothiazole analogues of dasatinib and 4-aminoimidazole derivatives were synthesized and tested against the SFKs Src, Fyn, Lyn, and Yes. Five hits were identified as the most promising compounds, with Ki values in the range of 90-480 nm. A combination of molecular docking, homology modeling, and molecular dynamics were then used to investigate the possible binding mode of such compounds within the ATP binding site of the SFKs. Finally, the antiproliferative activities of the best candidates were evaluated against SH-SY5Y and K562 cell lines. Compound 3 b [2-(4-{2-methyl-6-[(5-phenylthiazol-2-yl)amino]pyrimidin-4-yl}piperazin-1-yl)ethanol] was found to be the most active inhibitor. PMID:26514807

  2. Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family

    SciTech Connect

    Hanks, S.K.

    1987-01-01

    Mixed /sup 32/P-labeled oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identify with rabbit skeletal muscle phosphorylase kinase ..gamma..-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.

  3. Rab-family GTPase regulates TOR complex 2 signaling in fission yeast

    PubMed Central

    Tatebe, Hisashi; Morigasaki, Susumu; Murayama, Shinichi; Zeng, Cui Tracy; Shiozaki, Kazuhiro

    2010-01-01

    Summary Background From yeast to human, TOR (Target Of Rapamycin) kinase plays pivotal roles in coupling extracellular stimuli to cell growth and metabolism. TOR kinase functions in two distinct protein complexes, TOR complex 1 (TORC1) and 2 (TORC2), which phosphorylate and activate different AGC-family protein kinases. TORC1 is controlled by the small GTPase Rheb, but little is known about TORC2 regulators. Results We have identified the Ryh1 GTPase, a human Rab6 ortholog, as an activator of TORC2 signaling in the fission yeast Schizosaccharomyces pombe. Mutational inactivation of Ryh1 or its guanine nucleotide exchange factor compromises the TORC2-dependent phosphorylation of the AGC-family Gad8 kinase. In addition, the effector domain of Ryh1 is important for its physical interaction with TORC2 and for stimulation of TORC2 signaling. Thus, GTP-bound Ryh1 is likely to be the active form stimulatory to TORC2–Gad8 signaling. Consistently, expression of the GTP-locked mutant Ryh1 is sufficient to promote interaction between TORC2 and Gad8 and to induce Gad8 hyper-phosphorylation. The loss of functional Ryh1, TORC2 or Gad8 brings about similar vacuolar fragmentation and stress sensitivity, further corroborating their involvement in a common cellular process. Human Rab6 can substitute Ryh1 in S. pombe and therefore, Rab6 may be a potential activator of TORC2 in mammals. Conclusions In its GTP-bound form, Ryh1, an evolutionarily conserved Rab GTPase, activates TORC2 signaling to the AGC kinase Gad8. The Ryh1 GTPase and the TORC2–Gad8 pathway are required for vacuolar integrity and cellular stress resistance in S. pombe. PMID:21035342

  4. Members of a Novel Kinase Family (DUF1537) Can Recycle Toxic Intermediates into an Essential Metabolite.

    PubMed

    Thiaville, Jennifer J; Flood, Jake; Yurgel, Svetlana; Prunetti, Laurence; Elbadawi-Sidhu, Mona; Hutinet, Geoffrey; Forouhar, Farhad; Zhang, Xinshuai; Ganesan, Venkateswaran; Reddy, Patrick; Fiehn, Oliver; Gerlt, J A; Hunt, John F; Copley, Shelley D; de Crécy-Lagard, Valérie

    2016-08-19

    DUF1537 is a novel family of kinases identified by comparative genomic approaches. The family is widespread and found in all sequenced plant genomes and 16% of sequenced bacterial genomes. DUF1537 is not a monofunctional family and contains subgroups that can be separated by phylogenetic and genome neighborhood context analyses. A subset of the DUF1537 proteins is strongly associated by physical clustering and gene fusion with the PdxA2 family, demonstrated here to be a functional paralog of the 4-phosphohydroxy-l-threonine dehydrogenase enzyme (PdxA), a central enzyme in the synthesis of pyridoxal-5'-phosphate (PLP) in proteobacteria. Some members of this DUF1537 subgroup phosphorylate l-4-hydroxythreonine (4HT) into 4-phosphohydroxy-l-threonine (4PHT), the substrate of PdxA, in vitro and in vivo. This provides an alternative route to PLP from the toxic antimetabolite 4HT that can be directly generated from the toxic intermediate glycolaldehyde. Although the kinetic and physical clustering data indicate that these functions in PLP synthesis are not the main roles of the DUF1537-PdxA2 enzymes, genetic and physiological data suggest these side activities function has been maintained in diverse sets of organisms. PMID:27294475

  5. 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

  6. Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

    PubMed Central

    Vuori, K; Hirai, H; Aizawa, S; Ruoslahti, E

    1996-01-01

    Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion. PMID:8649368

  7. AGC-2 Specimen Post Irradiation Data Package Report

    SciTech Connect

    Windes, William Enoch; Swank, W. David; Rohrbaugh, David T.; Cottle, David L.

    2015-08-01

    This report documents results of the post-irradiation examination material property testing of the creep, control, and piggyback specimens from the irradiation creep capsule Advanced Graphite Creep (AGC)-2 are reported. This is the second of a series of six irradiation test trains planned as part of the AGC experiment to fully characterize the neutron irradiation effects and radiation creep behavior of current nuclear graphite grades. The AGC-2 capsule was irradiated in the Idaho National Laboratory Advanced Test Reactor at a nominal temperature of 600°C and to a peak dose of 5 dpa (displacements per atom). One-half of the creep specimens were subjected to mechanical stresses (an applied stress of either 13.8, 17.2, or 20.7 MPa) to induce irradiation creep. All post-irradiation testing and measurement results are reported with the exception of the irradiation mechanical strength testing, which is the last destructive testing stage of the irradiation testing program. Material property tests were conducted on specimens from 15 nuclear graphite grades using a similar loading configuration as the first AGC capsule (AGC-1) to provide easy comparison between the two capsules. However, AGC-2 contained an increased number of specimens (i.e., 487 total specimens irradiated) and replaced specimens of the minor grade 2020 with the newer grade 2114. The data reported include specimen dimensions for both stressed and unstressed specimens to establish the irradiation creep rates, mass and volume data necessary to derive density, elastic constants (Young’s modulus, shear modulus, and Poisson’s ratio) from ultrasonic time-of-flight velocity measurements, Young’s modulus from the fundamental frequency of vibration, electrical resistivity, and thermal diffusivity and thermal expansion data from 100–500°C. No data outliers were determined after all measurements were completed. A brief statistical analysis was performed on the irradiated data and a limited comparison between

  8. PK12, a plant dual-specificity protein kinase of the LAMMER family, is regulated by the hormone ethylene.

    PubMed Central

    Sessa, G; Raz, V; Savaldi, S; Fluhr, R

    1996-01-01

    The ethylene signal is transduced in plant cells via phosphorylation events. To identify protein kinases whose levels of expression are modulated by the plant hormone ethylene, we utilized a differential reverse transcriptase-polymerase chain reaction approach using mRNA extracted from ethylene-treated and untreated tobacco leaves. An ethylene-induced cDNA clone, PK12, encoding a protein kinase, was isolated. PK12 is a new member of the recently defined LAMMER family of protein kinases, which has been identified in mammals, flies, yeasts, and plants. The LAMMER kinases are related to the cell cycle-dependent CDC2-type kinases and are characterized by their similarity at kinase subdomain X. The recombinant PK12 protein autophosphorylates in vitro on serine, threonine, and tyrosine residues, thereby making it a member of the dual-specificity protein kinases. Immunoprecipitation of PK12 from plant extracts and kinase assay revealed that the apparent PK12 activity is rapidly and transiently increased when plants are treated with ethylene. By using in situ hybridization, we detected accumulation of the PK12 transcript in leaves after ethylene treatment and in the untreated flower abscission zone. The tissue in this zone is known to constitutively express ethylene-regulated genes. PMID:8989879

  9. Novel Small Molecule Activators of the Trk Family of Receptor Tyrosine Kinases

    PubMed Central

    Obianyo, Obiamaka; Ye, Keqiang

    2012-01-01

    The Tropomyosin-related kinase (Trk) receptors are a subset of the receptor tyrosine kinase family with an important functionality in the regulation of neurotrophic signaling in the peripheral and central nervous system. As the receptors are able to mediate neuronal survival by associating with their respective neurotrophin ligands, many studies have focused on the therapeutic potential of generating small-molecule mimetic compounds that elicit agonistic effects similar to those of the natural protein ligands. To this end, various structure-based studies have led to the generation of bivalent peptide-based agonists and antibodies that selectively initiate Trk receptor signaling; however, these compounds do not possess the ideal characteristics of a potential drug. Additionally, the reliance of structure-based data to generate the compound libraries, limits the potential identification of novel chemical structures with desirable activity. Therefore, subsequent investigations utilized a cell-based apoptotic screen to facilitate the analysis of large, diverse chemical libraries of small molecules and quickly identify compounds with Trk-dependent antiapoptotic activity. Herein, we describe the Trk agonists that have been identified by this screening methodology and summarize their in vitro and in vivo neurotrophic activity as well as their efficacy in various neurological disease models, implicating their future utility as therapeutic compounds. PMID:22982231

  10. Characterization of Mice Deficient in the Src Family Nonreceptor Tyrosine Kinase Frk/rak

    PubMed Central

    Chandrasekharan, Subhashini; Qiu, Ting Hu; Alkharouf, Nawal; Brantley, Kelly; Mitchell, James B.; Liu, Edison T.

    2002-01-01

    Frk/rak belongs to a novel family of Src kinases with epithelial tissue-specific expression. Although developmental expression patterns and functional overexpression in vitro have associated these kinases with growth suppression and differentiation, their physiological functions remain largely unknown. We therefore generated mice carrying a null mutation in iyk, the mouse homolog of Frk/rak. We report here that frk/rak−/− mice are viable, show similar growth rates to wild-type animals, and are fertile. Furthermore, a 2-year study of health and survival did not identify differences in the incidence and spectrum of spontaneous tumors or provide evidence of hyperplasias in frk/rak−/− epithelial tissues. Histological analysis of organs failed to reveal any morphological changes in epithelial tissues that normally express high levels of Frk/rak. Ultrastructural analysis of intestinal enterocytes did not identify defects in brush border morphology or structural polarization, demonstrating that Frk/rak is dispensable for intestinal cytodifferentiation. Additionally, frk/rak-null mice do not display altered sensitivity to intestinal damage induced by ionizing radiation. cDNA microarray analysis revealed an increase in c-src expression and identified subtle changes in the expression of genes regulated by thyroid hormones. Significant decreases in the circulating levels of T3 but not T4 hormone are consistent with this observation and reminiscent of euthyroid sick syndrome, a stress-associated clinical condition. PMID:12077350

  11. Src family kinase inhibitor PP2 accelerates differentiation in human intestinal epithelial cells.

    PubMed

    Seltana, Amira; Guezguez, Amel; Lepage, Manon; Basora, Nuria; Beaulieu, Jean-François

    2013-01-25

    The proto-oncogene Src is an important protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and survival. Here, we investigated the involvement of Src family kinases (SFKs) in human intestinal cell differentiation. We first observed that Src activity peaked in early stages of Caco-2/15 cell differentiation. Inhibition of SFKs with PP2, a selective SFK inhibitor, accelerated the overall differentiation program. Interestingly, all polarization and terminal differentiation markers tested, including sucrase-isomaltase, lactase-phlorizin hydrolase and E and Li-cadherins were found to be significantly up-regulated after only 3 days of treatment in the newly differentiating cells. Further investigation of the effects of PP2 revealed a significant up-regulation of the two main intestinal epithelial cell-specific transcription factors Cdx2 and HNF1α and a reduction of polycomb PRC2-related epigenetic repressing activity as measured by a decrease in H3K27me3, two events closely related to the control of cell terminal differentiation in the intestine. Taken together, these data suggest that SFKs play a key role in the control of intestinal epithelial cell terminal differentiation. PMID:23274493

  12. Dopamine agonists rescue Aβ-induced LTP impairment by Src-family tyrosine kinases.

    PubMed

    Yuan Xiang, PingAn; Janc, Oliwia; Grochowska, Katarzyna M; Kreutz, Michael R; Reymann, Klaus G

    2016-04-01

    Soluble forms of oligomeric amyloid beta (AβO) are involved in the loss of synaptic plasticity and memory, especially in early phases of Alzheimer's disease. Stimulation of dopamine D1/D5 receptors (D1R/D5R) is known to increase surface expression of synaptic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate subtype glutamate and N-methyl-D-aspartate subtype glutamate receptors and facilitates the induction of the late phase of long-term potentiation (LTP), probably via a related mechanism. In this study, we show that the D1/D5R agonist SKF38393 protects LTP of hippocampal CA1 synapses from the deleterious action of oligomeric amyloid beta. Unexpectedly, the D1R/D5R-mediated recovery of LTP is independent of protein kinase A or phospholipase C pathways. Instead, we found that the inhibition of Src-family tyrosine kinases completely abolished the protective effects of D1R/D5R stimulation in a cellular model of learning and memory. PMID:26973108

  13. SRC family kinase FYN promotes the neuroendocrine phenotype and visceral metastasis in advanced prostate cancer

    PubMed Central

    Sievert, Margarit; Duan, Peng; Lichterman, Jake; Huang, Jen-Ming; Smith, Bethany; You, Sungyong; Nandana, Srinivas; Chu, Gina Chia-Yi; Mink, Sheldon; Josson, Sajni; Liu, Chunyan; Morello, Matteo; Jones, Lawrence W. M.; Kim, Jayoung; Freeman, Michael R.; Bhowmick, Neil; Zhau, Haiyen E.; Chung, Leland W.K.; Posadas, Edwin M.

    2015-01-01

    FYN is a SRC family kinase (SFK) that has been shown to be up-regulated in human prostate cancer (PCa) tissues and cell lines. In this study, we observed that FYN is strongly up-regulated in human neuroendocrine PCa (NEPC) tissues and xenografts, as well as cells derived from a NEPC transgenic mouse model. In silico analysis of FYN expression in prostate cancer cell line databases revealed an association with the expression of neuroendocrine (NE) markers such as CHGA, CD44, CD56, and SYP. The loss of FYN abrogated the invasion of PC3 and ARCaPM cells in response to MET receptor ligand HGF. FYN also contributed to the metastatic potential of NEPC cells in two mouse models of visceral metastasis with two different cell lines (PC3 and TRAMPC2-RANKL). The activation of MET appeared to regulate neuroendocrine (NE) features as evidenced by increased expression of NE markers in PC3 cells with HGF. Importantly, the overexpression of FYN protein in DU145 cells was directly correlated with the increase of CHGA. Thus, our data demonstrated that the neuroendocrine differentiation that occurs in PCa cells is, at least in part, regulated by FYN kinase. Understanding the role of FYN in the regulation of NE markers will provide further support for ongoing clinical trials of SFK and MET inhibitors in castration-resistant PCa patients. PMID:26624980

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

    PubMed Central

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

    2012-01-01

    Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H2O2 at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immediate SFK and calcium signaling in epithelia was important for late epimorphic regeneration of amputated fins. Wound-induced activation of SFKs in epithelia was dependent on injury-generated H2O2. A SFK member, Fynb, was responsible for fin regeneration. This work provides a new link between early wound responses and late regeneration and suggests that redox, SFK, and calcium signaling are immediate “wound signals” that integrate early wound responses and late epimorphic regeneration. PMID:23045550

  15. CD45, CD148, and Lyp/Pep: Critical Phosphatases Regulating Src Family Kinase Signaling Networks in Immune Cells

    PubMed Central

    Hermiston, Michelle L.; Zikherman, Julie; Zhu, Jing W.

    2009-01-01

    Summary Reciprocal regulation of tyrosine phosphorylation by protein tyrosine kinases and phosphatases is central to normal immune cell function. Disruption of the equilibrium between protein tyrosine kinase and phosphatase activity can result in immunodeficiency, autoimmunity, or malignancy. Src family kinases play a central role in both immune cell function and disease due to their proximal position in numerous signal transduction cascades including those emanating from integrin, T and B cell antigen receptors, Fc, growth factor, and cytokine receptors. Given that tight regulation of Src family kinase activity is critical for appropriate responses to stimulation of these various signaling pathways, it is perhaps not surprising that multiple protein tyrosine phosphatases are involved in their regulation. Here, we focus on the role of three phosphatases, CD45, CD148, and LYP/PEP, which are critical regulators of src family kinase activity in hematopoietic cells. We review our current understanding of their structures, expression, functions in different hematopoietic cell subsets, regulation, and putative roles in disease. Finally, we discuss remaining questions that must be addressed if we are to have a clearer understanding of the coordinated regulation of tyrosine phosphorylation and signaling networks in hematopoietic cells and how they could potentially be manipulated therapeutically in disease. PMID:19290935

  16. Cyclin-dependent kinase 5 phosphorylation of familial prion protein mutants exacerbates conversion into amyloid structure.

    PubMed

    Rouget, Raphaël; Sharma, Gyanesh; LeBlanc, Andréa C

    2015-02-27

    Familial prion protein (PrP) mutants undergo conversion from soluble and protease-sensitive to insoluble and partially protease-resistant proteins. Cyclin-dependent kinase 5 (Cdk5) phosphorylation of wild type PrP (pPrP) at serine 43 induces a conversion of PrP into aggregates and fibrils. Here, we investigated whether familial PrP mutants are predisposed to Cdk5 phosphorylation and whether phosphorylation of familial PrP mutants increases conversion. PrP mutants representing three major familial PrP diseases and different PrP structural domains were studied. We developed a novel in vitro kinase reaction coupled with Thioflavin T binding to amyloid structure assay to monitor phosphorylation-dependent amyloid conversion. Although non-phosphorylated full-length wild type or PrP mutants did not convert into amyloid, Cdk5 phosphorylation rapidly converted these into Thioflavin T-positive structures following first order kinetics. Dephosphorylation partially reversed conversion. Phosphorylation-dependent conversion of PrP from α-helical structures into β-sheet structures was confirmed by circular dichroism. Relative to wild type pPrP, most PrP mutants showed increased rate constants of conversion. In contrast, non-phosphorylated truncated PrP Y145X (where X represents a stop codon) and Q160X mutants converted spontaneously into Thioflavin T-positive fibrils after a lag phase of over 20 h, indicating nucleation-dependent polymerization. Phosphorylation reduced the lag phase by over 50% and thus accelerated the formation of the nucleating event. Consistently, phosphorylated Y145X and phosphorylated Q160X exacerbated conversion in a homologous seeding reaction, whereas WT pPrP could not seed WT PrP. These results demonstrate an influence of both the N terminus and the C terminus of PrP on conversion. We conclude that post-translational modifications of the flexible N terminus of PrP can cause or exacerbate PrP mutant conversion. PMID:25572400

  17. Janus kinases and Src family kinases in the regulation of EGF-induced vimentin expression in MDA-MB-468 breast cancer cells.

    PubMed

    Stewart, Teneale A; Azimi, Iman; Brooks, Andrew J; Thompson, Erik W; Roberts-Thomson, Sarah J; Monteith, Gregory R

    2016-07-01

    Epithelial-mesenchymal transition (EMT) is an important process associated with the metastasis of breast cancer cells. Members of the Janus kinases (JAKs) and Src family kinases (SFKs) are implicated in the regulation of an invasive phenotype in various cancer cell types. Using the pharmacological inhibitors JAK Inhibitor I (a pan-JAK inhibitor) and PP2 we investigated the role of the JAKs and SFKs, respectively, in the regulation of EMT markers in the MDA-MB-468 breast cancer cell line model of epidermal growth factor (EGF)-induced EMT. We identified selective inhibition of EGF induction of the mesenchymal marker vimentin by PP2 and JAK Inhibitor I. The effect of JAK Inhibitor I on vimentin protein induction occurred at a concentration lower than that required to significantly inhibit EGF-mediated signal transducer and activator of transcription 3 (STAT3)-phosphorylation, suggesting involvement of a STAT3-independent mechanism of EGF-induced vimentin regulation by JAKs. Despite our identification of a role for the JAK family in EGF-induced vimentin protein expression, siRNA-mediated silencing of each member of the JAK family was unable to phenocopy pharmacological inhibition, indicating potential redundancy among the JAK family members in this pathway. While SFKs and JAKs do not represent global regulators of the EMT phenotype, our findings have identified a role for members of these signaling pathways in the regulation of EGF-induced vimentin expression in the MDA-MB-468 breast cancer cell line. PMID:27163529

  18. Identification and Characterization of the Cyclin-Dependent Kinases Gene Family in Silkworm, Bombyx mori.

    PubMed

    Li, Yinü; Jiang, Feng; Shi, Xiaofeng; Liu, Xingjian; Yang, Huipeng; Zhang, Zhifang

    2016-01-01

    Cyclin-dependent protein kinases (CDKs) play key roles at different checkpoint regulations of the eukaryotic cell cycle. However, only few studies of lepidoptera CDK family proteins have been reported so far. In this study, we performed the cDNA sequencing of 10 members of the CDK family in Bombyx mori. Gene structure analysis suggested that CDK12 and CDC2L1 owned two and three isoforms, respectively. Phylogenetic analysis showed that CDK genes in different species were highly conserved, implying that they evolved independently even before the split between vertebrates and invertebrates. We found that the expression levels of BmCDKs in 13 tissues of fifth-instar day 3 larvae were different: CDK1, CDK7, and CDK9 had a high level of expression, whereas CDK4 was low-level expressed and was detected only in the testes and fat body cells. Similar expression profiles of BmCDKs during embryo development were obtained. Among the variants of CDK12, CDK12 transcript variant A had the highest expression, and the expression of CDC2L1 transcript variant A was the highest among the variants of CDC2L1. It was shown from the RNAi experiments that the silencing of CDK1, CDK10, CDK12, and CDC2L1 could influence the cells from G0/G1 to S phase transition. PMID:26544066

  19. AGC-1 Pre-Irradiation Data Report Status

    SciTech Connect

    William Windes

    2011-08-01

    The Next Generation Nuclear Plant (NGNP) Graphite R&D program is currently measuring irradiated material property changes in several grades of nuclear graphite for predicting their behavior and operating performance within the core of new Very High Temperature Reactor (VHTR) designs. The Advanced Graphite Creep (AGC) experiment consisting of six irradiation capsules will generate this irradiated graphite performance data for NGNP reactor operating conditions. All samples in the experiment will be fully characterized before irradiation, irradiated in the Advanced Test Reactor (ATR), and then re-examined to determine the irradiation induced changes to key materials properties in the different graphite grades. The information generated during the AGC experiment will be utilized for NRC licensing of NGNP reactor designs, shared with international collaborators in the Generation IV Information Forum (GIF), and eventually utilized in ASME design code for graphite nuclear applications. This status report will describe the process the NGNP Graphite R&D program has developed to record the AGC1 pre-irradiation examination data.

  20. SRC family kinases are required for limb trajectory selection by spinal motor axons.

    PubMed

    Kao, Tzu-Jen; Palmesino, Elena; Kania, Artur

    2009-04-29

    Signal relay by guidance receptors at the axonal growth cone is a process essential for the assembly of a functional nervous system. We investigated the in vivo function of Src family kinases (SFKs) as growth cone guidance signaling intermediates in the context of spinal lateral motor column (LMC) motor axon projection toward the ventral or dorsal limb mesenchyme. Using in situ mRNA detection we determined that Src and Fyn are expressed in LMC motor neurons of chick and mouse embryos at the time of limb trajectory selection. Inhibition of SFK activity by C-terminal Src kinase (Csk) overexpression in chick LMC axons using in ovo electroporation resulted in LMC axons selecting the inappropriate dorsoventral trajectory within the limb mesenchyme, with medial LMC axon projecting into the dorsal and ventral limb nerve with apparently random incidence. We also detected LMC axon trajectory choice errors in Src mutant mice demonstrating a nonredundant role for Src in motor axon guidance in agreement with gain and loss of Src function in chick LMC neurons which led to the redirection of LMC axons. Finally, Csk-mediated SFK inhibition attenuated the retargeting of LMC axons caused by EphA or EphB over-expression, implying the participation of SFKs in Eph-mediated LMC motor axon guidance. In summary, our findings demonstrate that SFKs are essential for motor axon guidance and suggest that they play an important role in relaying ephrin:Eph signals that mediate the selection of motor axon trajectory in the limb. PMID:19403835

  1. Adenovirus E4 Open Reading Frame 4–Induced Apoptosis Involves Dysregulation of Src Family Kinases

    PubMed Central

    Lavoie, Josée N.; Champagne, Claudia; Gingras, Marie-Claude; Robert, Amélie

    2000-01-01

    The adenoviral early region 4 open reading frame 4 (E4orf4) death factor induces p53-independent apoptosis in many cell types and appears to kill selectively transformed cells. Here we show that expression of E4orf4 in transformed epithelial cells results in early caspase-independent membrane blebbing, associated with changes in the organization of focal adhesions and actin cytoskeleton. Evidence that E4orf4 can associate with and modulate Src family kinase activity, inhibiting Src-dependent phosphorylation of focal adhesion kinase (FAK) and paxillin while increasing phosphorylation of cortactin and some other cellular proteins, is presented. Furthermore, E4orf4 dramatically inhibited the ability of FAK and c-src to cooperate in induction of tyrosine phosphorylation of cellular substrates, suggesting that E4orf4 can interfere with the formation of a signaling complex at focal adhesion sites. Consistent with a functional role for E4orf4–Src interaction, overexpression of activated c-src dramatically potentiated E4orf4-induced membrane blebbing and apoptosis, whereas kinase dead c-src constructs inhibited E4orf4 effects on cell morphology and death. Moreover treatment of E4orf4-expressing cells with PP2, a selective Src kinase inhibitor, led to inhibition of E4orf4-dependent membrane blebbing and later to a marked decrease in E4orf4-induced nuclear condensation. Taken together, these observations indicate that expression of adenovirus 2 E4orf4 can initiate caspase-independent extranuclear manifestations of apoptosis through a modulation of Src family kinases and that these are involved in signaling E4orf4-dependent apoptosis. This study also suggests that Src family kinases are likely to play a role in the cytoplasmic execution of apoptotic programs. PMID:10973994

  2. Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint*

    PubMed Central

    Fukumoto, Yasunori; Morii, Mariko; Miura, Takahito; Kubota, Sho; Ishibashi, Kenichi; Honda, Takuya; Okamoto, Aya; Yamaguchi, Noritaka; Iwama, Atsushi; Nakayama, Yuji; Yamaguchi, Naoto

    2014-01-01

    The DNA damage checkpoint arrests cell cycle progression to allow time for repair. Once DNA repair is completed, checkpoint signaling is terminated. Currently little is known about the mechanism by which checkpoint signaling is terminated, and the disappearance of DNA lesions is considered to induce the end of checkpoint signaling; however, here we show that the termination of checkpoint signaling is an active process promoted by Src family tyrosine kinases. Inhibition of Src activity delays recovery from the G2 phase DNA damage checkpoint following DNA repair. Src activity is required for the termination of checkpoint signaling, and inhibition of Src activity induces persistent activation of ataxia telangiectasia mutated (ATM)- and Rad3-related (ATR) and Chk1 kinases. Src-dependent nuclear protein tyrosine phosphorylation and v-Src expression suppress the ATR-mediated Chk1 and Rad17 phosphorylation induced by DNA double strand breaks or DNA replication stress. Thus, Src family kinases promote checkpoint recovery through termination of ATR- and Chk1-dependent G2 DNA damage checkpoint. These results suggest a model according to which Src family kinases send a termination signal between the completion of DNA repair and the initiation of checkpoint termination. PMID:24634213

  3. Calcium-dependent protein kinase (CDPK) and CDPK-related kinase (CRK) gene families in tomato: genome-wide identification and functional analyses in disease resistance.

    PubMed

    Wang, Ji-Peng; Xu, You-Ping; Munyampundu, Jean-Pierre; Liu, Tian-Yu; Cai, Xin-Zhong

    2016-04-01

    Calcium-dependent protein kinases (CDPKs) and CDPK-related kinases (CRKs) play multiple roles in plant. Nevertheless, genome-wide identification of these two families is limited to several plant species, and role of CRKs in disease resistance remains unclear. In this study, we identified the CDPK and CRK gene families in genome of the economically important crop tomato (Solanum lycopersicum L.) and analyzed their function in resistance to various pathogens. Twenty-nine CDPK and six CRK genes were identified in tomato genome. Both SlCDPK and SlCRK proteins harbored an STKc_CAMK type protein kinase domain, while only SlCDPKs contained EF-hand type Ca(2+) binding domain(s). Phylogenetic analysis revealed that plant CRK family diverged early from CDPKs, and shared a common ancestor gene with subgroup IV CDPKs. Subgroup IV SlCDPK proteins were basic and their genes contained 11 introns, which were distinguished from other subgroups but similar to CRKs. Subgroup I SlCDPKs generally did not carry an N-terminal myristoylation motif while those of the remaining subgroups and SlCRKs universally did. SlCDPK and SlCRK genes were differently responsive to pathogenic stimuli. Furthermore, silencing analyses demonstrated that SlCDPK18 and SlCDPK10 positively regulated nonhost resistance to Xanthomonas oryzae pv. oryzae and host resistance to Pseudomonas syringae pv. tomato (Pst) DC3000, respectively, while SlCRK6 positively regulated resistance to both Pst DC3000 and Sclerotinia sclerotiorum in tomato. In conclusion, CRKs apparently evolved from CDPK lineage, SlCDPK and SlCRK genes regulate a wide range of resistance and SlCRK6 is the first CRK gene proved to function in plant disease resistance. PMID:26520101

  4. The Numerical Studies Program for the Atmospheric General Circulation Experiment (AGCE) for Spacelab Flights

    NASA Technical Reports Server (NTRS)

    Fowlis, W. W. (Editor); Davis, M. H. (Editor)

    1981-01-01

    The atmospheric general circulation experiment (AGCE) numerical design for Spacelab flights was studied. A spherical baroclinic flow experiment which models the large scale circulations of the Earth's atmosphere was proposed. Gravity is simulated by a radial dielectric body force. The major objective of the AGCE is to study nonlinear baroclinic wave flows in spherical geometry. Numerical models must be developed which accurately predict the basic axisymmetric states and the stability of nonlinear baroclinic wave flows. A three dimensional, fully nonlinear, numerical model and the AGCE based on the complete set of equations is required. Progress in the AGCE numerical design studies program is reported.

  5. 3-Phosphoinositide-Dependent protein Kinase-1 (PDK1) inhibitors: A review from patent literature

    PubMed Central

    Barile, Elisa; De, Surya K.; Pellecchia, Maurizio

    2016-01-01

    PDK1 (3-Phosphoinositide-dependent kinase 1) is a key member of the AGC protein kinase family. It plays an important role in a variety of cellular functions, leading to the activation of the PI3K signaling pathway, an event often associated with the onset and progression of several human cancers. Numerous recent observations suggest that PDK1 inhibitors may provide novel opportunities for the development of effective classes of therapeutics. On these premises, recent years have witnessed an increased effort by medicinal chemists to develop novel scaffolds to derive potent and selective PDK1 inhibitors. The intent of this review is to update the reader on the recent patent literature covering applications published between June 2008 and September 2011 that report on PDK1 inhibitors. PMID:24236780

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

    PubMed Central

    Meirelles, Gabriela Vaz; Perez, Arina Marina; de Souza, Edmárcia Elisa; Basei, Fernanda Luisa; Papa, Priscila Ferreira; Melo Hanchuk, Talita Diniz; Cardoso, Vanessa Bomfim; Kobarg, Jörg

    2014-01-01

    Aside from Polo and Aurora, a third but less studied kinase family involved in mitosis regulation is the never in mitosis-gene A (NIMA)-related kinases (Neks). The founding member of this family is the sole member NIMA of Aspergillus nidulans, which is crucial for the initiation of mitosis in that organism. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals: (1) centrioles/mitosis; (2) primary ciliary function/ciliopathies; and (3) DNA damage response (DDR). Recent findings, especially on Nek 1 and 8, showed however, that several Neks participate in parallel in at least two of these contexts: primary ciliary function and DDR. In the core section of this in-depth review, we report the current detailed functional knowledge on each of the 11 Neks. In the discussion, we return to the cross-connections among Neks and point out how our and other groups’ functional and interactomics studies revealed that most Neks interact with protein partners associated with two if not all three of the functional contexts. We then raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize the cellular events associated with these three core functions. The new and exciting findings on the Nek family open new perspectives and should allow the Neks to finally claim the attention they deserve in the field of kinases and cell cycle biology. PMID:24921005

  7. Mitogen Activated Protein Kinase Family Proteins and c-jun Signaling in Injury-induced Schwann Cell Plasticity.

    PubMed

    Lee, Hye Jeong; Shin, Yoon Kyung; Park, Hwan Tae

    2014-06-01

    Schwann cells (SCs) in the peripheral nerves myelinate axons during postnatal development to allow saltatory conduction of nerve impulses. Well-organized structures of myelin sheathes are maintained throughout life unless nerves are insulted. After peripheral nerve injury, unidentified signals from injured nerves drive SC dedifferentiation into an immature state. Dedifferentiated SCs participate in axonal regeneration by producing neurotrophic factors and removing degenerating nerve debris. In this review, we focus on the role of mitogen activated protein kinase family proteins (MAP kinases) in SC dedifferentiation. In addition, we will highlight neuregulin 1 and the transcription factor c-jun as upstream and downstream signals for MAP kinases in SC responses to nerve injury. PMID:24963277

  8. Casein Kinase Iδ Mutations in Familial Migraine and Advanced Sleep Phase

    PubMed Central

    Brennan, K. C.; Bates, Emily A.; Shapiro, Robert E.; Zyuzin, Jekaterina; Hallows, William C.; Huang, Yong; Lee, Hsien-Yang; Jones, Christopher R.; Fu, Ying-Hui; Charles, Andrew C.; Ptáček, Louis J.

    2014-01-01

    Migraine is a common disabling disorder with a significant genetic component, characterized by severe headache and often accompanied by nausea, vomiting, and light sensitivity. We identified two families, each with a distinct missense mutation in the gene encoding casein kinase Iδ (CKIδ), in which the mutation cosegregated with both the presence of migraine and advanced sleep phase. The resulting alterations (T44A and H46R) occurred in the conserved catalytic domain of CKIδ, where they caused reduced enzyme activity. Mice engineered to carry the CKIδ-T44A allele were more sensitive to pain after treatment with the migraine trigger nitroglycerin. CKIδ-T44A mice also exhibited a reduced threshold for cortical spreading depression (believed to be the physiological analog of migraine aura) and greater arterial dilation during cortical spreading depression. Astrocytes from CKIδ-T44A mice showed increased spontaneous and evoked calcium signaling. These genetic, cellular, physiological, and behavioral analyses suggest that decreases in CKIδ activity can contribute to the pathogenesis of migraine. PMID:23636092

  9. RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.

    PubMed

    Fritsch, Ralph; de Krijger, Inge; Fritsch, Kornelia; George, Roger; Reason, Beth; Kumar, Madhu S; Diefenbacher, Markus; Stamp, Gordon; Downward, Julian

    2013-05-23

    RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs. PMID:23706742

  10. Altered expression of PGK1 in a family with phosphoglycerate kinase deficiency.

    PubMed

    Svaasand, Eva K; Aasly, Jan; Landsem, Veslemøy Malm; Klungland, Helge

    2007-11-01

    The X-linked recessive disease phosphoglycerate kinase (PGK) deficiency is caused by altered expression of the PGK1 enzyme, which causes muscle stiffness, hemolytic anemia, and mental retardation. In this study we characterized the PGK1 gene in a family of two brothers, two sisters, and their parents. A single mutation in exon 6, which was associated with the pattern of inheritance of PGK1 deficiency, was observed. This silent G213G; c.639C>T mutation was localized to the conserved exon-intron boundary. We have developed a method for quantification of PGK1 mRNA and demonstrated a marked reduction in PGK1 mRNA in both brothers with the disease. A smaller decrease in PGK1 expression was observed in one sister with symptoms of PGK deficiency and in her mother. Only the normal PGK1 allele was expressed in the two heterozygous women. Whereas most known PGK1 mutations cause amino acid alterations, our study indicates that inhibition of the transcription mechanism is the cause of PGK deficiency. PMID:17661373

  11. Resequencing Analysis of the Candidate Tyrosine Kinase and RAS Pathway Gene Families in Multiple Myeloma

    PubMed Central

    Hucthagowder, Vishwanathan; Meyer, Rekha; Mullins, Chelsea; Nagarajan, Rakesh; DiPersio, John F.; Vij, Ravi; Tomasson, Michael H.; Kulkarni, Shashikant

    2012-01-01

    Multiple myeloma (MM) is an incurable, B-cell malignancy, characterized by the clonal proliferation and accumulation of malignant plasma cells in bone marrow. Despite recent advances in understanding of genomic aberrations, a comprehensive catalogue of clinically actionable mutations in MM is just beginning to emerge. The tyrosine kinase (TK) and RAS oncogenes, which encode important regulators of various signaling pathways, are among the most frequently altered gene families in cancer. To clarify the role of TK and RAS genes in pathogenesis of MM, we performed a systematic, targeted screening of mutations on prioritized RAS and TK genes, in CD138 sorted bone marrow specimens from 42 untreated patients. We identified a total of 24 mutations in KRAS, PIK3CA, INSR, LTK and MERTK genes. In particular, seven novel mutations in addition to known KRAS mutations were observed. Prediction analysis tools, PolyPhen and SIFT were used to assess the functional significance of these novel mutations. Our analysis predicted that these mutations may have a deleterious effect, resulting in functional alteration of proteins involved in the pathogenesis of myeloma. While further investigation is needed to determine the functional consequences of these proteins, mutational testing of the RAS and TK genes in larger myeloma cohorts might be also useful to establish the recurrent nature of these mutations. PMID:22939401

  12. Neuroprotection via pro-survival protein kinase C isoforms associated with Bcl-2 family members.

    PubMed

    Weinreb, Orly; Bar-Am, Orit; Amit, Tamar; Chillag-Talmor, Orly; Youdim, Moussa B H

    2004-09-01

    This study provides new insights into neuroprotection involving interaction of protein kinase C (PKC) pathway with Bcl-2 family proteins. Using a model of serum deprivation, we investigated the mechanism by which the anti-Parkinson/monoamine oxidase (MAO)-B inhibitor drug, rasagiline, exerts its neuroprotective effect in rat pheochromocytoma PC12 cells. Here, we report that rasagiline (0.1-10 microM) decreased apoptosis via multiple protection mechanisms, including the stimulation of PKC phosphorylation; up-regulation of PKCalpha and PKC mRNAs, induction of Bcl-xL, Bcl-w, and brain-derived neurotrophic factor (BDNF) mRNAs; and down-regulation of Bad and Bax mRNAs. Moreover, rasagiline inhibited the cleavage and activation of procaspase-3 and poly (ADP-ribose) polymerase (PARP), whereas the PKC inhibitor, GF109203X, reversed these actions. Similarly, rasagiline decreased serum-free-induced levels of the important regulator of cell death, Bad, which was also blocked by GF109203X, indicating the involvement of PKC in rasagiline-induced cell survival. Furthermore, these studies have established that PKC- and Bcl-2-dependent neuroprotective activity of rasagiline is dependent on its propargyl moiety, because propargylamine had similar effects with the same potency. PMID:15247150

  13. Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress

    PubMed Central

    Chaves-Sanjuan, Antonio; Sanchez-Barrena, Maria Jose; Gonzalez-Rubio, Juana Maria; Moreno, Maria; Ragel, Paula; Jimenez, Marta; Pardo, Jose M.; Martinez-Ripoll, Martin; Quintero, Francisco J.; Albert, Armando

    2014-01-01

    Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases. PMID:25288725

  14. Wortmannin and 1-butanol block activation of a novel family of protein kinases in neutrophils.

    PubMed

    Ding, J; Badwey, J A

    1994-07-11

    Neutrophils contain four uncharacterized protein kinases with molecular masses of ca. 69, 63, 49 and 40 kDa that are rapidly activated upon stimulation of these cells with the chemoattractant fMet-Leu-Phe [Ding, J. and Badwey, J.A. (1993) J. Biol. Chem. 268, 17326-17333]. We now report that wortmannin and 1-butanol block activation of all four of these kinases. These reagents are known to inhibit superoxide generation in neutrophils stimulated with this agonist. Wortmannin inhibits phosphatidylinositol 3-kinase and blocks activation of phospholipase D, whereas 1-butanol can reduce the generation of phosphatidate in cells by serving as a substrate for phospholipase D. These data suggest that phosphatidylinositol 3-kinase and phospholipase D may be involved in the activation of several novel protein kinases in neutrophils and that one or more of these kinases is/are involved in superoxide release. PMID:8034030

  15. Phosphorylation of ETS1 by Src family kinases prevents its recognition by the COP1 tumor suppressor.

    PubMed

    Lu, Gang; Zhang, Qing; Huang, Ying; Song, Jiaxi; Tomaino, Ross; Ehrenberger, Tobias; Lim, Elgene; Liu, Wenbin; Bronson, Roderick T; Bowden, Michaela; Brock, Jane; Krop, Ian E; Dillon, Deborah A; Gygi, Steven P; Mills, Gordon B; Richardson, Andrea L; Signoretti, Sabina; Yaffe, Michael B; Kaelin, William G

    2014-08-11

    Oncoproteins and tumor suppressors antagonistically converge on critical nodes governing neoplastic growth, invasion, and metastasis. We discovered that phosphorylation of the ETS1 and ETS2 transcriptional oncoproteins at specific serine or threonine residues creates binding sites for the COP1 tumor suppressor protein, which is an ubiquitin ligase component, leading to their destruction. In the case of ETS1, however, phosphorylation of a neighboring tyrosine residue by Src family kinases disrupts COP1 binding, thereby stabilizing ETS1. Src-dependent accumulation of ETS1 in breast cancer cells promotes anchorage-independent growth in vitro and tumor growth in vivo. These findings expand the list of potential COP1 substrates to include proteins whose COP1-binding sites are subject to regulatory phosphorylation and provide insights into transformation by Src family kinases. PMID:25117710

  16. Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

    PubMed Central

    Juo, P; Kuo, C J; Reynolds, S E; Konz, R F; Raingeaud, J; Davis, R J; Biemann, H P; Blenis, J

    1997-01-01

    The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades. PMID:8972182

  17. The rice kinase phylogenomics database: a guide for systematic analysis of the rice kinase super-family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Determination of gene function is particularly problematic when studying large-gene families because redundancy limits the ability to experimentally assess the contributions of individual genes. Phylogenomics, now referred to as phylomics, is a phylogenetic approach used in comparative genomics to ...

  18. Opa+ Neisseria gonorrhoeae Exhibits Reduced Survival in Human Neutrophils Via Src Family Kinase-Mediated Bacterial Trafficking Into Mature Phagolysosomes

    PubMed Central

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

    2015-01-01

    Summary During gonorrheal infection, there is a heterogeneous population of Neisseria gonorrhoeae (Gc) varied in their expression of opacity-associated (Opa) proteins. While Opa proteins are important for bacterial attachment and invasion of epithelial cells, Opa+ Gc has a survival defect after exposure to neutrophils. Here, we use constitutively Opa- and OpaD+ Gc in strain background FA1090 to show that Opa+ Gc is more sensitive to killing inside adherent, chemokine-treated primary human neutrophils due to increased bacterial residence in mature, degradative phagolysosomes that contain primary and secondary granule antimicrobial content. Although Opa+ Gc stimulates a potent oxidative burst, neutrophil killing of Opa+ Gc was instead attributable to non-oxidative components, particularly neutrophil proteases and the bactericidal/permeability-increasing protein. Blocking interaction of Opa+ Gc with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) or inhibiting Src family kinase signaling, which is downstream of CEACAM activation, enhanced the survival of Opa+ Gc in neutrophils. Src family kinase signaling was required for fusion of Gc phagosomes with primary granules to generate mature phagolysosomes. Conversely, ectopic activation of Src family kinases or coinfection with Opa+ Gc resulted in decreased survival of Opa- Gc in neutrophils. From these results, we conclude that Opa protein expression is an important modulator of Gc survival characteristics in neutrophils by influencing phagosome dynamics and thus bacterial exposure to neutrophils’ full antimicrobial arsenal. PMID:25346239

  19. Modes of Action and Functions of ERECTA-family Receptor-like Kinases in Plant Organ Growth and Development

    SciTech Connect

    TORII, Keiko U.

    2012-05-01

    Higher plants constitute the central resource for renewable lignocellulose biomass that can supplement for the world's depleting stores of fossil fuels. As such, understanding the molecular and genetic mechanisms of plant organ growth will provide key knowledge and genetic resources that enables manipulation of plant biomass feedstock for better growth and productivity. The goal of this proposal is to understand how cell proliferation and growth are coordinated during aboveground organ morphogenesis, and how cell-cell signaling mediated by a family of receptor kinases coordinates plant organogenesis. The well-established model plant Arabidopsis thaliana is used for our research to facilitate rapid progress. Specifically, we focus on how ERECTA-family leucine-rich repeat receptor kinases (LRR-RLKs) interact in a synergistic manner to promote organogenesis and pattern formation in Arabidopsis. This project was highly successful, resulted in fourteen publications including nine peer-reviewed original research articles. One provisional US patent has been filed through this DOE funding. We have addressed the critical roles for a family of receptor kinases in coordinating proliferation and differentiation of plants, and we successfully elucidated the downstream targets of this signaling pathway in specifying stomatal patterning.

  20. Regulation of polar auxin transport by protein and lipid kinases.

    PubMed

    Armengot, Laia; Marquès-Bueno, Maria Mar; Jaillais, Yvon

    2016-07-01

    The directional transport of auxin, known as polar auxin transport (PAT), allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima, and gradients that are instrumental in both organ initiation and shape determination. As such, PAT is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell to cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the 'non-genomic' regulation of auxin transport, placing an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability, and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK, and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some receptor-like kinases (RLKs) and two-component histidine kinase receptors in PAT, noting that there are probably RLKs involved in co-ordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition, as well as root gravitropism and shoot phototropism. PMID:27242371

  1. Rho kinase as a therapeutic target in cardiovascular disease

    PubMed Central

    Surma, Michelle; Wei, Lei; Shi, Jianjian

    2011-01-01

    Rho kinase (ROCK) belongs to the AGC (PKA/PKG/PKC) family of serine/threonine kinases and is a major downstream effector of the small GTPase RhoA. ROCK plays central roles in the organization of the actin cytoskeleton and is involved in a wide range of fundamental cellular functions such as contraction, adhesion, migration, proliferation and gene expression. Two ROCK isoforms, ROCK1 a n d ROCK2, are assumed to be functionally redundant, based largely on the major common activators, the high degree of homology within the kinase domain and studies from overexpression with kinase constructs a n d chemical inhibitors (e.g., Y27632 a n d fasudil), which inhibit both ROCK1 and ROCK2. Extensive experimental a n d clinical studies support a critical role for the RhoA/ROCK pathway in the vascular bed in the pathogenesis of cardiovascular diseases, in which increased ROCK activity mediates vascular smooth muscle cell hypercontraction, endothelial dysfunction, inflammatory cell recruitment and vascular remodeling. Recent experimental studies, using ROCK inhibitors or genetic mouse models, indicate that the RhoA/ROCK pathway in myocardium contributes to cardiac remodeling induced by ischemic injury or persistent hypertrophic stress, thereby leading to cardiac decompensation and heart failure. This article, based on recent molecular, cellular and animal studies, focuses on the current understanding of ROCK signaling in cardiovascular diseases and in the pathogenesis of heart failure. PMID:21929346

  2. Regulation of polar auxin transport by protein and lipid kinases

    PubMed Central

    Jaillais, Yvon

    2016-01-01

    The directional transport of auxin, known as polar auxin transport, allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima and gradients that are instrumental in both organ initiation and shape determination. As such, polar auxin transport is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell-to-cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the ‘non-genomic’ regulation of auxin transport, putting an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some Receptor-Like Kinases (RLK) and two-component histidine kinase receptors in polar auxin transport, noticing that there are likely RLKs involved in coordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition as well as root gravitropism and shoot phototropism. PMID:27242371

  3. Palmitoylation of G protein-coupled receptor kinase, GRK6. Lipid modification diversity in the GRK family.

    PubMed

    Stoffel, R H; Randall, R R; Premont, R T; Lefkowitz, R J; Inglese, J

    1994-11-11

    GRK6, a 66-kDa serine/threonine protein kinase, is a recently identified member of the G protein-coupled receptor kinase (GRK) family. GRKs are involved in the phosphorylation of seven-transmembrane receptors, a process mediating desensitization of signal transduction. An important feature of these enzymes is their membrane-associated nature, which for some members is stimulus-dependent. The structural basis for this membrane association previously has been shown in different members of the GRK family to include isoprenylation, G protein beta gamma-binding domains, and basic regions to provide electrostatic interactions with phospholipids. We provide evidence that another mechanism includes fatty acid acylation. GRK6, but not other GRKs tested, incorporated tritium after incubation with [3H]palmitate in Sf9 and in COS-7 cells overexpressing the kinase. The incorporated radioactivity was released from the protein by neutral hydroxylamine, indicating the presence of a thioester bond, and was confirmed as palmitic acid by high performance liquid chromatography analysis. Site-directed mutagenesis defined the region of palmitate attachment as a cluster of 3 cysteines (Cys561, Cys562, and Cys565) in the carboxyl-terminal domain of the kinase, consistent with the location of the membrane targeting domains of GRKs 1, 2, 3, and 5. Palmitoylation of GRK6 appears essential for membrane association, since palmitoylated kinase was found only in the membrane fraction. This lipid modification provides a structural basis for potential regulation of the subcellular distribution of GRK6 through acylation/deacylation cycles. PMID:7961702

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

    PubMed

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

    2013-12-01

    Glutamatergic neurotransmission in the dorsal hippocampus (DH) is necessary for drug context-induced reinstatement of cocaine-seeking behavior in an animal model of drug relapse. Furthermore, in vitro studies suggest that the Src family of tyrosine kinases critically regulates glutamatergic cellular functions within the DH. Thus, Src-family kinases in the DH may similarly control contextual cocaine-seeking behavior. To test this hypothesis, rats were trained to lever press for un-signaled cocaine infusions in a distinct context followed by extinction training in a different context. Cocaine-seeking behavior (non-reinforced active lever pressing) was then assessed in the previously cocaine-paired and extinction contexts after AP5 (N-methyl-D-aspartate glutamate (NMDA) receptor (NMDAR) antagonist; 0.25 or 2.5 μg/0.5 μl/hemisphere), PP2 (Src-family kinase inhibitor; 6.25 or 62.5 ng/0.5 μl/hemisphere), Ro25-6981 (NR2B subunit-containing NMDAR antagonist; 0.2 or 2 μg/0.5 μl/hemisphere), or vehicle administration into the DH. Administration of AP5, PP2, or Ro25-6981 into the DH dose-dependently impaired drug context-induced reinstatement of cocaine-seeking behavior relative to vehicle, without altering instrumental behavior in the extinction context or food-reinforced instrumental responding and general motor activity in control experiments. Cocaine-seeking behavior during the first 20 min of the test session in the cocaine-paired context was associated with an increase in NR2B subunit activation, and intra-DH PP2 pretreatment disrupted this relationship. Together, these findings suggest that Src-family kinase activation, NMDAR stimulation, and likely Src-family kinase-mediated NR2B subunit-containing NMDAR activation in the DH are necessary for incentive motivational and/or memory processes that promote contextual cocaine-seeking behavior. PMID:23872878

  5. SEMICONDUCTOR INTEGRATED CIRCUITS: A high-performance low-power CMOS AGC for GPS application

    NASA Astrophysics Data System (ADS)

    Qianqian, Lei; Qiming, Xu; Zhiming, Chen; Yin, Shi; Min, Lin; Hailong, Jia

    2010-02-01

    A wide tuning range, low power CMOS automatic gain control (AGC) with a simple architecture is proposed. The proposed AGC is composed of a variable gain amplifier (VGA), a comparator and a charge pump, and the dB-linear gain is controlled by the charge pump. The AGC was implemented in a 0.18 μm CMOS technology. The dynamic range of the VGA is more than 55 dB, the bandwidth is 30 MHz, and the gain error is lower than ±1.5 dB over the full temperature and gain ranges. It is designed for GPS application and is fed from a single 1.8 V power supply. The AGC power consumption is less than 5 mW, and the area of the AGC is 700 × 450 μm2.

  6. Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2).

    PubMed

    Min, Xiaoshan; Ungureanu, Daniela; Maxwell, Sarah; Hammarén, Henrik; Thibault, Steve; Hillert, Ellin-Kristina; Ayres, Merrill; Greenfield, Brad; Eksterowicz, John; Gabel, Chris; Walker, Nigel; Silvennoinen, Olli; Wang, Zhulun

    2015-11-01

    JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domain: a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2, demonstrating its central regulatory function. JH2s were considered catalytically inactive, but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with adenosine 5'-O-(thiotriphosphate) (ATP-γS) and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix αAL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, whereas the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications. PMID:26359499

  7. Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

    PubMed Central

    Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

    1998-01-01

    Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both

  8. The Structure of an NDR/LATS Kinase-Mob Complex Reveals a Novel Kinase-Coactivator System and Substrate Docking Mechanism.

    PubMed

    Gógl, Gergő; Schneider, Kyle D; Yeh, Brian J; Alam, Nashida; Nguyen Ba, Alex N; Moses, Alan M; Hetényi, Csaba; Reményi, Attila; Weiss, Eric L

    2015-05-01

    Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. "Hippo" pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with "Mob" coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1-Mob2, to our knowledge the first of an NDR/LATS kinase-Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1's regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides. PMID:25966461

  9. Navigating into the binding pockets of the HER family protein kinases: discovery of novel EGFR inhibitor as antitumor agent.

    PubMed

    Liu, Wei; Ning, Jin-Feng; Meng, Qing-Wei; Hu, Jing; Zhao, Yan-Bin; Liu, Chao; Cai, Li

    2015-01-01

    The epidermal growth factor receptor (EGFR) family has been validated as a successful antitumor drug target for decades. Known EGFR inhibitors were exposed to distinct drug resistance against the various EGFR mutants within non-small-cell lung cancer (NSCLC), particularly the T790M mutation. Although so far a number of studies have been reported on the development of third-generation EGFR inhibitors for overcoming the resistance issue, the design procedure largely depends on the intuition of medicinal chemists. Here we retrospectively make a detailed analysis of the 42 EGFR family protein crystal complexes deposited in the Protein Data Bank (PDB). Based on the analysis of inhibitor binding modes in the kinase catalytic cleft, we identified a potent EGFR inhibitor (compound A-10) against drug-resistant EGFR through fragment-based drug design. This compound showed at least 30-fold more potency against EGFR T790M than the two control molecules erlotinib and gefitinib in vitro. Moreover, it could exhibit potent HER2 inhibitory activities as well as tumor growth inhibitory activity. Molecular docking studies revealed a structural basis for the increased potency and mutant selectivity of this compound. Compound A-10 may be selected as a promising candidate in further preclinical studies. In addition, our findings could provide a powerful strategy to identify novel selective kinase inhibitors on the basis of detailed kinase-ligand interaction space in the PDB. PMID:26229444

  10. Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

    SciTech Connect

    Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.; Duquenne, Celine; Feng, Yanhong; Grant, Seth W.; Heerding, Dirk; Li, William H.; Miller, William H.; Romeril, Stuart P.; Scherzer, Daryl; Shu, Arthur; Bobko, Mark A.; Chadderton, Antony R.; Dumble, Melissa; Gardiner, Christine M.; Gilbert, Seth; Liu, Qi; Rabindran, Sridhar K.; Sudakin, Valery; Xiang, Hong; Brady, Pat G.; Campobasso, Nino; Ward, Paris; Axten, Jeffrey M.

    2014-10-02

    Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

  11. Linking phenotype to kinase: identification of a novel benzoxaborole hinge-binding motif for kinase inhibition and development of high-potency rho kinase inhibitors.

    PubMed

    Akama, Tsutomu; Dong, Chen; Virtucio, Charlotte; Sullivan, David; Zhou, Yasheen; Zhang, Yong-Kang; Rock, Fernando; Freund, Yvonne; Liu, Liang; Bu, Wei; Wu, Anne; Fan, Xiao-Qing; Jarnagin, Kurt

    2013-12-01

    Benzoxaboroles are a novel class of drug-like compounds that have been rich sources of novel inhibitors for various enzymes and of new drugs. While examining benzoxaborole activity in phenotypic screens, our attention was attracted by the (aminomethylphenoxy)benzoxaborole family, which potently inhibited Toll-like receptor-stimulated cytokine secretion from leukocytes. After considering their structure-activity relationships and the central role of kinases in leukocyte biology, we performed a kinome-wide screen to investigate the members of the (aminomethylphenoxy)benzoxaborole family. This technique identified Rho-activated kinase (ROCK) as a target. We showed competitive behavior, with respect to ATP, and then determined the ROCK2-drug cocrystal structure. The drug occupies the ATP site in which the oxaborole moiety provides hydrogen bond donors and acceptors to the hinge, and the aminomethyl group interacts with the magnesium/ATP-interacting aspartic acid common to protein kinases. The series exhibits excellent selectivity against most of the kinome, with greater than 15-fold selectivity against the next best member of the AGC protein kinase subfamily. Medicinal chemistry efforts with structure-based design resulted in a compound with a Ki of 170 nM. Cellular studies revealed strong enzyme inhibition rank correlation with suppression of intracellular phosphorylation of a ROCK substrate. The biochemical potencies of these compounds also translated to functional activity, causing smooth muscle relaxation in rat aorta and guinea pig trachea. The series exhibited oral availability and one member reduced rat blood pressure, consistent with ROCK's role in smooth muscle contraction. Thus, the benzoxaborole moiety represents a novel hinge-binding kinase scaffold that may have potential for therapeutic use. PMID:24049062

  12. The Src family kinases Hck, Fgr, and Lyn are critical for the generation of the in vivo inflammatory environment without a direct role in leukocyte recruitment

    PubMed Central

    Kovács, Miklós; Németh, Tamás; Jakus, Zoltán; Sitaru, Cassian; Simon, Edina; Futosi, Krisztina; Botz, Bálint; Helyes, Zsuzsanna; Lowell, Clifford A.

    2014-01-01

    Although Src family kinases participate in leukocyte function in vitro, such as integrin signal transduction, their role in inflammation in vivo is poorly understood. We show that Src family kinases play a critical role in myeloid cell–mediated in vivo inflammatory reactions. Mice lacking the Src family kinases Hck, Fgr, and Lyn in the hematopoietic compartment were completely protected from autoantibody-induced arthritis and skin blistering disease, as well as from the reverse passive Arthus reaction, with functional overlap between the three kinases. Though the overall phenotype resembled the leukocyte recruitment defect observed in β2 integrin–deficient (CD18−/−) mice, Hck−/−Fgr−/−Lyn−/− neutrophils and monocytes/macrophages had no cell-autonomous in vivo or in vitro migration defect. Instead, Src family kinases were required for the generation of the inflammatory environment in vivo and for the release of proinflammatory mediators from neutrophils and macrophages in vitro, likely due to their role in Fcγ receptor signal transduction. Our results suggest that infiltrating myeloid cells release proinflammatory chemokine, cytokine, and lipid mediators that attract further neutrophils and monocytes from the circulation in a CD18-dependent manner. Src family kinases are required for the generation of the inflammatory environment but not for the intrinsic migratory ability of myeloid cells. PMID:25225462

  13. The Src family kinases Hck, Fgr, and Lyn are critical for the generation of the in vivo inflammatory environment without a direct role in leukocyte recruitment.

    PubMed

    Kovács, Miklós; Németh, Tamás; Jakus, Zoltán; Sitaru, Cassian; Simon, Edina; Futosi, Krisztina; Botz, Bálint; Helyes, Zsuzsanna; Lowell, Clifford A; Mócsai, Attila

    2014-09-22

    Although Src family kinases participate in leukocyte function in vitro, such as integrin signal transduction, their role in inflammation in vivo is poorly understood. We show that Src family kinases play a critical role in myeloid cell-mediated in vivo inflammatory reactions. Mice lacking the Src family kinases Hck, Fgr, and Lyn in the hematopoietic compartment were completely protected from autoantibody-induced arthritis and skin blistering disease, as well as from the reverse passive Arthus reaction, with functional overlap between the three kinases. Though the overall phenotype resembled the leukocyte recruitment defect observed in β2 integrin-deficient (CD18(-/-)) mice, Hck(-/-)Fgr(-/-)Lyn(-/-) neutrophils and monocytes/macrophages had no cell-autonomous in vivo or in vitro migration defect. Instead, Src family kinases were required for the generation of the inflammatory environment in vivo and for the release of proinflammatory mediators from neutrophils and macrophages in vitro, likely due to their role in Fcγ receptor signal transduction. Our results suggest that infiltrating myeloid cells release proinflammatory chemokine, cytokine, and lipid mediators that attract further neutrophils and monocytes from the circulation in a CD18-dependent manner. Src family kinases are required for the generation of the inflammatory environment but not for the intrinsic migratory ability of myeloid cells. PMID:25225462

  14. Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

    SciTech Connect

    Tamm, Christoffer Galito, Sara Pijuan Anneren, Cecilia

    2012-02-15

    The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2. -- Highlights: Black-Right-Pointing-Pointer SFK inhibitor SU6656 induces senescence in mouse ES cells. Black-Right-Pointing-Pointer SU6656 inhibits mitosis in a SFK-independent manner via cross-selectivity for Aurora kinases. Black-Right-Pointing-Pointer SFK inhibitor PP2 impairs cell motility in various cell lines, including mouse ES cells. Black-Right-Pointing-Pointer Ensuing impeded motility, PP2 inhibits proliferation of various cells lines except for mouse ES cells. Black-Right-Pointing-Pointer SFK inhibitors PP2 and PD173952 impede spontaneous differentiation in standard mouse ES culture maintenance.

  15. On the Conservation of the Slow Conformational Dynamics within the Amino Acid Kinase Family: NAGK the Paradigm

    PubMed Central

    Marcos, Enrique; Crehuet, Ramon; Bahar, Ivet

    2010-01-01

    N-Acetyl-L-Glutamate Kinase (NAGK) is the structural paradigm for examining the catalytic mechanisms and dynamics of amino acid kinase family members. Given that the slow conformational dynamics of the NAGK (at the microseconds time scale or slower) may be rate-limiting, it is of importance to assess the mechanisms of the most cooperative modes of motion intrinsically accessible to this enzyme. Here, we present the results from normal mode analysis using an elastic network model representation, which shows that the conformational mechanisms for substrate binding by NAGK strongly correlate with the intrinsic dynamics of the enzyme in the unbound form. We further analyzed the potential mechanisms of allosteric signalling within NAGK using a Markov model for network communication. Comparative analysis of the dynamics of family members strongly suggests that the low-frequency modes of motion and the associated intramolecular couplings that establish signal transduction are highly conserved among family members, in support of the paradigm sequence→structure→dynamics→function. PMID:20386738

  16. The Extremely Metal-Poor Dwarf Galaxy AGC 198691

    NASA Astrophysics Data System (ADS)

    Hirschauer, Alec S.; Salzer, John Joseph; Cannon, John M.; Skillman, Evan D.; SHIELD II Team

    2016-01-01

    We present spectroscopic observations of the nearby dwarf irregular galaxy AGC 198691. This object is part of the Survey of HI in Extremely Low-Mass Dwarfs (SHIELD) sample, which consists of ultra-low HI mass galaxies discovered by the Arecibo Legacy Fast-Acting ALFA (ALFALFA) survey. SHIELD is a multi-configuration Expanded Very Large Array (EVLA) study of the neutral gas content and dynamics of galaxies with HI masses in the range of 106-107 M⊙. Our spectral data were obtained using the new high-throughput KPNO Ohio State Multi-Object Spectrograph (KOSMOS) on the Mayall 4-m telescope as part of a systematic study of the nebular abundances in the SHIELD galaxy sample. These observations enable measurement of the temperature sensitive [OIII]λ4363 line and hence the determination of a "direct" oxygen abundance for AGC 198691. We find this system to be an extremely metal-deficient (XMD) galaxy with an oxygen abundance comparable to such objects as I Zw 18, SBS 0335-052W, Leo P, and DDO 68 - the lowest metallicity star-forming systems known. It is worth noting that two of the five lowest-abundance galaxies currently recognized were discovered via the ALFALFA blind HI survey. These XMD galaxies are potential analogues to the first star-forming systems, which through hierarchical accretion processes built up the large galaxies we observe today in the local Universe. Detailed analysis of such XMD systems offers observational constraint to models of galactic evolution and star formation histories to allow a better understanding of the processes that govern the chemical evolution of low-mass galaxies.

  17. Status of the NGNP Graphite Creep Experiments AGC-1 and AGC-2 Irradiated in the Advanced Test Reactor

    SciTech Connect

    Blaine Grover

    2012-10-01

    The United States Department of Energy’s Next Generation Nuclear Plant (NGNP) Program will be irradiating six nuclear graphite creep experiments in the Advanced Test Reactor (ATR) located at the Idaho National Laboratory (INL). The graphite experiments will be irradiated over the next six to eight years to support development of a graphite irradiation performance data base on the new nuclear grade graphites now available for use in high temperature gas reactors. The goals of the irradiation experiments are to obtain irradiation performance data, including irradiation creep, at different temperatures and loading conditions to support design of the Next Generation Nuclear Plant (NGNP) Very High Temperature Gas Reactor, as well as other future gas reactors. The experiments will each consist of a single capsule that will contain six peripheral stacks of graphite specimens, with half of the graphite specimens in each stack under a compressive load, while the other half of the specimens will not be subjected to a compressive load during irradiation. The six peripheral stacks will have different compressive loads applied to the top half of each pair of specimen stacks, while a seventh stack will not have a compressive load. The specimens will be irradiated in an inert sweep gas atmosphere with on-line temperature and compressive load monitoring and control. There will also be sampling the sweep gas effluent to determine if any oxidation or off-gassing of the specimens occurs during irradiation of the experiment. The first experiment, AGC-1, started its irradiation in September 2009, and the irradiation was completed in January 2011. The second experiment, AGC-2, started its irradiation in April 2011 and completed its irradiation in May 2012. This paper will briefly discuss the design of the experiment and control systems, and then present the irradiation results for each experiment to date.

  18. 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

  19. 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

  20. Specific Oncogenic Activity of the Src-Family Tyrosine Kinase c-Yes in Colon Carcinoma Cells

    PubMed Central

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

    2011-01-01

    c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src. PMID:21390316

  1. THE CONTRIBUTION OF TYRO3 FAMILY RECEPTOR TYROSINE KINASES TO THE HETEROGENEITY OF APOPTOTIC CELL UPTAKE BY MONONUCLEAR PHAGOCYTES

    PubMed Central

    Curtis, Jeffrey L.; Todt, Jill C.; Hu, Bin; Osterholzer, John J.; Freeman, Christine M.

    2014-01-01

    Mononuclear phagocytes comprise a mobile, broadly dispersed and highly adaptable system that lies at the very epicenter of host defense against pathogens and the interplay of the innate and adaptive arms of immunity. Understanding the molecular mechanisms that control the response of mononuclear phagocytes to apoptotic cells and the anti-inflammatory consequences of that response is an important goal with implications for multiple areas of biomedical sciences. This review details current understanding of the heterogeneity of apoptotic cell uptake by different members of the mononuclear phagocyte family in humans and mice. It also recounts the unique role of the Tyro3 family of receptor tyrosine kinases, best characterized for Mertk, in the signal transduction leading both to apoptotic cell ingestion and the anti-inflammatory effects that result. PMID:19273223

  2. Nercc1, a mammalian NIMA-family kinase, binds the Ran GTPase and regulates mitotic progression

    PubMed Central

    Roig, Joan; Mikhailov, Alexei; Belham, Christopher; Avruch, Joseph

    2002-01-01

    The protein kinase NIMA is an indispensable pleiotropic regulator of mitotic progression in Aspergillus. Although several mammalian NIMA-like kinases (Neks) are known, none appears to have the broad importance for mitotic regulation attributed to NIMA. Nercc1 is a new NIMA-like kinase that regulates chromosome alignment and segregation in mitosis. Its NIMA-like catalytic domain is followed by a noncatalytic tail containing seven repeats homologous to those of the Ran GEF, RCC1, a Ser/Thr/Pro-rich segment, and a coiled-coil domain. Nercc1 binds to another NIMA-like kinase, Nek6, and also binds specifically to the Ran GTPase through both its catalytic and its RCC1-like domains, preferring RanGDP in vivo. Nercc1 exists as a homooligomer and can autoactivate in vitro by autophosphorylation. Nercc1 is a cytoplasmic protein that is activated during mitosis and is avidly phosphorylated by active p34Cdc2. Microinjection of anti-Nercc1 antibodies in prophase results in spindle abnormalities and/or chromosomal misalignment. In Ptk2 cells the outcome is prometaphase arrest or aberrant chromosome segregation and aneuploidy, whereas in CFPAC-1 cells prolonged arrest in prometaphase is the usual response. Nercc1 and its partner Nek6 represent a new signaling pathway that regulates mitotic progression. PMID:12101123

  3. Navigating into the binding pockets of the HER family protein kinases: discovery of novel EGFR inhibitor as antitumor agent

    PubMed Central

    Liu, Wei; Ning, Jin-Feng; Meng, Qing-Wei; Hu, Jing; Zhao, Yan-Bin; Liu, Chao; Cai, Li

    2015-01-01

    The epidermal growth factor receptor (EGFR) family has been validated as a successful antitumor drug target for decades. Known EGFR inhibitors were exposed to distinct drug resistance against the various EGFR mutants within non-small-cell lung cancer (NSCLC), particularly the T790M mutation. Although so far a number of studies have been reported on the development of third-generation EGFR inhibitors for overcoming the resistance issue, the design procedure largely depends on the intuition of medicinal chemists. Here we retrospectively make a detailed analysis of the 42 EGFR family protein crystal complexes deposited in the Protein Data Bank (PDB). Based on the analysis of inhibitor binding modes in the kinase catalytic cleft, we identified a potent EGFR inhibitor (compound A-10) against drug-resistant EGFR through fragment-based drug design. This compound showed at least 30-fold more potency against EGFR T790M than the two control molecules erlotinib and gefitinib in vitro. Moreover, it could exhibit potent HER2 inhibitory activities as well as tumor growth inhibitory activity. Molecular docking studies revealed a structural basis for the increased potency and mutant selectivity of this compound. Compound A-10 may be selected as a promising candidate in further preclinical studies. In addition, our findings could provide a powerful strategy to identify novel selective kinase inhibitors on the basis of detailed kinase–ligand interaction space in the PDB. PMID:26229444

  4. Src family protein tyrosine kinase (PTK) modulates the effect of SGK1 and WNK4 on ROMK channels.

    PubMed

    Yue, Peng; Lin, Dao-Hong; Pan, Chun-Yang; Leng, Qiang; Giebisch, Gerhard; Lifton, Richard P; Wang, Wen-Hui

    2009-09-01

    WNK4 (with no lysine kinase 4) inhibits ROMK channel activity in the distal nephron by stimulating clathrin-dependent endocytosis, an effect attenuated by SGK1 (serum-glucocorticoids-induced kinase)-mediated phosphorylation. It has been suggested that increased ROMK activity because of SGK1-mediated inhibition of WNK4 plays a role in promoting renal K secretion in response to elevated serum K or high K (HK) intake. In contrast, intravascular volume depletion also increases SGK1 activity but fails to stimulate ROMK channels and K secretion. Because HK intake decreases Src family protein tyrosine kinase (PTK) activity an inhibitor of ROMK channels, it is possible that Src family PTK may modulate the effects of SGK1 on WNK4. Here, we show that c-Src prevents SGK1 from attenuating WNK4's inhibition of ROMK activity. This effect of c-Src was WNK4-dependent because c-Src had no effect on ROMK harboring mutation at the site of c-Src phosphorylation (R1Y337A) in the absence of WNK4. Moreover, expression c-Src diminished the SGK1-mediated increase in serine phosphorylation of WNK4, suggesting that c-Src enhances WNK4-mediated inhibition of ROMK channels by suppressing the SGK1-induced phosphorylation. This notion is also supported by the observation that c-Src was not able to modulate the interaction between SGK1 and WNK4 mutants (WNK4(S1169A) or WNK4(S1169D)) in which an SGK1-phosphorylation site (serine 1169) was mutated by alanine or aspartate. We conclude that c-Src inhibits SGK1-mediated phosphorylation hereby restoring the WNK4-mediated inhibition of ROMK channels thus suppressing K secretion. PMID:19706464

  5. A Robust Distributed Multipoint Fiber Optic Gas Sensor System Based on AGC Amplifier Structure.

    PubMed

    Zhu, Cunguang; Wang, Rende; Tao, Xuechen; Wang, Guangwei; Wang, Pengpeng

    2016-01-01

    A harsh environment-oriented distributed multipoint fiber optic gas sensor system realized by automatic gain control (AGC) technology is proposed. To improve the photoelectric signal reliability, the electronic variable gain can be modified in real time by an AGC closed-loop feedback structure to compensate for optical transmission loss which is caused by the fiber bend loss or other reasons. The deviation of the system based on AGC structure is below 4.02% when photoelectric signal decays due to fiber bending loss for bending radius of 5 mm, which is 20 times lower than the ordinary differential system. In addition, the AGC circuit with the same electric parameters can keep the baseline intensity of signals in different channels of the distributed multipoint sensor system at the same level. This avoids repetitive calibrations and streamlines the installation process. PMID:27483267

  6. Genome-Wide Analysis and Evolution of the Pto-Like Protein Kinase (PLPK) Gene Family in Pepper.

    PubMed

    Venkatesh, Jelli; Jahn, Molly; Kang, Byoung-Cheorl

    2016-01-01

    The tomato Pto gene, which encodes a serine/threonine kinase (STK) domain-containing protein, confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato (Pst). In this study, in vivo recognition assays using PVX constructs showed that AvrPto was specifically recognized in the pepper genotypes. This AvrPto recognition caused a nonhost hypersensitive response (HR) and localization of the PVX::AvrPto fusion protein to inoculated pepper leaf tissues, which indicates the presence of a similar Pto recognition mechanism in pepper as in tomato. However, genome-wide analysis in pepper revealed no Pto clade corresponding to that in tomato, suggesting an alternative system for Pto recognition in pepper. Nevertheless, 25 Pto-like protein kinases (PLPKs) with a highly conserved STK domain have been identified in the pepper genome. For the majority of the amino acid sites in the STK domain of Ptos and PLPKs, nonsynonymous (dN) to synonymous (dS) nucleotide substitution ratios (ω) were less than one, suggesting that purifying selection played a predominant role in the evolutionary process. However, some amino acid sites were found to be subjected to episodic positive selection in the course of evolution of Pto homologs, and, thus, different evolutionary processes might have shaped the Pto gene family in plants. Based on RNA-seq data, PLPK genes and other Pto pathway genes, such as Prf, Pti1, Pti5, and Pti6 were expressed in all tested pepper genotypes. Therefore, the nonhost HR against Pst in pepper may be due to the recognition of the AvrPto effector by a PLPK homolog, and subsequent action of downstream components of the Pto signaling pathway. However, the possibility remains that the recognition of AvrPto in pepper plants may involve activities of other receptor like kinases (RLKs). The identification of the PLPKs in this study will serve as a foundation for further efforts to understand the roles of PLPKs in nonhost resistance. PMID:27536870

  7. Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK)

    PubMed Central

    Lin, Dao-Hong; Yue, Peng; Yarborough, Orlando; Scholl, Ute I.; Giebisch, Gerhard; Lifton, Richard P.; Rinehart, Jesse; Wang, Wen-Hui

    2015-01-01

    With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr1092, Tyr1094, and Tyr1143, and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr1092 or Tyr1143 to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4Y1092/1094F. Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser1196, an effect that was abrogated in the double mutant. WNK4Y1143F inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4Y1143F could not be reversed by SGK1. The failure to reverse WNK4Y1143F-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4–PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4Y1143F-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr1092 and Tyr1143 plays an important role in modulating the inhibitory effect of WNK4 on ROMK. PMID:25805816

  8. Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK).

    PubMed

    Lin, Dao-Hong; Yue, Peng; Yarborough, Orlando; Scholl, Ute I; Giebisch, Gerhard; Lifton, Richard P; Rinehart, Jesse; Wang, Wen-Hui

    2015-04-01

    With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr(1092), Tyr(1094), and Tyr(1143), and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr(1092) or Tyr(1143) to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4(Y1092/1094F). Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser(1196), an effect that was abrogated in the double mutant. WNK4(Y1143F) inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4(Y1143F) could not be reversed by SGK1. The failure to reverse WNK4(Y1143F)-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4-PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4(Y1143F)-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr(1092) and Tyr(1143) plays an important role in modulating the inhibitory effect of WNK4 on ROMK. PMID:25805816

  9. Genome-Wide Analysis and Evolution of the Pto-Like Protein Kinase (PLPK) Gene Family in Pepper

    PubMed Central

    Venkatesh, Jelli; Jahn, Molly; Kang, Byoung-Cheorl

    2016-01-01

    The tomato Pto gene, which encodes a serine/threonine kinase (STK) domain-containing protein, confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato (Pst). In this study, in vivo recognition assays using PVX constructs showed that AvrPto was specifically recognized in the pepper genotypes. This AvrPto recognition caused a nonhost hypersensitive response (HR) and localization of the PVX::AvrPto fusion protein to inoculated pepper leaf tissues, which indicates the presence of a similar Pto recognition mechanism in pepper as in tomato. However, genome-wide analysis in pepper revealed no Pto clade corresponding to that in tomato, suggesting an alternative system for Pto recognition in pepper. Nevertheless, 25 Pto-like protein kinases (PLPKs) with a highly conserved STK domain have been identified in the pepper genome. For the majority of the amino acid sites in the STK domain of Ptos and PLPKs, nonsynonymous (dN) to synonymous (dS) nucleotide substitution ratios (ω) were less than one, suggesting that purifying selection played a predominant role in the evolutionary process. However, some amino acid sites were found to be subjected to episodic positive selection in the course of evolution of Pto homologs, and, thus, different evolutionary processes might have shaped the Pto gene family in plants. Based on RNA-seq data, PLPK genes and other Pto pathway genes, such as Prf, Pti1, Pti5, and Pti6 were expressed in all tested pepper genotypes. Therefore, the nonhost HR against Pst in pepper may be due to the recognition of the AvrPto effector by a PLPK homolog, and subsequent action of downstream components of the Pto signaling pathway. However, the possibility remains that the recognition of AvrPto in pepper plants may involve activities of other receptor like kinases (RLKs). The identification of the PLPKs in this study will serve as a foundation for further efforts to understand the roles of PLPKs in nonhost resistance. PMID:27536870

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

    PubMed Central

    2015-01-01

    Phospholipids are highly conserved and essential components of biological membranes. The major phospholipids, phosphatidylethanolamine and phosphatidylcholine (PtdCho), are synthesized by the transfer of the phosphoethanolamine or phosphocholine polar head group, respectively, to the diacylglycerol backbone. The metabolism of the polar head group characterizing each phospholipid class is poorly understood; thus, the biosynthetic pathway of major phospholipids remains elusive in Arabidopsis thaliana. The choline/ethanolamine kinase (CEK) family catalyzes the initial steps of phospholipid biosynthesis. Here, we analyzed the function of the four CEK family members present in Arabidopsis. Knocking out of CEK4 resulted in defective embryo development, which was complemented by transformation of genomic CEK4. Reciprocal genetic crossing suggested that CEK4 knockout causes embryonic lethality, and microscopy analysis of the aborted embryos revealed developmental arrest after the heart stage, with no defect being found in the pollen. CEK4 is preferentially expressed in the vasculature, organ boundaries, and mature embryos, and CEK4 was mainly localized to the plasma membrane. Overexpression of CEK4 in wild-type Arabidopsis increased the levels of PtdCho in seedlings and mature siliques and of major membrane lipids in seedlings and triacylglycerol in mature siliques. CEK4 may be the plasma membrane-localized isoform of the CEK family involved in the rate-limiting step of PtdCho biosynthesis and appears to be required for embryo development in Arabidopsis. PMID:25966764

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

    PubMed

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

    2015-05-01

    Phospholipids are highly conserved and essential components of biological membranes. The major phospholipids, phosphatidylethanolamine and phosphatidylcholine (PtdCho), are synthesized by the transfer of the phosphoethanolamine or phosphocholine polar head group, respectively, to the diacylglycerol backbone. The metabolism of the polar head group characterizing each phospholipid class is poorly understood; thus, the biosynthetic pathway of major phospholipids remains elusive in Arabidopsis thaliana. The choline/ethanolamine kinase (CEK) family catalyzes the initial steps of phospholipid biosynthesis. Here, we analyzed the function of the four CEK family members present in Arabidopsis. Knocking out of CEK4 resulted in defective embryo development, which was complemented by transformation of genomic CEK4. Reciprocal genetic crossing suggested that CEK4 knockout causes embryonic lethality, and microscopy analysis of the aborted embryos revealed developmental arrest after the heart stage, with no defect being found in the pollen. CEK4 is preferentially expressed in the vasculature, organ boundaries, and mature embryos, and CEK4 was mainly localized to the plasma membrane. Overexpression of CEK4 in wild-type Arabidopsis increased the levels of PtdCho in seedlings and mature siliques and of major membrane lipids in seedlings and triacylglycerol in mature siliques. CEK4 may be the plasma membrane-localized isoform of the CEK family involved in the rate-limiting step of PtdCho biosynthesis and appears to be required for embryo development in Arabidopsis. PMID:25966764

  12. Interleukin-1β activates an Src family kinase to stimulate the plasma membrane Ca2+ pump in hippocampal neurons.

    PubMed

    Ghosh, Biswarup; Green, Matthew V; Krogh, Kelly A; Thayer, Stanley A

    2016-04-01

    The plasma membrane Ca(2+) ATPase (PMCA) plays a major role in clearing Ca(2+) from the neuronal cytoplasm. The cytoplasmic Ca(2+) clearance rate affects neuronal excitability, synaptic plasticity, and neurotransmission. Here, we examined the modulation of PMCA activity by PTKs in hippocampal neurons. PMCA-mediated Ca(2+) clearance slowed in the presence of pyrazolopyrimidine 2, an inhibitor of Src family kinases (SFKs), and accelerated in the presence of C2-ceramide, an activator of PTKs. Ca(2+) clearance kinetics were attenuated in cells expressing a dominant-negative Src mutant, suggesting that the pump is tonically stimulated by a PTK. Tonic stimulation was reduced in hippocampal neurons expressing short hairpin (sh)RNA directed to mRNA for Yes. shRNA-mediated knockdown of PMCA isoform 1 (PMCA1) removed tonic stimulation of Ca(2+) clearance, indicating that the kinase stimulates PMCA1. IL-1β accelerated Ca(2+) clearance in a manner blocked by an IL-1β receptor antagonist or by an inhibitor of neutral sphingomyelinase, the enzyme that produces ceramide. Thus IL-1β activates an SFK to stimulate the plasma membrane Ca(2+) pump, decreasing the duration of Ca(2+) transients in hippocampal neurons. PMID:26843596

  13. Anti-inflammatory effects of Perilla frutescens in activated human neutrophils through two independent pathways: Src family kinases and Calcium

    PubMed Central

    Chen, Chun-Yu; Leu, Yann-Lii; Fang, Yu; Lin, Chwan-Fwu; Kuo, Liang-Mou; Sung, Wei-Che; Tsai, Yung-Fong; Chung, Pei-Jen; Lee, Ming-Chung; Kuo, Yu-Ting; Yang, Hsuan-Wu; Hwang, Tsong-Long

    2015-01-01

    The leaves of Perilla frutescens (L.) Britt. have been traditionally used as an herbal medicine in East Asian countries to treat a variety diseases. In this present study, we investigated the inhibitory effects of P. frutescens extract (PFE) on N-formyl-Met-Leu-Phe (fMLF)-stimulated human neutrophils and the underlying mechanisms. PFE (1, 3, and 10 μg/ml) inhibited superoxide anion production, elastase release, reactive oxygen species formation, CD11b expression, and cell migration in fMLF-activated human neutrophils in dose-dependent manners. PFE inhibited fMLF-induced phosphorylation of the Src family kinases (SFKs), Src (Tyr416) and Lyn (Tyr396), and reduced their enzymatic activities. Both PFE and PP2 (a selective inhibitor of SFKs) reduced the phosphorylation of Burton’s tyrosine kinases (Tyr223) and Vav (Tyr174) in fMLF-activated human neutrophils. Additionally, PFE decreased intracellular Ca2+ levels ([Ca2+]i), whereas PP2 prolonged the time required for [Ca2+]i to return to its basal level. Our findings indicated that PFE effectively regulated the inflammatory activities of fMLF-activated human neutrophils. The anti-inflammatory effects of PFE on activated human neutrophils were mediated through two independent signaling pathways involving SFKs (Src and Lyn) and mobilization of intracellular Ca2+. PMID:26659126

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

    PubMed Central

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

    2016-01-01

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

  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. Anti-inflammatory effects of Perilla frutescens in activated human neutrophils through two independent pathways: Src family kinases and Calcium.

    PubMed

    Chen, Chun-Yu; Leu, Yann-Lii; Fang, Yu; Lin, Chwan-Fwu; Kuo, Liang-Mou; Sung, Wei-Che; Tsai, Yung-Fong; Chung, Pei-Jen; Lee, Ming-Chung; Kuo, Yu-Ting; Yang, Hsuan-Wu; Hwang, Tsong-Long

    2015-01-01

    The leaves of Perilla frutescens (L.) Britt. have been traditionally used as an herbal medicine in East Asian countries to treat a variety diseases. In this present study, we investigated the inhibitory effects of P. frutescens extract (PFE) on N-formyl-Met-Leu-Phe (fMLF)-stimulated human neutrophils and the underlying mechanisms. PFE (1, 3, and 10 μg/ml) inhibited superoxide anion production, elastase release, reactive oxygen species formation, CD11b expression, and cell migration in fMLF-activated human neutrophils in dose-dependent manners. PFE inhibited fMLF-induced phosphorylation of the Src family kinases (SFKs), Src (Tyr416) and Lyn (Tyr396), and reduced their enzymatic activities. Both PFE and PP2 (a selective inhibitor of SFKs) reduced the phosphorylation of Burton's tyrosine kinases (Tyr223) and Vav (Tyr174) in fMLF-activated human neutrophils. Additionally, PFE decreased intracellular Ca(2+) levels ([Ca(2+)]i), whereas PP2 prolonged the time required for [Ca(2+)]i to return to its basal level. Our findings indicated that PFE effectively regulated the inflammatory activities of fMLF-activated human neutrophils. The anti-inflammatory effects of PFE on activated human neutrophils were mediated through two independent signaling pathways involving SFKs (Src and Lyn) and mobilization of intracellular Ca(2+). PMID:26659126

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

    PubMed

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

    2016-01-01

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

  18. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    SciTech Connect

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  19. A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish.

    PubMed

    Liu, Shanming; Lu, Weining; Obara, Tomoko; Kuida, Shiei; Lehoczky, Jennifer; Dewar, Ken; Drummond, Iain A; Beier, David R

    2002-12-01

    The murine autosomal recessive juvenile cystic kidney (jck) mutation results in polycystic kidney disease. We have identified in jck mice a mutation in Nek8, a novel and highly conserved member of the Nek kinase family. In vitro expression of mutated Nek8 results in enlarged, multinucleated cells with an abnormal actin cytoskeleton. To confirm that a defect in the Nek8 gene can cause cystic disease, we performed a cross-species analysis: injection of zebrafish embryos with a morpholino anti-sense oligonucleotide corresponding to the ortholog of Nek8 resulted in the formation of pronephric cysts. These results demonstrate that comparative analysis of gene function in different model systems represents a powerful means to annotate gene function. PMID:12421721

  20. 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

  1. The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity

    PubMed Central

    Chan, Pamela Y.; Carrera Silva, Eugenio A.; De Kouchkovsky, Dimitri; Joannas, Leonel D.; Hao, Liming; Hu, Donglei; Huntsman, Scott; Eng, Celeste; Licona-Limón, Paula; Weinstein, Jason S.; Herbert, De’Broski R.; Craft, Joseph E.; Flavell, Richard A.; Repetto, Silvia; Correale, Jorge; Burchard, Esteban G.; Torgerson, Dara G.; Ghosh, Sourav; Rothlin, Carla V.

    2016-01-01

    Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown. Here, we show that genetic ablation of a receptor tyrosine kinase encoded by Tyro3 in mice or the functional neutralization of its ortholog in human dendritic cells resulted in enhanced type 2 immunity. Furthermore, the TYRO3 agonist PROS1 was induced in T cells by the quintessential type 2 cytokine, interleukin-4. T cell–specific Pros1 knockouts phenocopied the loss of Tyro3. Thus, a PROS1-mediated feedback from adaptive immunity engages a rheostat, TYRO3, on innate immune cells to limit the intensity of type 2 responses. PMID:27034374

  2. The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity.

    PubMed

    Chan, Pamela Y; Carrera Silva, Eugenio A; De Kouchkovsky, Dimitri; Joannas, Leonel D; Hao, Liming; Hu, Donglei; Huntsman, Scott; Eng, Celeste; Licona-Limón, Paula; Weinstein, Jason S; Herbert, De'Broski R; Craft, Joseph E; Flavell, Richard A; Repetto, Silvia; Correale, Jorge; Burchard, Esteban G; Torgerson, Dara G; Ghosh, Sourav; Rothlin, Carla V

    2016-04-01

    Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown. Here, we show that genetic ablation of a receptor tyrosine kinase encoded byTyro3in mice or the functional neutralization of its ortholog in human dendritic cells resulted in enhanced type 2 immunity. Furthermore, the TYRO3 agonist PROS1 was induced in T cells by the quintessential type 2 cytokine, interleukin-4. T cell-specificPros1knockouts phenocopied the loss ofTyro3 Thus, a PROS1-mediated feedback from adaptive immunity engages a rheostat, TYRO3, on innate immune cells to limit the intensity of type 2 responses. PMID:27034374

  3. [p53 activation by PI-3K family kinases after DNA double-strand breaks].

    PubMed

    Pernin, D; Uhrhammer, N; Verrelle, P; Bignon, Y J; Bay, J O

    2000-09-01

    p53 plays a central role in the cellular response to DNA double-strand breaks (DSBs), and to DNA damage in general. The protein kinases ATM, ATR and DNA-PK detect DSBs and transmit this information to p53 by phosphorylation. This phosphorylation dissociates p53 from its negative regulator, mdm2. p53 then undergoes further modification and activates transcription of the genes responsible for cell cycle arrest. In certain circumstances, p53 also activates transcription of the genes responsible for apoptosis. The dysfunction of this cascade of events is oncogenic, with P53 itself being the most commonly mutated gene in malignant cells, although mutations in both the DNA damage sensors and cell cycle checkpoint and apoptosis effectors are frequent. A more complete understanding of p53 and the proteins it interacts with may allow the development of new cancer treatments. PMID:11038413

  4. p70 S6 kinase and actin dynamics

    PubMed Central

    Ip, Carman K.M.; Wong, Alice S.T.

    2012-01-01

    p70 S6 kinase (p70S6K), a member of the AGC serine/threonine kinase family, was initially identified as a key player, together with its downstream effector S6, in the regulation of cellular growth and survival. The p70S6K protein has emerged in recent years as a multifunctional protein which also regulates the actin cytoskeleton and thus plays a role in cell migration. This new function is through two important activities of p70S6K, namely actin cross-linking and Rac1 and Cdc42 activation. The testis is critically dependent on an intricate balance of fundamental cellular processes such as adhesion, migration, and differentiation. It is increasingly evident that Rho GTPases and actin binding proteins play fundamental roles in regulating spermatogenesis within the testis. In this review, we will discuss current findings of p70S6K in the control of actin cytoskeleton dynamics. In addition, the potential role of p70S6K in spermatogenesis and testicular function will be highlighted. PMID:22553489

  5. Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity.

    PubMed

    Weilinger, Nicholas L; Lohman, Alexander W; Rakai, Brooke D; Ma, Evelyn M M; Bialecki, Jennifer; Maslieieva, Valentyna; Rilea, Travis; Bandet, Mischa V; Ikuta, Nathan T; Scott, Lucas; Colicos, Michael A; Teskey, G Campbell; Winship, Ian R; Thompson, Roger J

    2016-03-01

    Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca(2+)-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg(2+) or a Ca(2+)-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology. PMID:26854804

  6. REQUIREMENT OF THE TEC FAMILY TYROSINE KINASE BTK29A FOR COURTSHIP MEMORY IN Drosophila MALES.

    PubMed

    Sunouchi, Kazuya; Koganezawa, Masayuki; Yamamoto, Daisuke

    2016-03-01

    A male Drosophila that is not successful in courtship will reduce his courtship efforts in the next encounter with a female. This courtship suppression persists for more than 1 h in wild-type males. The Btk29A(ficP) mutant males null for the Btk29A type 2 isoform, a fly homolog of the nonreceptor tyrosine kinase Btk, show no courtship suppression, while Btk29A hypomorphic males exhibit a rapid decline in courtship suppression, leading to its complete loss within 30 min. The males of a revertant stock or Btk29A(ficP) males that are also mutant for parkas, a gene encoding the presumptive negative regulator of Btk29A, exhibit normal courtship suppression. Since another behavioral assay has shown that Btk29A(ficP) mutants are sensitization-defective, we hypothesize that the mutant flies are unable to maintain the neural excitation state acquired by experience, resulting in the rapid loss of courtship suppression. PMID:26782301

  7. Detection of a novel missense mutation in the mevalonate kinase gene in one Chinese family with DSAP

    PubMed Central

    Lu, Wen-Sheng; Zheng, Xiao-Dong; Yao, Xiu-Hua; Zhang, Lan-Fang; Hu, Bai; Lu, Yao-Juan

    2014-01-01

    Disseminated superficial actinic porokeratosis (DSAP) is the most common form of porokeratosis and a severe chronic autosomal dominant cutaneous disorder with high genetic heterogeneity. Recently, the mevalonate kinase (MVK) gene has been identified as a candidate gene responsible for DSAP and multiple mutations have been reported. Here, we report identification of a novel missense mutation in the MVK gene in a Chinese family with DSAP. A 50-year-old male was diagnosed as proband of DSAP based on the clinical and histological findings, which show numerous hyperpigmented macules by physical examination and cornoid lamella by skin biopsy. Similar skin symptoms were also observed in his father, who died many years ago. We prepared genomic DNA from the proband, unaffected individuals from his family members, as well as 100 unrelated healthy controls. PCR was then conducted using the above genomic DNA as template and the MVK gene-specific primers. The PCR product was subjected to direct sequencing and the sequence was compared to that of MVK gene within the NCBI database. We detected a heterozygous C to G transition at nucleotide 643 in exon 7 of MVK gene of the proband. This will result in an amino acid change at codon 215 (P.Arg215Gly.), which is from an arginine codon (CGA) to a Glycine codon (GGA). We did not detect any mutation in the unaffected family members or the 100 unrelated healthy controls, demonstrating that this is a novel missense mutation in MVK gene and therefore, contributes to the molecular diagnosis of DSAP. PMID:24551296

  8. Design and performance test of a MEMS vibratory gyroscope with a novel AGC force rebalance control

    NASA Astrophysics Data System (ADS)

    Sung, Woon-Tahk; Sung, Sangkyung; Lee, Jang Gyu; Kang, Taesam

    2007-10-01

    In this paper, the development and performance test results of a laterally oscillating MEMS gyroscope using a novel force rebalance control strategy are presented. The micromachined structure and electrodes are fabricated using the deep reactive ion etching (DRIE) and anodic wafer bonding process. The high quality factor required for the resonance-based sensor is achieved using a vacuum-sealed device package. A systematic design approach of the force rebalance control is applied via a modified automatic gain control (AGC) method. The rebalance control design takes advantages of a novel AGC loop modification, which allows the approximation of the system's dynamics into a simple linear form. Using the proposed modification of AGC and the rebalance strategy that maintains a biased oscillation, a number of performance improvements including bandwidth extension and widened operating range were observed to be achieved. Finally, the experimental results of the gyroscope's practical application verify the feasibility and performance of the developed sensor.

  9. Evolution of the Phosphoenolpyruvate Carboxylase Protein Kinase Family in C3 and C4 Flaveria spp.1[W][OPEN

    PubMed Central

    Aldous, Sophia H.; Weise, Sean E.; Sharkey, Thomas D.; Waldera-Lupa, Daniel M.; Stühler, Kai; Mallmann, Julia; Groth, Georg; Gowik, Udo; Westhoff, Peter; Arsova, Borjana

    2014-01-01

    The key enzyme for C4 photosynthesis, Phosphoenolpyruvate Carboxylase (PEPC), evolved from nonphotosynthetic PEPC found in C3 ancestors. In all plants, PEPC is phosphorylated by Phosphoenolpyruvate Carboxylase Protein Kinase (PPCK). However, differences in the phosphorylation pattern exist among plants with these photosynthetic types, and it is still not clear if they are due to interspecies differences or depend on photosynthetic type. The genus Flaveria contains closely related C3, C3-C4 intermediate, and C4 species, which are evolutionarily young and thus well suited for comparative analysis. To characterize the evolutionary differences in PPCK between plants with C3 and C4 photosynthesis, transcriptome libraries from nine Flaveria spp. were used, and a two-member PPCK family (PPCKA and PPCKB) was identified. Sequence analysis identified a number of C3- and C4-specific residues with various occurrences in the intermediates. Quantitative analysis of transcriptome data revealed that PPCKA and PPCKB exhibit inverse diel expression patterns and that C3 and C4 Flaveria spp. differ in the expression levels of these genes. PPCKA has maximal expression levels during the day, whereas PPCKB has maximal expression during the night. Phosphorylation patterns of PEPC varied among C3 and C4 Flaveria spp. too, with PEPC from the C4 species being predominantly phosphorylated throughout the day, while in the C3 species the phosphorylation level was maintained during the entire 24 h. Since C4 Flaveria spp. evolved from C3 ancestors, this work links the evolutionary changes in sequence, PPCK expression, and phosphorylation pattern to an evolutionary phase shift of kinase activity from a C3 to a C4 mode. PMID:24850859

  10. Two Arabidopsis AGC kinases are critical for the polarized growth of pollen tubes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reproduction of flowering plants requires the growth of pollen tubes to deliver immotile sperm for fertilization. Pollen tube growth resembles that of polarized metazoan cells, in that some molecular mechanisms underlying cell polarization and growth are evolutionarily conserved, including the funct...

  11. Multi-area adaptive LFC developed for a comprehensive AGC simulator

    SciTech Connect

    Shoults, R.R. . Energy Systems Research Center); Ibarra, J.A.J. )

    1993-05-01

    A multi-area adaptive Load Frequency Control (LFC) based on the Self-Tuning Regulator (STR) for a comprehensive Automatic Generator Control Simulator (AGCS) is investigated. A multi-area AGCS comprising the dynamic as well as the static solutions of an interconnected power system has been implemented to accommodate a control center with adaptive LFC features. Simulation results from a three-area system driven by realistic load compositions show that the STR presents better tracking properties than the integral or the combination of integral and STR controllers.

  12. Data Report on Post-Irradiation Dimensional Change of AGC-1 Samples

    SciTech Connect

    William Windes

    2012-06-01

    This report summarizes the initial dimensional changes for loaded and unloaded AGC-1 samples. The dimensional change for all samples is presented as a function of dose. The data is further presented by graphite type and applied load levels to illustrate the differences between graphite forming processes and stress levels within the graphite components. While the three different loads placed on the samples have been verified [ ref: Larry Hull’s report] verification of the AGC-1 sample temperatures and dose levels are expected in the summer of 2012. Only estimated dose and temperature values for the samples are presented in this report to allow a partial analysis of the results.

  13. Activation of the lutropin/choriogonadotropin receptor (LHR) in MA-10 cells leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) by a pathway that involves Src family kinases*

    PubMed Central

    Mizutani, Tetsuya; Shiraishi, Koji; Welsh, Toni; Ascoli, Mario

    2006-01-01

    We show that activation of the endogenous or recombinant LHR in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) and one of its substrates (paxillin). Using specific antibodies to the five tyrosine residues of FAK that become phosphorylated we show that activation of the LHR increases the phosphorylation of Tyr576 and Tyr577 but it does not affect the phosphorylation of Tyr397, Tyr861 or Tyr925. Because FAK is a prominent substrate for the Src family of tyrosine kinases (SFKs) we tested for their involvement in the LHR-mediated phosphorylation of FAK-Tyr576. Src is not detectable in MA-10 cells, but two other prominent members of this family (Fyn and Yes) are present. The LHR-mediated phosphorylation of FAK-Tyr576 is readily inhibited by PP2 (a pharmacological inhibitor of SFKs) and by dominant-negative mutants of SKFs. Moreover, activation of the LHR in MA-10 cells results in the stimulation of the activity of Fyn and Yes and overexpression of either of these two tyrosine kinases enhances the LHR-mediate phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different Gα subunits, and the use of second messenger analogs suggest that the LHR-induced phosphorylation of FAK-Tyr576 in MA-10 cells is mediated by SFKs, and that this family of kinases is, in turn, independently or cooperatively activated by the LHR-induced stimulation of Gs and Gq/11-mediated pathways. PMID:16293639

  14. A small molecule inhibitor of SRC family kinases promotes simple epithelial differentiation of human pluripotent stem cells.

    PubMed

    Lian, Xiaojun; Selekman, Joshua; Bao, Xiaoping; Hsiao, Cheston; Zhu, Kexian; Palecek, Sean P

    2013-01-01

    Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs, the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of β-catenin during retinoic acid (RA)--induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated β-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8), which are expressed in simple epithelial cells, while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4- simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications. PMID:23527294

  15. The role of the Janus kinase family/signal transducer and activator of transcription signaling pathway in fibrotic renal disease

    PubMed Central

    Matsui, Futoshi; Meldrum, Kirstan K.

    2012-01-01

    Over the past several years, a number of cytokines and growth factors including transforming growth factor β1, tumor necrosis factor α, and angiotensin II have been shown to play a crucial role in renal fibrosis. The Janus kinase family (JAK) and signal transducers and activators of transcription (STATs) constitute one of the primary signaling pathways that regulate cytokine expression, and the JAK/STAT signaling pathway has increasingly been implicated in the pathophysiology of renal disease. This review examines the role of the JAK/STAT signaling pathway in fibrotic renal disease. The JAK/STAT signaling pathway is activated in a variety of renal diseases and has been implicated in the pathophysiology of renal fibrosis. Experimental evidence suggests that inhibition of the JAK/STAT signaling pathway, in particular JAK2 and STAT3, may suppress renal fibrosis and protect renal function. However, it is incompletely understood which cells activate the JAK/STAT signaling pathway and which JAK/STAT signaling pathway is activated in each renal disease. Research regarding JAK/STAT signaling and its contribution to renal disease is still ongoing in humans. Future studies are required to elucidate the potential role of JAK/STAT signaling inhibition as a therapeutic strategy in the attenuation of renal fibrosis. PMID:22883438

  16. Genome-wide survey and expression analysis of the calcium-dependent protein kinase gene family in cassava.

    PubMed

    Hu, Wei; Hou, Xiaowan; Xia, Zhiqiang; Yan, Yan; Wei, Yunxie; Wang, Lianzhe; Zou, Meiling; Lu, Cheng; Wang, Wenquan; Peng, Ming

    2016-02-01

    Calcium-dependent protein kinases (CPKs) play important roles in regulating plant tolerance to abiotic stress and signal transduction; however, no data are currently available regarding the CPK family in cassava. Herein, we identified 27 CPK genes from cassava based on our previous genome sequencing data. Phylogenetic analysis showed that cassava CPKs could be clustered into three groups, which was further supported by gene structure and conserved protein motif analyses. Global expression analysis suggested that MeCPK genes showed distinct expression patterns in different tissues between wild subspecies and cultivated varieties, indicating their involvement in the functional diversity of different varieties. Transcriptomics, interaction networks, and co-expression assays revealed a broad transcriptional response of cassava CPKs and CPK-mediated networks to drought stress and their differential expression profiles in different varieties, implying their contribution to drought stress tolerance in cassava. Expression analysis of eight MeCPK genes suggested a comprehensive response to osmotic stress, salt, cold, abscisic acid, and H2O2, which indicated that cassava CPKs might be convergence points for different signaling pathways. This study provides a basis for crop improvements and understanding of abiotic stress responses and signal transduction mediated by CPKs in cassava. PMID:26272723

  17. The kinesin I family member KIF5C is a novel substrate for protein kinase CK2

    SciTech Connect

    Schaefer, Barbara; Goetz, Claudia; Montenarh, Mathias

    2008-10-17

    Protein kinase CK2 is ubiquitously expressed. The holoenzyme is composed of two catalytic {alpha}- or {alpha}'-subunits and two regulatory {beta}-subunits but evidence is accumulating that the subunits can function independently. The composition of the holoenzyme as well as the expression of the individual subunits varies in different tissues, with high expression of CK2{alpha}' in testis and brain. CK2 phosphorylates a number of different substrates which are implicated in basal cellular processes such as proliferation and survival of cells. Here, we report a new substrate, KIF5C, which is a member of the kinesin 1 family of motor neuron proteins. Phosphorylation of KIF5C was demonstrated in vitro and in vivo. Using deletion mutants, a peptide library, and mutation analysis a phosphorylation site for CK2 was mapped to amino acid 338 which is located in the non-motor domain of KIF5C. Interestingly, KIF5C is phosphorylated by holoenzymes composed of CK2{alpha}/CK2{beta} and CK2{alpha}'/CK2{beta} as well as by CK2{alpha}' alone but not by CK2{alpha} alone.

  18. Identification of a BET family Bromodomain / Casein Kinase II / TAF-containing complex as a regulator of mitotic condensin function

    PubMed Central

    Kim, Hyun-Soo; Mukhopadhyay, Rituparna; Rothbart, Scott B.; Silva, Andrea C.; Vanoosthuyse, Vincent; Radovani, Ernest; Kislinger, Thomas; Roguev, Assen; Ryan, Colm J.; Xu, Jiewei; Jahari, Harlizawati; Hardwick, Kevin G.; Greenblatt, Jack F.; Krogan, Nevan J.; Fillingham, Jeffrey S.; Strahl, Brian D.; Bouhassira, Eric E.; Edelmann, Winfried; Keogh, Michael-Christopher

    2014-01-01

    SUMMARY Condensin is a central regulator of mitotic genome structure, with mutants showing poorly condensed chromosomes and profound segregation defects. Here we identify NCT complex, comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), Casein Kinase II (CKII) and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions, but only briefly co-localize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell cycle-directed manner to modulate the activity of condensin during chromosome condensation and decondensation. PMID:24565511

  19. SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2 (SNRK2): A FAMILY OF PROTEIN KINASES INVOLVED IN HYPEROSMOTIC STRESS SIGNALING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our understanding of plant adaptation to abiotic stresses, which include drought, salinity, non-optimal temperatures and poor soil nutrition, is still limiting although significant strides have been made in identifying some of the gene players and signaling partners. Several protein kinases get acti...

  20. RF Variable-Gain Amplifiers and AGC Loops for Digital TV Receivers

    NASA Astrophysics Data System (ADS)

    Iizuka, Kunihiko; Koutani, Masato; Mitsunaka, Takeshi; Kawamura, Hiroshi; Toyoyama, Shinji; Miyamoto, Masayuki; Matsuzawa, Akira

    RF Variable Gain Amplifiers (RF-VGA) are important components for integrated TV broadcast receivers. Analog and digital controlled RF-VGAs are compared in terms of linearity and an AGC loop architecture suitable for digitally controlled RF-VGA is proposed. Further linearity enhancement applicable for CMOS implementation is also discussed.

  1. ASASSN-16ke: Discovery of A Probable Supernova in AGC 331536

    NASA Astrophysics Data System (ADS)

    Brimacombe, J.; Brown, J. S.; Stanek, K. Z.; Holoien, T. W.-S.; Kochanek, C. S.; Shields, J.; Shappee, B. J.; Prieto, J. L.; Bersier, D.; Dong, Subo; Bose, S.; Chen, Ping; Bock, G.; Cruz, I.; Marples, P.; Post, R. S.

    2016-09-01

    During the ongoing All Sky Automated Survey for SuperNovae (ASAS-SN or "Assassin"), using data from the quadruple 14-cm "Brutus" telescope in Haleakala, Hawaii, we discovered a new transient source, most likely a supernova, in the galaxy AGC 331536.

  2. Bifunctional Ag/C3N4.5 composite nanobelts for photocatalysis and antibacterium.

    PubMed

    Lei, Renbo; Jian, Jikang; Zhang, Zhihua; Song, Bo; Wu, Rong

    2016-09-30

    Multiple functions can be achieved in carbon nitride-based composite nanomaterials by tuning their components and structures. Here, we report on a large-scale synthesis of novel bifunctional Ag/C3N4.5 composite nanobelts (CNBs) with efficient photocatalytic and antibacterial activity. The Ag/C3N4.5 CNBs were synthesized in high yield by a two-step route including a homogeneous precipitation process and a subsequent calcination treatment. The structural, morphological, compositional, and spectroscopic characterizations revealed that the Ag/C3N4.5 CNBs are composed of N-deficient melem ultrathin nanobelts and crystalline Ag nanoparticles attached to the surface of the nanobelts with good contact. The band gap of the Ag/C3N4.5 CNBs is determined to be about 3.04 eV. The efficient photocatalytic and antibacterial activities of the composite nanomaterials are verified by testing the degradation of Rhodamine B (RhB) and the inhibition zone to bacterium E. coli. The work provides a facile route to bifunctional carbon nitride-based composites with potential applications in the fields of the environment and biology. PMID:27560165

  3. SHIELD II: AGC 198507 - An Extremely Rare Low-Mass Galaxy Interaction?

    NASA Astrophysics Data System (ADS)

    Nikolina Borg Stevens, Karin; Cannon, John M.; McNichols, Andrew; McQuinn, Kristen B.; Teich, Yaron; SHIELD II Team

    2016-01-01

    The "Survey of HI in Extremely Low-mass Dwarfs II" ("SHIELD II") is a multiwavelength, legacy-class observational campaign that is facilitating the study of both internal and global evolutionary processes in low-mass dwarf galaxies discovered by the Arecibo Legacy Fast ALFA (ALFALFA) survey. New HST imaging of one of these sample galaxies, AGC 198507, has revealed it to be a very rare interacting system; to our knowledge this is one of only a few known interactions in this extreme mass range. WSRT imaging indicates that the bulk of the HI is associated with the more luminous AGC 198507, while low surface brightness gas extends toward and coincides with the less luminous companion, which is separated by roughly 1.5 kpc from AGC 198507. Here we present new VLA B configuration HI imaging that allows us to localize the HI gas, to examine the rotational dynamics of AGC 198507, and to study the nature of star formation in this unique low-mass interacting system.Support for this work was provided by NSF grant AST-1211683 to JMC at Macalester College, and by NASA through grant GO-13750 from the Space Telescope Science Institute, which is operated by AURA, Inc., under NASA contract NAS5-26555.

  4. ALFALFA Discovery of the Most Metal-poor Gas-rich Galaxy Known: AGC 198691

    NASA Astrophysics Data System (ADS)

    Hirschauer, Alec S.; Salzer, John J.; Skillman, Evan D.; Berg, Danielle; McQuinn, Kristen B. W.; Cannon, John M.; Gordon, Alex J. R.; Haynes, Martha P.; Giovanelli, Riccardo; Adams, Elizabeth A. K.; Janowiecki, Steven; Rhode, Katherine L.; Pogge, Richard W.; Croxall, Kevin V.; Aver, Erik

    2016-05-01

    We present spectroscopic observations of the nearby dwarf galaxy AGC 198691. This object is part of the Survey of H i in Extremely Low-Mass Dwarfs project, which is a multi-wavelength study of galaxies with H i masses in the range of 106–107.2 M ⊙, discovered by the Arecibo Legacy Fast ALFA (ALFALFA) survey. We have obtained spectra of the lone H ii region in AGC 198691 with the new high-throughput KPNO Ohio State Multi-Object Spectrograph on the Mayall 4 m, as well as with the Blue Channel spectrograph on the MMT 6.5 m telescope. These observations enable the measurement of the temperature-sensitive [O iii]λ4363 line and hence the determination of a “direct” oxygen abundance for AGC 198691. We find this system to be an extremely metal-deficient (XMD) system with an oxygen abundance of 12+log(O/H) = 7.02 ± 0.03, making AGC 198691 the lowest-abundance star-forming galaxy known in the local universe. Two of the five lowest-abundance galaxies known have been discovered by the ALFALFA blind H i survey; this high yield of XMD galaxies represents a paradigm shift in the search for extremely metal-poor galaxies.

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

    PubMed Central

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

    2016-01-01

    Summary In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality. PMID:26774483

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

    PubMed

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

    2016-01-26

    In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality. PMID:26774483

  7. Expression and function of the Ror-family receptor tyrosine kinases during development: lessons from genetic analyses of nematodes, mice, and humans.

    PubMed

    Yoda, Akinori; Oishi, Isao; Minami, Yasuhiro

    2003-02-01

    Receptor tyrosine kinases (RTKs) play crucial roles in various developmental processes. Ror-family RTKs are characterized by the intracellular tyrosine kinase domains, highly related to those of the Trk-family RTKs, and by the extracellular Frizzled-like cysteine-rich domains (CRDs) and Kringle domains. Rors are evolutionally conserved among Caenorhabditis elegans, Aplysia, Drosophila melanogaster, Xenopus, mice, and humans. In D. melanogaster and mammals, pairs of structurally related Rors are found, while a single Ror protein is identified in C. elegans or Aplysia. In Aplysia and D. melanogaster, Rors are expressed exclusively in developing nervous systems. On the other hand, rather widespread expression of Rors was observed in C. elegans and mammals. Mutations in Ror of C. elegans cause inappropriate axon outgrowth as well as defects in cell migration and asymmetric cell division. It has also been reported that the nematode Ror possesses kinase-dependent and kinase-independent functions. Mouse Rors, Ror1, and Ror2, are expressed mainly in migrating neural crest cells and mesenchymal cells, and Ror2-deficient mice exhibit skeletal abnormalities and ventricular septal defects in the heart. Although Ror1-deficient mice exhibit no apparent skeletal or cardiac abnormalities, Ror1/Ror2 double mutant mice show markedly enhanced skeletal and cardiac abnormalities compared with Ror2 mutant mice, indicating genetic interaction of Ror1 and Ror2. In humans, mutations within Ror2 have been found in two genetic skeletal disorders, recessive Robinow syndrome and dominant Brachydactyly type B (BDB), further emphasizing critical functions of Ror2 during developmental morphogenesis. In this article, we also discuss the signaling machinery mediated by Ror-family RTKs with a particular emphasis on our recent structure-function analyses of Ror-family RTKs. PMID:12680586

  8. Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway*

    PubMed Central

    Cohen, Adiel; Kupiec, Martin; Weisman, Ronit

    2014-01-01

    The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling. PMID:24928510

  9. A New Family of Receptor Tyrosine Kinases with a Venus Flytrap Binding Domain in Insects and Other Invertebrates Activated by Aminoacids

    PubMed Central

    Ahier, Arnaud; Rondard, Philippe; Gouignard, Nadège; Khayath, Naji; Huang, Siluo; Trolet, Jacques; Donoghue, Daniel J.; Gauthier, Monique; Pin, Jean-Philippe; Dissous, Colette

    2009-01-01

    Background Tyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. Methods and Findings Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABAB receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. Conclusion The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases. PMID:19461966

  10. Theoretical studies of the structure and stability of (Ag(C 6H 6) n+ ( n = 1 and 2)

    NASA Astrophysics Data System (ADS)

    Ma, N. L.; Ng, K. M.; Tsang, C. W.

    1997-10-01

    Ab initio molecular orbital techniques are used to probe the structure and stability of Ag(C 6H 6) + and Ag(C 6H 6) 2+ recently observed in electrospray ionization mass spectra. Of all the Ag(C 6H 6) + structures investigated, the C 6v complex is the most stable. Our calculated first binding energy of benzene to Ag + is in agreement with the experimental value and the second binding enegy of benzene to Ag + is estimated to be 114 (10) kJ mol -1.

  11. Mutations in the protein kinase A R1α regulatory subunit cause familial cardiac myxomas and Carney complex

    PubMed Central

    Casey, Mairead; Vaughan, Carl J.; He, Jie; Hatcher, Cathy J.; Winter, Jordan M.; Weremowicz, Stanislawa; Montgomery, Kate; Kucherlapati, Raju; Morton, Cynthia C.; Basson, Craig T.

    2000-01-01

    Cardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1α gene encoding the R1α regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1α frameshift mutations in three unrelated families result in haploinsufficiency of R1α and cause Carney complex. We did not detect any truncated R1α protein encoded by mutant PRKAR1α. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1α deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1α alleles and that wild-type R1α protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1α to R2β regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1α haploinsufficiency on PKA activity and the role of PKA in cardiac growth and differentiation. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org. J. Clin. Invest. 106:R31–R38 (2000). PMID:10974026

  12. Selective targeting of MAPK family kinases JNK over p38 by rationally designed peptides as potential therapeutics for neurological disorders and epilepsy.

    PubMed

    Zhuo, Zhi-Hong; Sun, Yi-Zhen; Jin, Pei-Na; Li, Feng-Yan; Zhang, Yi-Le; Wang, Huai-Li

    2016-07-19

    Human mitogen-activated protein kinase (MAPK) family members JNK and p38 are two homologous protein-serine/threonine kinases but play distinct roles in the pathological process of neurological disorders. Selective targeting of JNK over p38 has been established as a potential therapeutic approach to epilepsy and other nervous system diseases. Herein, we describe an integrated in vitro-in silico protocol to rationally design kinase-peptide interaction specificity based on crystal structure data. In the procedure, a simulated annealing (SA) iteration optimization strategy is described to improve peptide selectivity between the two kinases. The optimization accepts moderate compromise in peptide affinity to JNK in order to maximize the affinity difference between peptide interactions with JNK and p38. The structural basis, energetic properties and dynamic behavior of SA-improved peptides bound with the peptide-docking sites of JNK and p38 kinase domains are investigated in detail using atomistic molecular dynamics (MD) simulations and post binding free energy analysis. The theoretical findings and computational designs are then confirmed by fluorescence polarization assays. Using the integrated protocol we successfully obtain three decapeptide ligands, namely RLHPSMTDFL, RAKLPTSVDY and KPSRPWNLEI, that exhibit both potent affinity to JNK (K = 8.0, 5.4 and 12.1 μM, respectively) and high selectivity for JNK over p38 (K/K = 9.2, 17.9 and 6.3 fold, respectively). We also demonstrate that a JNK-over-p38 selective peptide should have a positively charged N-terminus, a polar central region and a negatively charged C-terminus, in which a number of hydrophobic residues distribute randomly along the peptide sequence. In particular, the residue positions 1, 6 and 9 play a crucial role in shaping peptide selectivity; the presence of, respectively, Arg, Thr and Asp at the three positions confers high specificity to kinase-peptide interactions. PMID:27263470

  13. Src family kinases are required for WNT5 signaling through the Derailed/RYK receptor in the Drosophila embryonic central nervous system.

    PubMed

    Wouda, Rene R; Bansraj, Monique R K S; de Jong, Anja W M; Noordermeer, Jasprina N; Fradkin, Lee G

    2008-07-01

    Members of the RYK/Derailed family have recently been shown to regulate axon guidance in both Drosophila and mammals by acting as Wnt receptors. Little is known about how the kinase activity-deficient RYKs transduce Wnt signals. Here, we show that the non-receptor Src family tyrosine kinases, SRC64B and SRC42A, are involved in WNT5-mediated signaling through Derailed in the Drosophila embryonic central nervous system. Analysis of animals lacking SRC64B and SRC42A reveals defects in commissure formation similar to those observed in Wnt5 and derailed mutants. Reductions in SRC64B expression levels suppress a Wnt5/derailed-dependent dominant gain-of-function phenotype, and increased levels of either SRC64B or SRC42A enhance Wnt5/derailed-mediated axon commissure switching. Derailed and SRC64B form a complex, which contains catalytically active SRC64B, the formation or stability of which requires SRC64B kinase activity. Furthermore, Derailed is phosphorylated in a SRC64B-dependent manner and coexpression of Derailed and SRC64B results in the activation of SRC64B. The mammalian orthologs of Derailed and SRC64B also form complexes, suggesting that Src roles in RYK signaling are conserved. Finally, we show that coexpression of WNT5 and Derailed has no apparent effect upon TCF/LEF-dependent transcription, suggesting that the WNT5/Derailed signaling pathway is unlikely to directly regulate canonical Wnt pathway targets. Together, these findings indicate that the Src family kinases play novel roles in WNT5/Derailed-mediated signaling. PMID:18539923

  14. The human gene for neurotrophic tyrosine kinase receptor type 2 (NTRK2) is located on chromosome 9 but is not the familial dysautonomia gene

    SciTech Connect

    Slaugenhaupt, S.A. |; Liebert, C.B.; Lucente, D.E.

    1995-02-10

    The neurotrophic tyrosine kinase receptor type 2 (NTRK2) gene is a member of the trk family of tyrosine protein kinases, which encode receptors for the nerve growth factor-related proteins known as neurotrophins. The neurotrophins and their receptors have long been considered candidate genes for familial dysautonomia (FD), a hereditary sensory neuropathy resulting from the congenital loss of both sensory and autonomic neurons. The DYS gene has recently been mapped to human chromosome 9q31-q33, and therefore we set out to determine the chromosomal localization of the candidate gene NTRK2. A mouse trkB probe was hybridized to both somatic cell hybrids containing human chromosome 9 and a human chromosome 9 flow-sorted cosmid library. The human homologue of trkB, NTRK2, was assigned to chromosome 9. To localize the NTRK2 gene further, a dinucleotide repeat polymorphism was identified within a cosmid that contains NTRK2 exon sequences. This marker was genotyped in the CEPH reference pedigrees and places the NTRK2 gene near D9S1 on the proximal long arm of human chromosome 9. The NTRK2 gene is located approximately 22 cm proximal to DYS and shows several recombinants in disease families. Therefore, the NTRK2 gene can now be excluded as a candidate gene for familial dysautonomia. 18 refs., 1 fig.

  15. RAFTK, a Novel Member of the Focal Adhesion Kinase Family, Is Phosphorylated and Associates with Signaling Molecules upon Activation of Mature T Lymphocytes

    PubMed Central

    Ganju, Ramesh K.; Hatch, William C.; Avraham, Hava; Ona, Mel A.; Druker, Brian; Avraham, Shalom; Groopman, Jerome E.

    1997-01-01

    The related adhesion focal tyrosine kinase (RAFTK), a recently discovered member of the focal adhesion kinase family, has previously been reported to participate in signal transduction in neuronal cells, megakaryocytes, and B lymphocytes. We have found that RAFTK is constitutively expressed in human T cells and is rapidly phosphorylated upon the activation of the T cell receptor (TCR). This activation also results in an increase in the autophosphorylation and kinase activity of RAFTK. After its stimulation, there was an increase in the association of the src cytoplasmic tyrosine kinase Fyn and the adapter protein Grb2. This association was mediated through the SH2 domains of Fyn and Grb2. RAFTK also co-immunoprecipitates with the SH2 domain of Lck and with the cytoskeletal protein paxillin through its COOH-terminal proline-rich domain. The tyrosine phosphorylation of RAFTK after T cell receptor-mediated stimulation was reduced by the pretreatment of cells with cytochalasin D, suggesting the role of the cytoskeleton in this process. These observations indicate that RAFTK participates in T cell receptor signaling and may act to link signals from the cell surface to the cytoskeleton and thereby affect the host immune response. PMID:9091579

  16. Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modelling of aspartokinase.

    PubMed

    Marco-Marín, Clara; Ramón-Maiques, Santiago; Tavárez, Sandra; Rubio, Vicente

    2003-11-28

    We test, using site-directed mutagenesis, predictions based on the X-ray structure of N-acetyl-L-glutamate kinase (NAGK), the paradigm of the amino acid kinase protein family, about the roles of specific residues on substrate binding and catalysis. The mutations K8R and D162E decreased V([sustrate]= infinity ) 100-fold and 1000-fold, respectively, in agreement with the predictions that K8 catalyzes phosphoryl transfer and D162 organizes the catalytic groups. R66K and N158Q increased selectively K(m)(Asp) three to four orders of magnitude, in agreement with the binding of R66 and N158 to the C(alpha) substituents of NAG. Mutagenesis in parallel of aspartokinase III (AKIII phosphorylates aspartate instead of acetylglutamate), another important amino acid kinase family member of unknown 3-D structure, identified in AKIII two residues, K8 and D202, that appear to play roles similar to those of K8 and D162 of NAGK, and supports the involvement of E119 and R198, similarly to R66 and N158 of NAGK, in the binding of the amino acid substrate, apparently interacting, respectively, with the alpha-NH(3)(+) and alpha-COO(-) of aspartate. These results and an improved alignment of the NAGK and AKIII sequences have guided us into 3-D modelling of the amino acid kinase domain of AKIII using NAGK as template. The model has good stereochemistry and validation parameters. It provides insight into substrate binding and catalysis, agreeing with mutagenesis results with another aspartokinase that were not considered when building the model.AKIII is homodimeric and is inhibited by lysine. Lysine may bind to a regulatory region that is C-terminal to the amino acid kinase domain. We make a C-terminally truncated AKIII (AKIIIt) and show that the C-region is involved in intersubunit interactions, since AKIIIt is found to be monomeric. Further, it is inactive, as demanded if dimer formation is essential for activity. Models for AKIII architecture are proposed that account for these findings

  17. Design and realization of an AEC&AGC system for the CCD aerial camera

    NASA Astrophysics Data System (ADS)

    Liu, Hai ying; Feng, Bing; Wang, Peng; Li, Yan; Wei, Hao yun

    2015-08-01

    An AEC and AGC(Automatic Exposure Control and Automatic Gain Control) system was designed for a CCD aerial camera with fixed aperture and electronic shutter. The normal AEC and AGE algorithm is not suitable to the aerial camera since the camera always takes high-resolution photographs in high-speed moving. The AEC and AGE system adjusts electronic shutter and camera gain automatically according to the target brightness and the moving speed of the aircraft. An automatic Gamma correction is used before the image is output so that the image is better for watching and analyzing by human eyes. The AEC and AGC system could avoid underexposure, overexposure, or image blurring caused by fast moving or environment vibration. A series of tests proved that the system meet the requirements of the camera system with its fast adjusting speed, high adaptability, high reliability in severe complex environment.

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

    PubMed Central

    Beilina, Alexandria; Rudenko, Iakov N.; Kaganovich, Alice; Civiero, Laura; Chau, Hien; Kalia, Suneil K.; Kalia, Lorraine V.; Lobbestael, Evy; Chia, Ruth; Ndukwe, Kelechi; Ding, Jinhui; Nalls, Mike A.; Olszewski, Maciej; Hauser, David N.; Kumaran, Ravindran; Lozano, Andres M.; Baekelandt, Veerle; Greene, Lois E.; Taymans, Jean-Marc; Greggio, Elisa; Cookson, Mark R.; Nalls, Mike A.; Plagnol, Vincent; Martinez, Maria; Hernandez, Dena G; Sharma, Manu; Sheerin, Una-Marie; Saad, Mohamad; Simón-Sánchez, Javier; Schulte, Claudia; Lesage, Suzanne; Sveinbjörnsdóttir, Sigurlaug; Arepalli, Sampath; Barker, Roger; Ben-Shlomo, Yoav; Berendse, Henk W; Berg, Daniela; Bhatia, Kailash; de Bie, Rob M A; Biffi, Alessandro; Bloem, Bas; Bochdanovits, Zoltan; Bonin, Michael; Bras, Jose M; Brockmann, Kathrin; Brooks, Janet; Burn, David J; Charlesworth, Gavin; Chen, Honglei; Chong, Sean; Clarke, Carl E; Cookson, Mark R; Cooper, J Mark; Corvol, Jean Christophe; Counsell, Carl; Damier, Philippe; Dartigues, Jean-François; Deloukas, Panos; Deuschl, Günther; Dexter, David T; van Dijk, Karin D; Dillman, Allissa; Durif, Frank; Dürr, Alexandra; Edkins, Sarah; Evans, Jonathan R; Foltynie, Thomas; Gao, Jianjun; Gardner, Michelle; Gibbs, J Raphael; Goate, Alison; Gray, Emma; Guerreiro, Rita; Gústafsson, Ómar; Harris, Clare; van Hilten, Jacobus J; Hofman, Albert; Hollenbeck, Albert; Holton, Janice; Hu, Michele; Huang, Xuemei; Huber, Heiko; Hudson, Gavin; Hunt, Sarah E; Huttenlocher, Johanna; Illig, Thomas; München, Helmholtz Zentrum; Jónsson, Pálmi V; Lambert, Jean-Charles; Langford, Cordelia; Lees, Andrew; Lichtner, Peter; München, Helmholtz Zentrum; Limousin, Patricia; Lopez, Grisel; Lorenz, Delia; McNeill, Alisdair; Moorby, Catriona; Moore, Matthew; Morris, Huw R; Morrison, Karen E; Mudanohwo, Ese; O’Sullivan, Sean S; Pearson, Justin; Perlmutter, Joel S; Pétursson, Hjörvar; Pollak, Pierre; Post, Bart; Potter, Simon; Ravina, Bernard; Revesz, Tamas; Riess, Olaf; Rivadeneira, Fernando; Rizzu, Patrizia; Ryten, Mina; Sawcer, Stephen; Schapira, Anthony; Scheffer, Hans; Shaw, Karen; Shoulson, Ira; Sidransky, Ellen; Smith, Colin; Spencer, Chris C A; Stefánsson, Hreinn; Steinberg, Stacy; Stockton, Joanna D; Strange, Amy; Talbot, Kevin; Tanner, Carlie M; Tashakkori-Ghanbaria, Avazeh; Tison, François; Trabzuni, Daniah; Traynor, Bryan J; Uitterlinden, André G; Velseboer, Daan; Vidailhet, Marie; Walker, Robert; van de Warrenburg, Bart; Wickremaratchi, Mirdhu; Williams, Nigel; Williams-Gray, Caroline H; Winder-Rhodes, Sophie; Stefánsson, Kári; Hardy, John; Heutink, Peter; Brice, Alexis; Gasser, Thomas; Singleton, Andrew B; Wood, Nicholas W; Chinnery, Patrick F; Arepalli, Sampath; Cookson, Mark R; Dillman, Allissa; Ferrucci, Luigi; Gibbs, J Raphael; Hernandez, Dena G; Johnson, Robert; Longo, Dan L; Majounie, Elisa; Nalls, Michael A; O’Brien, Richard; Singleton, Andrew B; Traynor, Bryan J; Troncoso, Juan; van der Brug, Marcel; Zielke, H Ronald; Zonderman, Alan B

    2014-01-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein–protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G–associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy–lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms. PMID:24510904

  19. CagA Phosphorylation in Helicobacter pylori-Infected B Cells Is Mediated by the Nonreceptor Tyrosine Kinases of the Src and Abl Families.

    PubMed

    Krisch, Linda M; Posselt, Gernot; Hammerl, Peter; Wessler, Silja

    2016-09-01

    CagA is one of the most important virulence factors of the human pathogen Helicobacter pylori CagA expression can be associated with the induction of severe gastric disorders such as gastritis, ulceration, gastric cancer, or mucosa-associated lymphoid tissue (MALT) lymphoma. After translocation through a type IV secretion system into epithelial cells, CagA is tyrosine phosphorylated by kinases of the Src and Abl families, leading to drastic cell elongation and motility. While the functional role of CagA in epithelial cells is well investigated, knowledge about CagA phosphorylation and its associated signal transduction pathways in B cells is only marginal. Here, we established the B cell line MEC1 derived from a B cell chronic lymphocytic leukemia (B-CLL) patient as a new infection model to study the signal transduction in B cells controlled by H. pylori We observed that CagA was rapidly injected, strongly tyrosine phosphorylated, and cleaved into a 100-kDa N-terminal and a 40-kDa C-terminal fragment. To identify upstream signal transduction pathways of CagA phosphorylation in MEC1 cells, pharmacological inhibitors were employed to specifically target Src and Abl kinases. We observed that CagA phosphorylation was strongly inhibited upon treatment with an Src inhibitor and slightly diminished when the Abl kinase inhibitor imatinib mesylate (Gleevec) was applied. The addition of dasatinib to block c-Abl and Src kinases led to a complete loss of CagA phosphorylation. In conclusion, these results demonstrate an important role for Src and Abl tyrosine kinases in CagA phosphorylation in B cells, which represent druggable targets in H. pylori-mediated gastric MALT lymphoma. PMID:27382024

  20. CagA Phosphorylation in Helicobacter pylori-Infected B Cells Is Mediated by the Nonreceptor Tyrosine Kinases of the Src and Abl Families

    PubMed Central

    Krisch, Linda M.; Posselt, Gernot; Hammerl, Peter

    2016-01-01

    CagA is one of the most important virulence factors of the human pathogen Helicobacter pylori. CagA expression can be associated with the induction of severe gastric disorders such as gastritis, ulceration, gastric cancer, or mucosa-associated lymphoid tissue (MALT) lymphoma. After translocation through a type IV secretion system into epithelial cells, CagA is tyrosine phosphorylated by kinases of the Src and Abl families, leading to drastic cell elongation and motility. While the functional role of CagA in epithelial cells is well investigated, knowledge about CagA phosphorylation and its associated signal transduction pathways in B cells is only marginal. Here, we established the B cell line MEC1 derived from a B cell chronic lymphocytic leukemia (B-CLL) patient as a new infection model to study the signal transduction in B cells controlled by H. pylori. We observed that CagA was rapidly injected, strongly tyrosine phosphorylated, and cleaved into a 100-kDa N-terminal and a 40-kDa C-terminal fragment. To identify upstream signal transduction pathways of CagA phosphorylation in MEC1 cells, pharmacological inhibitors were employed to specifically target Src and Abl kinases. We observed that CagA phosphorylation was strongly inhibited upon treatment with an Src inhibitor and slightly diminished when the Abl kinase inhibitor imatinib mesylate (Gleevec) was applied. The addition of dasatinib to block c-Abl and Src kinases led to a complete loss of CagA phosphorylation. In conclusion, these results demonstrate an important role for Src and Abl tyrosine kinases in CagA phosphorylation in B cells, which represent druggable targets in H. pylori-mediated gastric MALT lymphoma. PMID:27382024

  1. Research of AGC technology in a digital optical fiber sensing system with PGC modulation and demodulation

    NASA Astrophysics Data System (ADS)

    Tang, Jianfeng; Xiong, Shuidong; Zhang, Yan

    2014-11-01

    The magnitude of light intensity on the photo-to-electric detector fluctuates all the time in an optic fiber sensing system, because of the influence of various factors in the fiber optic sensing system and from the external environment. As a result of the excessive intensity, the electric signal will be overload after the amplifier circuit with constant enlargement factor, and when the light intensity becames too small, it will reduce the signal-to-noise ratio of the electric signal. Therefore, it is necessary to introduce an automatic gain control (AGC) module into the system, which can insure the electric signal in a reasonable magnitude. In order to solve the problem of optic intensity fluctuating in the optical fiber sensing system with PGC modulation and demodulation, in this paper, firstly, it is analyzed that the impact of different magnitudes of interferential intensity to the PGC demodulation in theory. Secondly, a reasonable control method is put forward and an AGC module based on the AD602 chip is designed and produced. Finally, it is proved that the optic fiber sensor system with an AGC module has strong ability to resist fluctuation of light intensity within 40dB.

  2. Capacitive deionization of seawater effected by nano Ag and Ag@C on graphene.

    PubMed

    Cai, P-F; Su, C-J; Chang, W-T; Chang, F-C; Peng, C-Y; Sun, I-W; Wei, Y-L; Jou, C-J; Wang, H Paul

    2014-08-30

    Drinking water shortage has become worse in recent decades. A new capacitive deionization (CDI) method for increasing water supplies through the effective desalination of seawater has been developed. Silver as nano Ag and Ag@C which was prepared by carbonization of the Ag(+)-β-cyclodextrin complex at 573 K for 30 min can add the antimicrobial function into the CDI process. The Ag@C and Ag nanoparticles dispersed on reduced graphene oxide (Ag@C/rGO and nano Ag/rGO) were used as the CDI electrodes. The nano Ag/rGO and Ag@C/rGO electrodes can reduce the charging resistant, and enhance the electrosorption capability. Better CDI efficiencies with the nano Ag/rGO and Ag@C/rGO electrodes can therefore be obtained. When reversed the voltage, the electrodes can be recovered up to 90% within 5 min. This work presents the feasibility for the nano Ag and Ag@C on rGO electrodes applied in CDI process to produce drinking water from seawater or saline water. PMID:24928455

  3. NV Energy Large-Scale Photovoltaic Integration Study: Intra-Hour Dispatch and AGC Simulation

    SciTech Connect

    Lu, Shuai; Etingov, Pavel V.; Meng, Da; Guo, Xinxin; Jin, Chunlian; Samaan, Nader A.

    2013-01-02

    The uncertainty and variability with photovoltaic (PV) generation make it very challenging to balance power system generation and load, especially under high penetration cases. Higher reserve requirements and more cycling of conventional generators are generally anticipated for large-scale PV integration. However, whether the existing generation fleet is flexible enough to handle the variations and how well the system can maintain its control performance are difficult to predict. The goal of this project is to develop a software program that can perform intra-hour dispatch and automatic generation control (AGC) simulation, by which the balancing operations of a system can be simulated to answer the questions posed above. The simulator, named Electric System Intra-Hour Operation Simulator (ESIOS), uses the NV Energy southern system as a study case, and models the system’s generator configurations, AGC functions, and operator actions to balance system generation and load. Actual dispatch of AGC generators and control performance under various PV penetration levels can be predicted by running ESIOS. With data about the load, generation, and generator characteristics, ESIOS can perform similar simulations and assess variable generation integration impacts for other systems as well. This report describes the design of the simulator and presents the study results showing the PV impacts on NV Energy real-time operations.

  4. Human biliverdin reductase: a member of the insulin receptor substrate family with serine/threonine/tyrosine kinase activity.

    PubMed

    Lerner-Marmarosh, Nicole; Shen, Jenny; Torno, Michael D; Kravets, Anatoliy; Hu, Zhenbo; Maines, Mahin D

    2005-05-17

    We describe here the tyrosine kinase activity of human biliverdin reductase (BVR) and its potential role in the insulin-signaling pathway. BVR is both a substrate for insulin receptor (IR) tyrosine kinase (IRK) activity and a kinase for serine phosphorylation of IR substrate 1 (IRS-1). Our previous studies have revealed serine/threonine kinase activity of BVR. Y198, in the YMKM motif found in the C-terminal domain of BVR, is shown to be a substrate for insulin-activated IRK. This motif in IRS proteins provides a docking site for proteins that contain a Src homology 2 domain. Additionally, Y228 in the YLSF sequence and Y291 are IRK substrates; the former sequence provides optimum recognition motif in the tyrosine phosphatase, SHP-1, and for SHC (Src homology 2 domain containing transfroming protein 1). BVR autophosphorylates N-terminal tyrosines Y72 and Y83. Serine residues in IRS-1 are targets for BVR phosphorylation, and point mutation of serine residues in the kinase domain of the reductase inhibits phosphotransferase activity. Because tyrosine phosphorylation of IRS-1 activates the insulin signaling pathway and serine phosphorylation of IRS-1 blocks insulin action, our findings that insulin increases BVR tyrosine phosphorylation and that there is an increase in glucose uptake in response to insulin when expression of BVR is "knocked down" by small interfering RNA suggest a potential role for BVR in the insulin signaling pathway. PMID:15870194

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

    PubMed

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

    2016-08-01

    The design and synthesis of new pyrido[3,4-g]quinazoline derivatives is described as well as their protein kinase inhibitory potencies toward five CMGC family members (CDK5, CK1, GSK3, CLK1 and DYRK1A). The interest for this original tricyclic heteroaromatic scaffold as modulators of CLK1/DYRK1A activity was validated by nanomolar potencies (compounds 12 and 13). CLK1 co-crystal structures with two inhibitors revealed the binding mode of these compounds within the ATP-binding pocket. PMID:27128181

  6. 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

  7. The DYRK-family kinase Pom1 phosphorylates the F-BAR protein Cdc15 to prevent division at cell poles

    PubMed Central

    Ullal, Pranav; McDonald, Nathan A.; Chen, Jun-Song; Lo Presti, Libera; Roberts-Galbraith, Rachel H.; Gould, Kathleen L.

    2015-01-01

    Division site positioning is critical for both symmetric and asymmetric cell divisions. In many organisms, positive and negative signals cooperate to position the contractile actin ring for cytokinesis. In rod-shaped fission yeast Schizosaccharomyces pombe cells, division at midcell is achieved through positive Mid1/anillin-dependent signaling emanating from the central nucleus and negative signals from the dual-specificity tyrosine phosphorylation-regulated kinase family kinase Pom1 at the cell poles. In this study, we show that Pom1 directly phosphorylates the F-BAR protein Cdc15, a central component of the cytokinetic ring. Pom1-dependent phosphorylation blocks Cdc15 binding to paxillin Pxl1 and C2 domain protein Fic1 and enhances Cdc15 dynamics. This promotes ring sliding from cell poles, which prevents septum assembly at the ends of cells with a displaced nucleus or lacking Mid1. Pom1 also slows down ring constriction. These results indicate that a strong negative signal from the Pom1 kinase at cell poles converts Cdc15 to its closed state, destabilizes the actomyosin ring, and thus promotes medial septation. PMID:26553932

  8. The sucrose non-fermenting-1-related (SnRK) family of protein kinases: potential for manipulation to improve stress tolerance and increase yield.

    PubMed

    Coello, Patricia; Hey, Sandra J; Halford, Nigel G

    2011-01-01

    Sucrose non-fermenting-1 (SNF1)-related protein kinases (SnRKs) take their name from their fungal homologue, SNF1, a global regulator of carbon metabolism. The plant family has burgeoned to comprise 38 members which can be subdivided into three sub-families: SnRK1, SnRK2, and SnRK3. There is now good evidence that this has occurred to allow plants to link metabolic and stress signalling in a way that does not occur in other organisms. The role of SnRKs, focusing in particular on abscisic acid-induced signalling pathways, salinity tolerance, responses to nutritional stress and disease, and the regulation of carbon metabolism and, therefore, yield, is reviewed here. The key role that SnRKs play at the interface between metabolic and stress signalling make them potential candidates for manipulation to improve crop performance in extreme environments. PMID:20974737

  9. Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars.

    PubMed

    Zhang, Xinshuai; Carter, Michael S; Vetting, Matthew W; San Francisco, Brian; Zhao, Suwen; Al-Obaidi, Nawar F; Solbiati, Jose O; Thiaville, Jennifer J; de Crécy-Lagard, Valérie; Jacobson, Matthew P; Almo, Steven C; Gerlt, John A

    2016-07-19

    Using a large-scale "genomic enzymology" approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for d-threonate, l-threonate, and d-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including d-erythronate and l-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-l-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of d-threonate to dihydroxyacetone phosphate and CO2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of d-erythronate and l-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO2 The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses. PMID:27402745

  10. Syk and Src Family Kinases Regulate C-type Lectin Receptor 2 (CLEC-2)-mediated Clustering of Podoplanin and Platelet Adhesion to Lymphatic Endothelial Cells*

    PubMed Central

    Pollitt, Alice Y.; Poulter, Natalie S.; Gitz, Eelo; Navarro-Nuñez, Leyre; Wang, Ying-Jie; Hughes, Craig E.; Thomas, Steven G.; Nieswandt, Bernhard; Douglas, Michael R.; Owen, Dylan M.; Jackson, David G.; Dustin, Michael L.; Watson, Steve P.

    2014-01-01

    The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development. PMID:25368330

  11. Inhibitors of the Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N)

    SciTech Connect

    Sueyoshi, Noriyuki; Takao, Toshihiko; Nimura, Takaki; Sugiyama, Yasunori; Numano, Takamasa; Shigeri, Yasushi; Taniguchi, Takanobu; Kameshita, Isamu Ishida, Atsuhiko

    2007-11-23

    Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca{sup 2+}/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP family phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid. When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro.

  12. PP2, a potent inhibitor of Src family kinases, protects against hippocampal CA1 pyramidal cell death after transient global brain ischemia.

    PubMed

    Hou, Xiao-Yu; Liu, Yong; Zhang, Guang-Yi

    2007-06-15

    It has been indicated that Src family protein tyrosine kinases (SrcPTKs) potentiate N-methyl-D-aspartate (NMDA) receptor function by phosphorylating NR2A subunits and that postsynaptic density protein 95 (PSD-95) facilitates this regulation. In this paper, we define the role of SrcPTKs in delayed neuronal damage following transient brain ischemia and explore the underlying mechanisms involved in this event. Transient global brain ischemia was induced by the four-vessel occlusion method. A specific Src family kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine) and a PP2 negative control PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyramidine) were infused into rat cerebroventricule 30 min before occlusion. Hematoxylin and eosine staining showed that the number of surviving pyramidal neurons in rat hippocampal CA1 subfield increased markedly in PP2-treated rats comparing to PP3-treated groups after 5 days of reperfusion following ischemia. Additionally, immunoprecipitation and immunoblot analysis revealed that preadministration of PP2, but not PP3, attenuated not only the increased tyrosine phosphorylation of NR2A but also the enhanced interactions among Src, NR2A and PSD-95 induced by ischemia/reperfusion. In conclusion, SrcPTKs promote binding of the kinases and their substrate NR2A attributed to the scaffolding effect of PSD-95 during transient brain ischemia and reperfusion, which are responsible for the elevation of NR2A tyrosine phosphorylation and consequent delayed neuronal cell death. PMID:17556100

  13. A digitally controlled AGC loop circuitry for GNSS receiver chip with a binary weighted accurate dB-linear PGA

    NASA Astrophysics Data System (ADS)

    Gang, Jin; Yiqi, Zhuang; Yue, Yin; Miao, Cui

    2015-03-01

    A novel digitally controlled automatic gain control (AGC) loop circuitry for the global navigation satellite system (GNSS) receiver chip is presented. The entire AGC loop contains a programmable gain amplifier (PGA), an AGC circuit and an analog-to-digital converter (ADC), which is implemented in a 0.18 μm complementary metal-oxide-semiconductor (CMOS) process and measured. A binary-weighted approach is proposed in the PGA to achieve wide dB-linear gain control with small gain error. With binary-weighted cascaded amplifiers for coarse gain control, and parallel binary-weighted trans-conductance amplifier array for fine gain control, the PGA can provide a 64 dB dynamic range from -4 to 60 dB in 1.14 dB gain steps with a less than 0.15 dB gain error. Based on the Gaussian noise statistic characteristic of the GNSS signal, a digital AGC circuit is also proposed with low area and fast settling. The feed-backward AGC loop occupies an area of 0.27 mm2 and settles within less than 165 μs while consuming an average current of 1.92 mA at 1.8 V.

  14. IP6K Structure and the Molecular Determinants of Catalytic Specificity in an Inositol Phosphate Kinase Family

    PubMed Central

    Wang, Huanchen; DeRose, Eugene F.; London, Robert E.; Shears, Stephen B.

    2014-01-01

    IP3Ks and IP6Ks each regulate specialized signaling activities by phosphorylating either InsP3 or InsP6 respectively; what are the molecular determinants of these different kinase activities? We address this question by determining the crystal structure of an enzymatic parallel to a “living fossil”: an Entamoeba histolytica hybrid IP6K/IP3K. Through molecular modeling and mutagenesis, we also extrapolated our findings to human IP6K2, which retains vestigial IP3K activity. Two structural elements, an α-helical pair and a rare, two-turn 310 helix, together forge a substrate-binding pocket with an open-clamshell geometry. InsP6 forms substantial contacts with both structural elements. Relative to InsP6, enzyme-bound InsP3 rotates 55° closer to the α-helices, which provide most of the protein’s interactions with InsP3. These non-overlapping substrate orientations are unprecedented for an inositol phosphate kinase. This arrangement also suggests an unusual evolutionary trajectory for a primordial kinase that could have favored efficient bi-functionality, prior to propagation of separate IP3Ks and IP6Ks. PMID:24956979

  15. AGC System after Deregulation Considering TCPS in Series with the Tie-Line

    NASA Astrophysics Data System (ADS)

    Abraham, Rajesh Joseph; Das, D.; Patra, A.

    2015-06-01

    This paper presents the study of automatic generation control (AGC) of two area interconnected power system after deregulation, considering a thyristor controlled phase shifter (TCPS) in series with the tie-line. It is possible to minimize the system frequency and tie-power oscillations by controlling the phase angle of TCPS which is expected to provide a new ancillary service for the future power system. Effect of TCPS is examined for three different cases, i.e. (1) unilateral contract, (2) bilateral contract and (3) contract violation. Analysis reveals that a TCPS is quite capable of suppressing the frequency and tie-power oscillations effectively as compared to that obtained without TCPS.

  16. Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone.

    PubMed

    Yang, Xiudong; Yan, Wenjuan; Tian, Ye; Ma, Pan; Opperman, Lynne A; Wang, Xiaofang

    2016-01-01

    Recent studies have identified family with sequence similarity member 20C (FAM20C) as a kinase that phosphorylates the Ser in Ser-X-Glu/phospho-Ser (pSer) motifs in the small-integrin-binding ligand N-linked glycoproteins (SIBLINGs). There is no in vivo evidence that validates this finding, and it is unclear whether FAM20C is the only kinase for SIBLINGs. We extracted bone noncollagenous proteins (NCPs) from Fam20C-knockout (KO) mice and analyzed the phosphorylation levels. The total NCPs were separated into osteopontin-, bone sialoprotein-, and dentin matrix protein-1-enriched fractions by anion-exchange chromatography and analyzed by SDS-PAGE, native PAGE, and Western immunoblot analysis. The NCP phosphorylation level in the KO mice was lower than that in the wild-type (WT). On the native gel, the SIBLINGs from KO mice showed a lower migration rate (Mr) than those from the WT. Calf intestine phosphatase treatment shifted SIBLINGs from the WT mice to the level adjacent to the KO, but failed to shift the latter, suggesting a phosphorylation loss of SIBLINGs in the KO mice. Mass spectrometry identified less pSers in the SIBLINGs from the KO mice [including the region of the acidic Ser- and aspartate-rich motif (ASARM) peptides]. In an intriguing finding, several pSers in the Ser-X-Glu motifs in the KO mice maintained their phosphorylation, whereas several others in non-Ser-X-Glu motifs did not. Phospho-Tyrs and phospho-Thrs in the SIBLINGs did not appear to be associated with FAM20C. Our results indicate that FAM20C is the primary, but not the only, kinase for the SIBLINGs.-Yang, X., Yan, W., Tian, Y., Ma, P., Opperman, L. A., Wang, X. Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone. PMID:26324849

  17. Src Family Kinases Modulate the Loss of Endothelial Barrier Function in Response to TNF-α: Crosstalk with p38 Signaling.

    PubMed

    Adam, Alejandro P; Lowery, Anthony M; Martino, Nina; Alsaffar, Hiba; Vincent, Peter A

    2016-01-01

    Activation of Src Family Kinase (SFK) signaling is required for the increase in endothelial permeability induced by a variety of cytokines and growth factors. However, we previously demonstrated that activation of endogenous SFKs by expression of dominant negative C-terminal Src Kinase (DN-Csk) is not sufficient to decrease endothelial adherens junction integrity. Basal SFK activity has been observed in normal venular endothelia and was not associated with increased basal permeability. The basal SFK activity however was found to contribute to increased sensitivity of the venular endothelium to inflammatory mediator-induced leakage. How SFK activation achieves this is still not well understood. Here, we show that SFK activation renders human dermal microvascular endothelial cells susceptible to low doses of TNF-α. Treatment of DN-Csk-expressing cells with 50 pg/ml TNF-α induced a loss of TEER as well as drastic changes in the actin cytoskeleton and focal adhesion proteins. This synergistic effect was independent of ROCK or NF-κB activity. TNF-α-induced p38 signaling was required for the synergistic effect on barrier function, and activation of the p38 MAPK alone was also able to induce changes in permeability only in monolayers with active SFKs. These results suggest that the activation of endogenous levels of SFK renders the endothelial barrier more susceptible to low, physiologic doses of TNF-α through activation of p38 which leads to a loss of endothelial tight junctions. PMID:27603666

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

    PubMed Central

    Yonesaka, Kimio; Kudo, Keita; Nishida, Satomi; Takahama, Takayuki; Iwasa, Tsutomu; Yoshida, Takeshi; Tanaka, Kaoru; Takeda, Masayuki; Kaneda, Hiroyasu; Okamoto, Isamu; Nishio, Kazuto; Nakagawa, Kazuhiko

    2015-01-01

    Afatinib is a second generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) characterized as an irreversible pan-human EGFR (HER) family inhibitor. Afatinib remains effective for a subpopulation of patients with non-small cell lung cancer (NSCLC) with acquired resistance to first generation EGFF-TKIs such as erlotinib. Heregulin activates HER3 in an autocrine fashion and causes erlotinib resistance in NSCLC. Here we examine whether afatinib is effective against heregulin-overexpressing NSCLCs harboring EGFR activating mutations. Afatinib but not erlotinib decreased EGFR mutant NSCLC PC9HRG cell proliferation in vitro and in mouse xenografts. Afatinib inhibited phosphorylation of the cell signaling pathway proteins HER3, EGFR, HER2, and HER4, likely by prevention of trans-phosphorylation as HER3 kinase activity is inadequate for auto-phosphorylation. Afatinib, unlike erlotinib, inhibited AKT activation, resulting in elevated apoptosis in PC9HRG cells. Clinically, a subpopulation of 33 patients with EGFR mutations and NSCLC who had received first generation EGFR-TKIs exhibited elevated plasma heregulin levels compared to healthy volunteers; one of these achieved a response with afatinib therapy despite having previously developed erlotinib resistance. Afatinib can overcome heregulin-mediated resistance to erlotinib in EGFR mutant NSCLC. Further studies are necessary to determine whether heregulin can predict afatinib efficacy after development offirst generation EGFR-TKI resistance. PMID:26418897

  19. MAP4K family kinases act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway

    PubMed Central

    Meng, Zhipeng; Moroishi, Toshiro; Mottier-Pavie, Violaine; Plouffe, Steven W.; Hansen, Carsten G.; Hong, Audrey W.; Park, Hyun Woo; Mo, Jung-Soon; Lu, Wenqi; Lu, Shicong; Flores, Fabian; Yu, Fa-Xing; Halder, Georg; Guan, Kun-Liang

    2015-01-01

    The Hippo pathway plays a central role in tissue homoeostasis, and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include a kinase cascade of MST1/2 and LATS1/2 and the transcription co-activators YAP/TAZ. In response to stimulation, LATS1/2 phosphorylate and inhibit YAP/TAZ, the main effectors of the Hippo pathway. Accumulating evidence suggests that MST1/2 are not required for the regulation of YAP/TAZ. Here we show that deletion of LATS1/2 but not MST1/2 abolishes YAP/TAZ phosphorylation. We have identified MAP4K family members—Drosophila Happyhour homologues MAP4K1/2/3 and Misshapen homologues MAP4K4/6/7—as direct LATS1/2-activating kinases. Combined deletion of MAP4Ks and MST1/2, but neither alone, suppresses phosphorylation of LATS1/2 and YAP/TAZ in response to a wide range of signals. Our results demonstrate that MAP4Ks act in parallel to and are partially redundant with MST1/2 in the regulation of LATS1/2 and YAP/TAZ, and establish MAP4Ks as components of the expanded Hippo pathway. PMID:26437443

  20. Genome-wide identification and expression analysis of the mitogen-activated protein kinase gene family from banana suggest involvement of specific members in different stages of fruit ripening.

    PubMed

    Asif, Mehar Hasan; Lakhwani, Deepika; Pathak, Sumya; Bhambhani, Sweta; Bag, Sumit K; Trivedi, Prabodh Kumar

    2014-03-01

    Mitogen-activated protein kinases (MAPKs) are important components of the tripartite mitogen-activated protein kinase signaling cascade and play an important role in plant growth and development. Although members of the MAPK gene family have been identified in model plants, little information is available regarding this gene family in fruit crops. In this study, we carried out a computational analysis using the Musa Genome database to identify members of the MAPK gene family in banana, an economically important crop and the most popular fruit worldwide. Our analysis identified 25 members of the MAP kinase (MAPK or MPK) gene family. Phylogenetic analyses of MPKs in Arabidopsis, Oryza, and Populus have classified these MPKs into four subgroups. The presence of conserved domains in the deduced amino acid sequences, phylogeny, and genomic organization strongly support their identity as members of the MPK gene family. Expression analysis during ethylene-induced banana fruit ripening suggests the involvement of several MPKs in the ethylene signal transduction pathway that are necessary for banana fruit ripening. Analysis of the cis-regulatory elements in the promoter regions and the involvement of the identified MPKs in various cellular processes, as analyzed using Pathway Studio, suggest a role for the banana MPK gene family in diverse functions related to growth, development, and the stress response. This report is the first concerning the identification of members of a gene family and the elucidation of their role in various processes using the Musa Genome database. PMID:24275941

  1. Synergistic Activity of the Src Family Kinase Inhibitor Dasatinib and Oxaliplatin in Colon Carcinoma Cells is Mediated by Oxidative Stress

    PubMed Central

    Kopetz, Scott; Lesslie, Donald P.; Dallas, Nikolas A.; Park, Serk I.; Johnson, Marjorie; Parikh, Nila U.; Kim, Michael P.; Abbruzzese, James L.; Ellis, Lee M.; Chandra, Joya; Gallick, Gary E.

    2009-01-01

    Chemotherapeutic regimens for the treatment of colorectal cancer generally include oxaliplatin, although inherent and acquired resistance is common. One potential mediator of oxaliplatin sensitivity is the non-receptor protein tyrosine kinase, Src, the activity of which correlates with disease stage and patient survival. Therefore, we investigated the effects of Src inhibition using the tyrosine kinase inhibitor dasatinib on oxaliplatin sensitivity. We demonstrate that oxaliplatin acutely activates Src and that combination treatment with dasatinib is synergistic in a cell-line dependent manner, with the level of Src activation correlating with extent of synergy in a panel of six cell lines. Intracellular reactive oxygen species (ROS) are generated after oxaliplatin treatment, and ROS potently activates Src. Pretreatment with antioxidants inhibits oxaliplatin-induced Src activation. In oxaliplatin resistant cell lines, Src activity is constitutively increased. In a mouse model of colorectal liver metastases, treatment with oxaliplatin also results in chronic Src activation. The combination of dasatinib and oxaliplatin results in significantly smaller tumors compared to single agent treatment, corresponding with reduced proliferation and angiogenesis. Therefore, we conclude that oxaliplatin activates Src through a ROS-dependent mechanism. Src inhibition increases oxaliplatin activity both in vitro and in vivo. These results suggest that Src inhibitors combined with oxaliplatin may have efficacy in metastatic colon cancer, and may provide the first indication of a molecular phenotype that might be susceptible to such combinations. PMID:19383922

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

    PubMed Central

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

    2016-01-01

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

  3. Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases.

    PubMed Central

    Sudbeck, B D; Baumann, P; Ryan, G J; Breitkopf, K; Nischt, R; Krieg, T; Mauch, C

    1999-01-01

    Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation. PMID:10085241

  4. The N-terminal SH4 region of the Src family kinase Fyn is modified by methylation and heterogeneous fatty acylation: role in membrane targeting, cell adhesion, and spreading.

    PubMed

    Liang, Xiquan; Lu, Yun; Wilkes, Meredith; Neubert, Thomas A; Resh, Marilyn D

    2004-02-27

    The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function. PMID:14660555

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

    SciTech Connect

    Hagemann, T.L.; Kwan, Sau-Ping; Assa`ad, A.H.

    1995-11-06

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

  6. Genome-Wide Identification of Mitogen-Activated Protein Kinase Gene Family across Fungal Lineage Shows Presence of Novel and Diverse Activation Loop Motifs

    PubMed Central

    Mohanta, Tapan Kumar; Mohanta, Nibedita; Parida, Pratap; Panda, Sujogya Kumar; Ponpandian, Lakshmi Narayanan; Bae, Hanhong

    2016-01-01

    The mitogen-activated protein kinase (MAPK) is characterized by the presence of the T-E-Y, T-D-Y, and T-G-Y motifs in its activation loop region and plays a significant role in regulating diverse cellular responses in eukaryotic organisms. Availability of large-scale genome data in the fungal kingdom encouraged us to identify and analyse the fungal MAPK gene family consisting of 173 fungal species. The analysis of the MAPK gene family resulted in the discovery of several novel activation loop motifs (T-T-Y, T-I-Y, T-N-Y, T-H-Y, T-S-Y, K-G-Y, T-Q-Y, S-E-Y and S-D-Y) in fungal MAPKs. The phylogenetic analysis suggests that fungal MAPKs are non-polymorphic, had evolved from their common ancestors around 1500 million years ago, and are distantly related to plant MAPKs. We are the first to report the presence of nine novel activation loop motifs in fungal MAPKs. The specificity of the activation loop motif plays a significant role in controlling different growth and stress related pathways in fungi. Hence, the presences of these nine novel activation loop motifs in fungi are of special interest. PMID:26918378

  7. 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

  8. A wide dynamic range laser rangefinder with cm-level resolution based on AGC amplifier structure

    NASA Astrophysics Data System (ADS)

    Zhu, Feihu; Gong, Ke; Huo, Yujing

    2012-03-01

    A pulsed time-of-flight (TOF) laser rangefinder with a pulsed laser diode and an avalanche photo diode (APD) receiver is constructed and tested. Trigged by an avalanche transistor, the laser diode can emit a periodic pulse with rise time of ˜2 ns. A new structure with auto gain control (AGC) circuits both in the pre-amplifier and the post-amplifier is presented. Through this technology, not only the dynamic range of the receiver is extended, but also the walk error of timing discriminators is reduced. Large measurement range from 5 m to 500 m is achieved without any cooperative target. The single-shot precision is 3 cm for the weakest signal. Compared with previous laser rangefinders, the complexity of this system is greatly simplified.

  9. A CMOS low power, process/temperature variation tolerant RSSI with an integrated AGC loop

    NASA Astrophysics Data System (ADS)

    Qianqian, Lei; Min, Lin; Yin, Shi

    2013-03-01

    A low voltage low power CMOS limiter and received signal strength indicator (RSSI) with an integrated automatic gain control (AGC) loop for a short-distance receiver are implemented in SMIC 0.13 μm CMOS technology. The RSSI has a dynamic range of more than 60 dB and the RSSI linearity error is within ±0.5 dB for an input power from -65 to -8 dBm. The RSSI output voltage is from 0.15 to 1 V and the slope of the curve is 14.17 mV/dB while consuming 1.5 mA (I and Q paths) from a 1.2 V supply. Auto LNA gain mode selection with a combined RSSI function is also presented. Furthermore, with the compensation circuit, the proposed RSSI shows good temperature-independent and good robustness against process variation characteristics.

  10. Coexpression of the long and short forms of CheA, the chemotaxis histidine kinase, by members of the family Enterobacteriaceae.

    PubMed Central

    McNamara, B P; Wolfe, A J

    1997-01-01

    CheA is the histidine protein kinase of a two-component signal transduction system required for bacterial chemotaxis. Motile cells of the enteric species Escherichia coli and Salmonella typhimurium synthesize two forms of CheA by utilizing in-frame initiation sites within the gene cheA. The full-length protein, CheAL, plays an essential role in the chemotactic signaling pathway. In contrast, the function of the short form, CheAs, remains elusive. Although CheAs lacks the histidine residue that becomes phosphorylated in CheAL, it exhibits both kinase activity and the ability to interact with and enhance the activity of CheZ, a chemotaxis protein that accelerates dephosphorylation of the two-component response regulator CheY. To determine whether other members of the family Enterobacteriaceae express CheAs and CheZ, we analyzed immunoblots of proteins from clinical isolates of a variety of enteric species. All motile, chemotactic isolates that we tested coexpressed CheAL, CheAs, and CheZ. The only exceptions were closely related plant pathogens of the genus Erwinia, which expressed CheAL and CheZ but not CheAs. We also analyzed nucleotide sequences of the cheA loci from isolates of Serratia marcescens and Enterobacter cloacae, demonstrating the presence of in-frame translation initiation sites similar to those observed in the cheA loci of E. coli and S. typhimurium. Since coexpression of CheAs and CheZ appears to be limited to motile, chemotactic enteric bacteria, we propose that CheAs may play an important role in chemotactic responses in some environmental niches encountered by enteric species. PMID:9045846

  11. The Protein Kinase CK2 Mediates Cross-Talk between Auxin- and Salicylic Acid-Signaling Pathways in the Regulation of PINOID Transcription.

    PubMed

    Armengot, Laia; Caldarella, Eleonora; Marquès-Bueno, Maria Mar; Martínez, M Carmen

    2016-01-01

    The protein kinase CK2 is a ubiquitous and highly conserved enzyme, the activity of which is vital for eukaryotic cells. We recently demonstrated that CK2 modulates salicylic acid (SA) homeostasis in Arabidopsis thaliana, and that functional interplay between CK2 and SA sustains transcriptional expression of PIN-FORMED (PIN) genes. In this work, we show that CK2 also plays a key role in the transcriptional regulation of PINOID (PID), an AGC protein kinase that modulates the apical/basal localization of auxin-efflux transporters. We show that PID transcription is up-regulated by auxin and by SA and that CK2 is involved in both pathways. On the one hand, CK2 activity is required for proteosome-dependent degradation of AXR3, a member of the AUX/IAA family of auxin transcriptional repressors that must be degraded to activate auxin-responsive gene expression. On the other hand, the role of CK2 in SA homeostasis and, indirectly, in SA-driven PID transcription, was confirmed by using Arabidopsis NahG transgenic plants, which cannot accumulate SA. In conclusion, our results evidence a role for CK2 as a functional link in the negative cross-talk between auxin- and SA-signaling. PMID:27275924

  12. The Protein Kinase CK2 Mediates Cross-Talk between Auxin- and Salicylic Acid-Signaling Pathways in the Regulation of PINOID Transcription

    PubMed Central

    Armengot, Laia; Caldarella, Eleonora; Marquès-Bueno, Maria Mar; Martínez, M. Carmen

    2016-01-01

    The protein kinase CK2 is a ubiquitous and highly conserved enzyme, the activity of which is vital for eukaryotic cells. We recently demonstrated that CK2 modulates salicylic acid (SA) homeostasis in Arabidopsis thaliana, and that functional interplay between CK2 and SA sustains transcriptional expression of PIN-FORMED (PIN) genes. In this work, we show that CK2 also plays a key role in the transcriptional regulation of PINOID (PID), an AGC protein kinase that modulates the apical/basal localization of auxin-efflux transporters. We show that PID transcription is up-regulated by auxin and by SA and that CK2 is involved in both pathways. On the one hand, CK2 activity is required for proteosome-dependent degradation of AXR3, a member of the AUX/IAA family of auxin transcriptional repressors that must be degraded to activate auxin-responsive gene expression. On the other hand, the role of CK2 in SA homeostasis and, indirectly, in SA-driven PID transcription, was confirmed by using Arabidopsis NahG transgenic plants, which cannot accumulate SA. In conclusion, our results evidence a role for CK2 as a functional link in the negative cross-talk between auxin- and SA-signaling. PMID:27275924

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

    PubMed

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

    2008-01-01

    It is generally considered mammals and birds have five Tec family kinases (TFKs): Btk, Bmx (also known as Etk), Itk, Tec, and Txk (also known as Rlk). Here, we discuss the domains and their functions and regulation in TFKs. Over the last few years, a large number of genomes from various phyla have been sequenced making it possible to study evolutionary relationships at the molecular and sequence level. Using bioinformatics tools, we for the first time demonstrate that a TFK ancestor exists in the unicellular choanoflagellate Monosiga brevicollis, which is the closest known relative to metazoans with a sequenced genome. The analysis of the genomes for sponges, insects, hagfish, and frogs suggests that these species encode a single TFK. The insect form has a divergent and unique N-terminal region. Duplications generating the five members took place prior to the emergence of vertebrates. Fishes have two or three forms and the platypus, Ornithorhynchus anatinus, has four (lacks Txk). Thus, not all mammals have all five TFKs. The single identified TFK in frogs is an ortholog of Tec. Bmx seems to be unique to mammals and birds. SH3BP5 is a negative regulator of Btk. It is conserved in choanoflagellates and interestingly exists also in nematodes, which do not express TFKs, suggesting a broader function in addition to Btk regulation. The related SH3BP5-like protein is not found in Nematodes. PMID:19161832

  14. Identification of a BET family bromodomain/casein kinase II/TAF-containing complex as a regulator of mitotic condensin function.

    PubMed

    Kim, Hyun-Soo; Mukhopadhyay, Rituparna; Rothbart, Scott B; Silva, Andrea C; Vanoosthuyse, Vincent; Radovani, Ernest; Kislinger, Thomas; Roguev, Assen; Ryan, Colm J; Xu, Jiewei; Jahari, Harlizawati; Hardwick, Kevin G; Greenblatt, Jack F; Krogan, Nevan J; Fillingham, Jeffrey S; Strahl, Brian D; Bouhassira, Eric E; Edelmann, Winfried; Keogh, Michael-Christopher

    2014-03-13

    Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation. PMID:24565511

  15. Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment.

    PubMed

    Maza, Paloma K; Suzuki, Erika

    2016-01-01

    Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation. PMID:27148251

  16. Elevated Src family kinase activity stabilizes E-cadherin-based junctions and collective movement of head and neck squamous cell carcinomas

    PubMed Central

    Veracini, Laurence; Grall, Dominique; Schaub, Sébastien; Divonne, Stéphanie Beghelli-de la Forest; Etienne-Grimaldi, Marie-Christine; Milano, Gérard; Bozec, Alexandre; Babin, Emmanuel; Sudaka, Anne; Thariat, Juliette; Van Obberghen-Schilling, Ellen

    2015-01-01

    EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results. Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype. Robust expression of total and activated Src was observed in advanced stage head and neck tumors (N=60) and in head and neck squamous cell carcinoma lines. In cultured cancer cells Src co-localized with E-cadherin in cell-cell junctions and its phosphorylation on Y419 was both constitutive and independent of EGFR activation. Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes. These findings reveal an EGFR-independent role for SFKs in the maintenance of intercellular junctions, which likely contributes to the cohesive invasion E-cadherin-positive cells in advanced tumors. Further, they highlight the need for a deeper comprehension of molecular pathways that drive collective cell invasion, in absence of mesenchymal transition, in order to combat tumor spread. PMID:25779657

  17. Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment

    PubMed Central

    Maza, Paloma K.; Suzuki, Erika

    2016-01-01

    Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation. PMID:27148251

  18. Elevated Src family kinase activity stabilizes E-cadherin-based junctions and collective movement of head and neck squamous cell carcinomas.

    PubMed

    Veracini, Laurence; Grall, Dominique; Schaub, Sébastien; Beghelli-de la Forest Divonne, Stéphanie; Etienne-Grimaldi, Marie-Christine; Milano, Gérard; Bozec, Alexandre; Babin, Emmanuel; Sudaka, Anne; Thariat, Juliette; Van Obberghen-Schilling, Ellen

    2015-04-10

    EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results. Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype. Robust expression of total and activated Src was observed in advanced stage head and neck tumors (N=60) and in head and neck squamous cell carcinoma lines. In cultured cancer cells Src co-localized with E-cadherin in cell-cell junctions and its phosphorylation on Y419 was both constitutive and independent of EGFR activation. Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes. These findings reveal an EGFR-independent role for SFKs in the maintenance of intercellular junctions, which likely contributes to the cohesive invasion E-cadherin-positive cells in advanced tumors. Further, they highlight the need for a deeper comprehension of molecular pathways that drive collective cell invasion, in absence of mesenchymal transition, in order to combat tumor spread. PMID:25779657

  19. SRC family kinase inhibitors antagonize the toxicity of multiple serotypes of botulinum neurotoxin in human embryonic stem cell-derived motor neurons.

    PubMed

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

    2015-05-01

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

  20. Functional characterization of a member of alanine or glycine: cation symporter family in halotolerant cyanobacterium Aphanothece halophytica.

    PubMed

    Bualuang, Aporn; Kageyama, Hakuto; Tanaka, Yoshito; Incharoensakdi, Aran; Takabe, Teruhiro

    2015-01-01

    Membrane proteins of amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play important roles in the regulation of cellular processes. The alanine or glycine: cation symporter (AGCS) family belongs to APC superfamily and is found in prokaryotes, but its substrate specificity remains to be clarified. In this study, we found that a halotolerant cyanobacterium, Aphanothece halophytica has two putative ApagcS genes. The deduced amino acid sequence of one of genes, ApagcS1, exhibited high homology to Pseudomonas AgcS. The ApagcS1 gene was expressed in Escherichia coli JW4166 which is deficient in glycine uptake. Kinetics studies in JW4166 revealed that ApAgcS1 is a sodium-dependent glycine transporter. Competition experiments showed the significant inhibition by glutamine, asparagine, and glycine. The level of mRNA for ApagcS1 was induced by NaCl and nitrogen-deficient stresses. Uptake of glutamine by ApAgcS1 was also observed. Based on these data, the physiological role of ApAgcS1 was discussed. PMID:25421789

  1. The inhibition of tube formation in a collagen-fibrinogen, three-dimensional gel by cleaved kininogen (HKa) and HK domain 5 (D5) is dependent on Src family kinases

    SciTech Connect

    Liu Yuchuan; Sainz, Irma M.; Wu Yi; Pixley, Robin; Espinola, Ricardo G.; Hassan, Sarmina; Khan, Mohammad M.; Colman, Robert W.

    2008-02-15

    Cleaved high molecular weight kininogen (HKa), as well as its domain 5 (D5), inhibits migration and proliferation induced by angiogenic factors and induces apoptosis in vitro. To study its effect on tube formation we utilized a collagen-fibrinogen, three-dimensional gel, an in vitro model of angiogenesis. HKa, GST-D5 and D5 had a similar inhibitory effect of tube length by 90 {+-} 4.5%, 86 {+-} 5.5% and 77 {+-} 12.9%, respectively. D5-derived synthetic peptides: G440-H455 H475-H485 and G486-K502 inhibited tube length by 51 {+-} 3.7%, 54 {+-} 3.8% and 77 {+-} 1.7%, respectively. By a comparison of its inhibitory potency and its sequences, a functional sequence of HKa was defined to G486-G496. PP2, a Src family kinase inhibitor, prevented tube formation in a dose-dependent manner (100-400 nM), but PP3 at 5 {mu}M, an inactive analogue of PP2, did not. HKa and D5 inhibited Src 416 phosphorylation by 62 {+-} 12.3% and 83 {+-} 6.1%, respectively. The C-terminal Src kinase (Csk) inhibits Src kinase activity. Using a siRNA to Csk, expression of Csk was down-regulated by 86 {+-} 7.0%, which significantly increased tube length by 27 {+-} 5.8%. The addition of HKa and D5 completely blocked this effect. We further showed that HKa inhibited Src family kinase activity by disrupting the complex of uPAR, {alpha}v{beta}3 integrin and Src. Our results indicate that the anti-angiogenic effect of HKa and D5 is mediated at least in part through Src family kinases and identify a potential novel target for therapeutic inhibition of neovascularization in cancer and inflammatory arthritis.

  2. 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

  3. Ag/C nanoparticles as an cathode catalyst for a zinc-air battery with a flowing alkaline electrolyte

    NASA Astrophysics Data System (ADS)

    Han, Jia-Jun; Li, Ning; Zhang, Tian-Yun

    The cyclic voltammetry indicated that the oxygen reduction reaction (ORR) proceeded by the four-electron pathway mechanism on larger Ag particles (174 nm), and that the ORR proceeded by the four-electron pathway and the two-electron pathway mechanisms on finer Ag particles (4.1 nm), simultaneously. The kinetics towards ORR was measured at a rotating disk electrode (RDE) with Ag/C electrode. The number of exchanged electrons for the ORR was found to be close to four on larger Ag particles (174 nm) and close to three on finer Ag particles (4.1 nm). The zinc-air battery with Ag/C catalysts (25.9 nm) was fabricated and examined.

  4. PPARδ Activation Acts Cooperatively with 3-Phosphoinositide-Dependent Protein Kinase-1 to Enhance Mammary Tumorigenesis

    PubMed Central

    Pollock, Claire B.; Yin, Yuzhi; Yuan, Hongyan; Zeng, Xiao; King, Sruthi; Li, Xin; Kopelovich, Levy; Albanese, Chris; Glazer, Robert I.

    2011-01-01

    Peroxisome proliferator-activated receptorδ (PPARδ) is a transcription factor that is associated with metabolic gene regulation and inflammation. It has been implicated in tumor promotion and in the regulation of 3-phosphoinositide-dependent kinase-1 (PDK1). PDK1 is a key regulator of the AGC protein kinase family, which includes the proto-oncogene AKT/PKB implicated in several malignancies, including breast cancer. To assess the role of PDK1 in mammary tumorigenesis and its interaction with PPARδ, transgenic mice were generated in which PDK1 was expressed in mammary epithelium under the control of the MMTV enhancer/promoter region. Transgene expression increased pT308AKT and pS9GSK3β, but did not alter phosphorylation of mTOR, 4EBP1, ribosomal protein S6 and PKCα. The transgenic mammary gland also expressed higher levels of PPARδ and a gene expression profile resembling wild-type mice maintained on a diet containing the PPARδ agonist, GW501516. Both wild-type and transgenic mice treated with GW501516 exhibited accelerated rates of tumor formation that were more pronounced in transgenic animals. GW501516 treatment was accompanied by a distinct metabolic gene expression and metabolomic signature that was not present in untreated animals. GW501516-treated transgenic mice expressed higher levels of fatty acid and phospholipid metabolites than treated wild-type mice, suggesting the involvement of PDK1 in enhancing PPARδ-driven energy metabolism. These results reveal that PPARδ activation elicits a distinct metabolic and metabolomic profile in tumors that is in part related to PDK1 and AKT signaling. PMID:21297860

  5. EXTL2, a Member of the EXT Family of Tumor Suppressors, Controls Glycosaminoglycan Biosynthesis in a Xylose Kinase-dependent Manner*

    PubMed Central

    Nadanaka, Satomi; Zhou, Shaobo; Kagiyama, Shoji; Shoji, Naoko; Sugahara, Kazuyuki; Sugihara, Kazushi; Asano, Masahide; Kitagawa, Hiroshi

    2013-01-01

    Mutant alleles of EXT1 or EXT2, two members of the EXT gene family, are causative agents in hereditary multiple exostoses, and their gene products function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2, one of three EXT-like genes in the human genome that are homologous to EXT1 and EXT2, encodes a transferase that adds not only GlcNAc but also N-acetylgalactosamine to the glycosaminoglycan (GAG)-protein linkage region via an α1,4-linkage. However, both the role of EXTL2 in the biosynthesis of GAGs and the biological significance of EXTL2 remain unclear. Here we show that EXTL2 transfers a GlcNAc residue to the tetrasaccharide linkage region that is phosphorylated by a xylose kinase 1 (FAM20B) and thereby terminates chain elongation. We isolated an oligosaccharide from the mouse liver, which was not detected in EXTL2 knock-out mice. Based on structural analysis by a combination of glycosidase digestion and 500-MHz 1H NMR spectroscopy, the oligosaccharide was found to be GlcNAcα1-4GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), which was considered to be a biosynthetic intermediate of an immature GAG chain. Indeed, EXTL2 specifically transferred a GlcNAc residue to a phosphorylated linkage tetrasaccharide, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate). Remarkably, the phosphorylated linkage pentasaccharide generated by EXTL2 was not used as an acceptor for heparan sulfate or chondroitin sulfate polymerases. Moreover, production of GAGs was significantly higher in EXTL2 knock-out mice than in wild-type mice. These results indicate that EXTL2 functions to suppress GAG biosynthesis that is enhanced by a xylose kinase and that the EXTL2-dependent mechanism that regulates GAG biosynthesis might be a “quality control system” for proteoglycans. PMID:23395820

  6. Controlling Wind Turbines for Secondary Frequency Regulation: An Analysis of AGC Capabilities Under New Performance Based Compensation Policy: Preprint

    SciTech Connect

    Aho, J.; Pao, L. Y.; Fleming, P.; Ela, E.

    2015-02-01

    As wind energy becomes a larger portion of the world's energy portfolio there has been an increased interest for wind turbines to control their active power output to provide ancillary services which support grid reliability. One of these ancillary services is the provision of frequency regulation, also referred to as secondary frequency control or automatic generation control (AGC), which is often procured through markets which recently adopted performance-based compensation. A wind turbine with a control system developed to provide active power ancillary services can be used to provide frequency regulation services. Simulations have been performed to determine the AGC tracking performance at various power schedule set-points, participation levels, and wind conditions. The performance metrics used in this study are based on those used by several system operators in the US. Another metric that is analyzed is the damage equivalent loads (DELs) on turbine structural components, though the impacts on the turbine electrical components are not considered. The results of these single-turbine simulations show that high performance scores can be achieved when there is sufficient wind resource available. The capability of a wind turbine to rapidly and accurately follow power commands allows for high performance even when tracking rapidly changing AGC signals. As the turbine de-rates to meet decreased power schedule set-points there is a reduction in the DELs, and the participation in frequency regulation has a negligible impact on these loads.

  7. Comparison between the Strength Levels of Baseline Nuclear-Grade Graphite and Graphite Irradiated in AGC-2

    SciTech Connect

    Carroll, Mark Christopher

    2015-07-01

    This report details the initial comparison of mechanical strength properties between the cylindrical nuclear-grade graphite specimens irradiated in the second Advanced Graphite Creep (AGC-2) experiment with the established baseline, or unirradiated, mechanical properties compiled in the Baseline Graphite Characterization program. The overall comparative analysis will describe the development of an appropriate test protocol for irradiated specimens, the execution of the mechanical tests on the AGC-2 sample population, and will further discuss the data in terms of developing an accurate irradiated property distribution in the limited amount of irradiated data by leveraging the considerably larger property datasets being captured in the Baseline Graphite Characterization program. Integrating information on the inherent variability in nuclear-grade graphite with more complete datasets is one of the goals of the VHTR Graphite Materials program. Between “sister” specimens, or specimens with the same geometry machined from the same sub-block of graphite from which the irradiated AGC specimens were extracted, and the Baseline datasets, a comprehensive body of data will exist that can provide both a direct and indirect indication of the full irradiated property distributions that can be expected of irradiated nuclear-grade graphite while in service in a VHTR system. While the most critical data will remain the actual irradiated property measurements, expansion of this data into accurate distributions based on the inherent variability in graphite properties will be a crucial step in qualifying graphite for nuclear use as a structural material in a VHTR environment.

  8. Polymer Assisted Core-shell Ag-C nanoparticles Synthesis via Green hydrothermal Technique

    NASA Astrophysics Data System (ADS)

    Williams, James; Mishra, Sanjay

    2009-03-01

    Core-Shell Ag-C nanoparticles were synthesized in the presence of glucose through a one-pot green hydrothermal wet chemical process. An aqueous solution of glucose and Ag nitrate was hydrothermally treated to produce porous carbonaceous shell over silver core nanoparticles. The growth of carbon shells was regulated by either of the polymers (poly) vinyl pyrrolidone (PVP) or poly vinyl alcohol (PVA). The two polymers were compared to take a measure of different tunable sizes of cores, and shells. The effects of hydrothermal temperature, time, and concentration of reagents on the final formation of nanostructures were studied using UV-vis extinction spectra, transmission electron microscope, and Raman spectroscopy. The polymer molecules were found to be incorporated into carbonaceous shell. The resulting opacity of the shell was found to be hydrothermal time and temperature dependent. The shell structure was found to be more uniform with PVP than PVA. Furthermore, the polymer concentration was found to influence size and shape of the core-silver particles as well. The core-shelled nanoparticles have surfaces with organic groups capable of assembling with different reagents that could be useful in drug-delivery, optical nanodevices or biochemistry.

  9. DIDS inhibits Na-K-ATPase activity in porcine nonpigmented ciliary epithelial cells by a Src family kinase-dependent mechanism.

    PubMed

    Shahidullah, Mohammad; Wei, Guojun; Delamere, Nicholas A

    2013-09-01

    The anion transport inhibitor DIDS is known to reduce aqueous humor secretion but questions remain about anion dependence of the effect. In some tissues, DIDS is reported to cause Na-K-ATPase inhibition. Here, we report on the ability of DIDS to inhibit Na-K-ATPase activity in nonpigmented ciliary epithelium (NPE) and investigate the underlying mechanism. Porcine NPE cells were cultured to confluence on permeable supports, treated with drugs added to both sides of the membrane, and then used for (86)Rb uptake measurements or homogenized to measure Na-K-ATPase activity or to detect protein phosphorylation. DIDS inhibited ouabain-sensitive (86)Rb uptake, activated Src family kinase (SFK), and caused a reduction of Na-K-ATPase activity. PP2, an SFK inhibitor, prevented the DIDS responses. In BCECF-loaded NPE, DIDS was found to reduce cytoplasmic pH (pHi). PP2-sensitive Na-K-ATPase activity inhibition, (86)Rb uptake suppression, and SFK activation were observed when a similar reduction of pHi was imposed by low-pH medium or an ammonium chloride withdrawal maneuver. PP2 and the ERK inhibitor U0126 prevented robust ERK1/2 activation in cells exposed to DIDS or subjected to pHi reduction, but U0126 did not prevent SFK activation or the Na-K-ATPase activity response. The evidence points to an inhibitory influence of DIDS on NPE Na-K-ATPase activity by a mechanism that hinges on SFK activation associated with a reduction of cytoplasmic pH. PMID:23677800

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

    PubMed Central

    Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa. PMID:26173020

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

    PubMed

    Lu, Kun; Guo, Wenjin; Lu, Junxing; Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa. PMID:26173020

  12. 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

  13. A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development.

    PubMed Central

    Zhou, K; Takegawa, K; Emr, S D; Firtel, R A

    1995-01-01

    Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate. These phosphoinositides have been shown to function in vesicle-mediated protein sorting, and they serve as second-messenger signaling molecules for regulating cell growth. To further elucidate the mechanism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Database analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 are most closely related to the mammalian p110 PI 3-kinase, DdPIK5 is closest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous to PI 4-kinases. Together with other known PI kinases, a superfamily of PI kinase genes has been defined, with all of the encoded proteins sharing a common highly conserved catalytic core domain. DdPIK1, -2, and -3 may have redundant functions because disruption of any single gene had no effect on D. discoideum growth or development. However, strains in which both of the two most highly related genes, DdPIK1 and DdPIK2, were disrupted showed both growth and developmental defects, while double knockouts of DdPIK1 and DdPIK3 and DdPIK2 and DdPIK3 appear to be lethal. The delta Ddpik1 delta Ddpik2 null cells were smaller than wild-type cells and grew slowly both in association with bacteria and in axenic medium when attached to petri plates but were unable to grow in suspension in axenic medium. When delta Ddpik1 delta Ddpik2 null cells were plated for multicellular development, they formed aggregates having multiple tips and produced abnormal fruiting bodies. Antisense expression of DdPIK5 (a putative homolog of the Saccharomyces cerevisiae VPS34) led to a defect in the growth of D. discoideum cells on bacterial lawns and abnormal development. DdPIK5 complemented the temperature-sensitive growth defect of a Schizosaccharomyces pombe delta Svps34 mutant strain, suggesting DdPIK5

  14. Transient response and stability of the AGC-PI closed-loop controlled MEMS vibratory gyroscopes

    NASA Astrophysics Data System (ADS)

    Cui, J.; Chi, X. Z.; Ding, H. T.; Lin, L. T.; Yang, Z. C.; Yan, G. Z.

    2009-12-01

    This paper presents a detailed study on the transient response and stability of the automatic gain control (AGC) with a proportion-integral (PI) controller for a MEMS vibratory gyroscope, which constructs a closed-loop control system to make the gyroscope achieve a constant amplitude vibration at its resonant frequency. The nonlinear mathematical model for the control system is established by applying the averaging and linearization method, which is evaluated through numerical simulations. The stability and convergence characteristics of the whole loop are investigated by using the phase plane method and Routh-Hurwitz criterion. The analysis provides a quantitative methodology for selecting the system parameters to approach stability and an optimal transient response. The negative impact induced by drift of the resonant frequency and Q-factor is also discussed. Simulation results predicted by the model are shown to be in close agreement with the experimental results carried out on a doubly decoupled bulk micromachined gyroscope. By optimizing the control parameters, the measured rising time is less than 100 ms without obvious overshoot. The setting time of the whole loop is less than 200 ms with the relative fluctuation of velocity amplitude within approximately 16 ppm for an hour. The resulting overall performance of the gyroscope is tested under atmospheric pressure. The resonant frequencies and the Q-factor of the drive mode and sense mode are 2.986 kHz, 213 and 3.199 kHz, 233, respectively. The gyroscope achieves a scale factor of 27.6 mV/deg/s with nonlinearity less than 120 ppm in the full-scale range of 800° s-1. The threshold of sensitivity is measured to be about 0.005° s-1 with noise equivalent angular rate evaluated to be 0.001°/s/Hz1/2.

  15. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  16. Multi-kinase inhibitors, AURKs and cancer.

    PubMed

    Cicenas, Jonas; Cicenas, Erikas

    2016-05-01

    Inhibitors that impact function of kinases are valuable both for the biological research as well as therapy of kinase-associated diseases, such as different cancers. There are quite a number of inhibitors, which are quite specific for certain kinases and several of them are either already approved for the cancer therapy or are in clinical studies of various phases. However, that does not mean that each single kinase inhibitor is suitable for targeted therapy. Some of them are not effective others might be toxic or fail some other criteria for the use in vivo. On the other hand, even in case of successful therapy, many responders eventually develop resistance to the inhibitors. The limitations of various single kinase inhibitors can be fought using compounds which target multiple kinases. This tactics can increase effectiveness of the inhibitors by the synergistic effect or help to diminish the likelihood of drug resistance. To date, several families of kinases are quite popular targets of the inhibition in cancers, such as tyrosine kinases, cycle-dependent kinases, mitogen-activated protein kinases, phosphoinositide 3-kinases as well as their pathway "players" and aurora kinases. Aurora kinases play an important role in the control of the mitosis and are often altered in diverse human cancers. Here, we will describe the most interesting multi-kinase inhibitors which inhibit aurora kinases among other targets and their use in preclinical and clinical cancer studies. PMID:27038473

  17. Searching for Optical Counterparts to Ultra-compact High Velocity Clouds: Possible Detection of a Counterpart to AGC 198606

    NASA Astrophysics Data System (ADS)

    Janesh, William; Rhode, Katherine L.; Salzer, John J.; Janowiecki, Steven; Adams, Elizabeth A. K.; Haynes, Martha P.; Giovanelli, Riccardo; Cannon, John M.; Muñoz, Ricardo R.

    2015-09-01

    We report initial results from a campaign to obtain optical imaging of Ultra Compact High Velocity Clouds (UCHVCs) discovered by the Arecibo Legacy Fast ALFA (ALFALFA) Hi survey. UCHVCs have properties consistent with their being low-mass dwarf galaxies in the Local Volume, but do not have identified optical counterparts. We are using the WIYN 3.5 m telescope to image these objects and search for an associated stellar population. Here we present our observational strategy and method for searching for resolved stellar counterparts to the UCHVCs. We combine careful photometry, a color-magnitude filter, and spatial smoothing techniques to search for stellar overdensities in the g- and i-band images. We also run statistical tests to quantify the likelihood that detected overdensities are real and not chance superpositions of sources. We demonstrate the method by applying it to WIYN imaging of two objects: Leo P, a UCHVC discovered by ALFALFA and shown to be a star-forming dwarf galaxy in the Local Volume and AGC 198606, an ALFALFA source near in position and velocity to the Local Group dwarf galaxy Leo T. Applying the search method to the Leo P data yields an unambiguous detection (>99% confidence) of the galaxy’s stellar population. Applying the method to the AGC 198606 imaging yields a possible detection (92% confidence) of an optical counterpart located ˜2.5 arcmin from the centroid of AGC 198606's Hi distribution and within the Hi disk. We estimate a distance to the stellar counterpart of 373-393 kpc, an absolute magnitude Mi = -4.67 ± 0.09, and an Hi-to-stellar mass ratio of ˜45-110.

  18. Searching for Optical Counterparts to Ultra-compact High Velocity Clouds: Possible Detection of a Counterpart to AGC 198606

    NASA Astrophysics Data System (ADS)

    Janesh, William; Rhode, Katherine L.; Salzer, John J.; Janowiecki, Steven; Adams, Elizabeth A. K.; Haynes, Martha P.; Giovanelli, Riccardo; Cannon, John M.; Muñoz, Ricardo R.

    2015-09-01

    We report initial results from a campaign to obtain optical imaging of Ultra Compact High Velocity Clouds (UCHVCs) discovered by the Arecibo Legacy Fast ALFA (ALFALFA) Hi survey. UCHVCs have properties consistent with their being low-mass dwarf galaxies in the Local Volume, but do not have identified optical counterparts. We are using the WIYN 3.5 m telescope to image these objects and search for an associated stellar population. Here we present our observational strategy and method for searching for resolved stellar counterparts to the UCHVCs. We combine careful photometry, a color–magnitude filter, and spatial smoothing techniques to search for stellar overdensities in the g- and i-band images. We also run statistical tests to quantify the likelihood that detected overdensities are real and not chance superpositions of sources. We demonstrate the method by applying it to WIYN imaging of two objects: Leo P, a UCHVC discovered by ALFALFA and shown to be a star-forming dwarf galaxy in the Local Volume and AGC 198606, an ALFALFA source near in position and velocity to the Local Group dwarf galaxy Leo T. Applying the search method to the Leo P data yields an unambiguous detection (>99% confidence) of the galaxy’s stellar population. Applying the method to the AGC 198606 imaging yields a possible detection (92% confidence) of an optical counterpart located ∼2.5 arcmin from the centroid of AGC 198606's Hi distribution and within the Hi disk. We estimate a distance to the stellar counterpart of 373–393 kpc, an absolute magnitude Mi = ‑4.67 ± 0.09, and an Hi-to-stellar mass ratio of ∼45–110.

  19. Hybrid Stochastic Search Technique based Suboptimal AGC Regulator Design for Power System using Constrained Feedback Control Strategy

    NASA Astrophysics Data System (ADS)

    Ibraheem, Omveer, Hasan, N.

    2010-10-01

    A new hybrid stochastic search technique is proposed to design of suboptimal AGC regulator for a two area interconnected non reheat thermal power system incorporating DC link in parallel with AC tie-line. In this technique, we are proposing the hybrid form of Genetic Algorithm (GA) and simulated annealing (SA) based regulator. GASA has been successfully applied to constrained feedback control problems where other PI based techniques have often failed. The main idea in this scheme is to seek a feasible PI based suboptimal solution at each sampling time. The feasible solution decreases the cost function rather than minimizing the cost function.

  20. Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina

    PubMed Central

    Lindsay, Ken J.; Du, Jianhai; Sloat, Stephanie R.; Contreras, Laura; Linton, Jonathan D.; Turner, Sally J.; Sadilek, Martin; Hurley, James B.

    2014-01-01

    Symbiotic relationships between neurons and glia must adapt to structures, functions, and metabolic roles of the tissues they are in. We show here that Müller glia in retinas have specific enzyme deficiencies that can enhance their ability to synthesize Gln. The metabolic cost of these deficiencies is that they impair the Müller cell’s ability to metabolize Glc. We show here that the cells can compensate for this deficiency by using metabolites produced by neurons. Müller glia are deficient for pyruvate kinase (PK) and for aspartate/glutamate carrier 1 (AGC1), a key component of the malate-aspartate shuttle. In contrast, photoreceptor neurons express AGC1 and the M2 isoform of pyruvate kinase, which is commonly associated with aerobic glycolysis in tumors, proliferating cells, and some other cell types. Our findings reveal a previously unidentified type of metabolic relationship between neurons and glia. Müller glia compensate for their unique metabolic adaptations by using lactate and aspartate from neurons as surrogates for their missing PK and AGC1. PMID:25313047

  1. A 6 μW per channel analog biomimetic cochlear implant processor filterbank architecture with across channels AGC.

    PubMed

    Yang, Guang; Lyon, Richard F; Drakakis, Emmanuel M

    2015-02-01

    A new analog cochlear implant processor filterbank architecture of increased biofidelity, enhanced across-channel contrast and very low power consumption has been designed and prototyped. Each channel implements a biomimetic, asymmetric bandpass-like One-Zero-Gammatone-Filter (OZGF) transfer function, using class-AB log-domain techniques. Each channel's quality factor and suppression are controlled by means of a new low power Automatic Gain Control (AGC) scheme which is coupled across the neighboring channels and emulates lateral inhibition (LI) phenomena in the auditory system. Detailed measurements from a five-channel silicon IC prototype fabricated in a 0.35 μm AMS technology confirm the operation of the coupled AGC scheme and its ability to enhance contrast among channel outputs. The prototype is characterized by an input dynamic range of 92 dB while consuming only 28 μW of power in total ( ∼ 6 μW per channel) under a 1.8 V power supply. The architecture is well-suited for fully-implantable cochlear implants. PMID:25069120

  2. Cyclin-dependent kinases

    PubMed Central

    2014-01-01

    Summary Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a separate subunit - a cyclin - that provides domains essential for enzymatic activity. CDKs play important roles in the control of cell division and modulate transcription in response to several extra- and intracellular cues. The evolutionary expansion of the CDK family in mammals led to the division of CDKs into three cell-cycle-related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20). Unlike the prototypical Cdc28 kinase of budding yeast, most of these CDKs bind one or a few cyclins, consistent with functional specialization during evolution. This review summarizes how, although CDKs are traditionally separated into cell-cycle or transcriptional CDKs, these activities are frequently combined in many family members. Not surprisingly, deregulation of this family of proteins is a hallmark of several diseases, including cancer, and drug-targeted inhibition of specific members has generated very encouraging results in clinical trials. PMID:25180339

  3. The Src family kinase inhibitors PP2 and PP1 block TGF-beta1-mediated cellular responses by direct and differential inhibition of type I and type II TGF-beta receptors.

    PubMed

    Ungefroren, Hendrik; Sebens, Susanne; Groth, Stephanie; Gieseler, Frank; Fändrich, Fred

    2011-05-01

    Both the nonreceptor tyrosine kinase Src and the receptors for transforming growth factor (TGF)-β (TβRI, TβRII) play major roles during tumorigenesis by regulating cell growth, migration/invasion and metastasis. The common Src family kinase inhibitors PP2 and PP1 effectively block Src activity in vitro and in vivo, however, they may exert non-specific effects on other kinases. In this study, we have evaluated PP2 and PP1 for their ability to inhibit TGFβ1-mediated responses in the TGF-β-responsive pancreatic adenocarcinoma cell line Panc1. We show that PP2 and PP1 but not the more specific Src inhibitor SU6656 effectively relieved TGF-b1-induced growth arrest and p21(WAF1) induction, while basal growth was enhanced by PP2 and PP1, and suppressed by SU6656. PP2 and PP1 but not SU6656 also suppressed TGF-β1-induced epithelial-to-mesenchymal transition (EMT) as evidenced by their ability to inhibit downregulation of the epithelial marker E-cadherin, and upregulation of the EMT-associated transcription factor Slug. Likewise, PP2 and PP1 but not SU6656 effectively blocked TGF-β1-induced activation of Smad2 and p38 MAPK and partially suppressed Smad activation and transcriptional activity on TGF-β/Smad-responsive reporters of a kinase-active TβRI mutant ectopically expressed in Panc1 cells. Interestingly, PP2 and PP1 strongly inhibited recombinant TβRI in an in vitro kinase assay, with PP1 being more potent and PP2 being nearly as potent as the established TβRI inhibitor SB431542. PP2 but not PP1 also weakly inhibited the TβRII kinase. Together, these data provide evidence that PP2 and PP1 are powerful inhibitors of TβR function that can block TGF-β/Smad signaling in a Src-unrelated fashion. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics of late stage metastatic disease. PMID:21395548

  4. Decorin induces rapid secretion of thrombospondin-1 in basal breast carcinoma cells via inhibition of Ras homolog gene family, member A/Rho-associated coiled-coil containing protein kinase 1.

    PubMed

    Neill, Thomas; Jones, Holly R; Crane-Smith, Zoe; Owens, Rick T; Schaefer, Liliana; Iozzo, Renato V

    2013-05-01

    Pathological neovascularization relies on an imbalance between potent proangiogenic agents and equally effective antiangiogenic cues. Collectively, these factors contribute to an angiogenic niche within the tumor microenvironment. Oncogenic events and hypoxia contribute to augmented levels of angiokines, and thereby activate the so-called angiogenic switch to promote aggressive tumorigenic and metastatic growth. Soluble decorin functions as a paracrine pan-inhibitor of receptor tyrosine kinases, such as Met and epidermal growth factor receptor, and thus is capable of suppressing angiogenesis under normoxia. This leads to noncanonical repression of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor A (VEGFA), and concurrent induction of thrombospondin-1. The substantial induction of endogenous tumor cell-derived thrombospondin-1, a potent antiangiogenic effector, led us to the discovery of an unexpected secretory phenotype occurring very rapidly (within 5 min) after decorin treatment of the triple-negative basal breast carcinoma cell line MDA-MB-231. Surprisingly, the effect was not mediated by Met receptor antagonism, as initially hypothesized, but required epidermal growth factor receptor signaling to achieve swift and robust thrombospondin-1 release. Furthermore, this effect was ultimately dependent on the prompt degradation of Ras homolog gene family member A, via the 26S proteasome, leading to direct inactivation of Rho-associated coiled-coil containing protein kinase 1. The latter led to derepression of thrombospondin-1 secretion. Collectively, these data provide a novel mechanistic role for Rho-associated coiled-coil containing protein kinase 1, in addition to providing the first conclusive evidence of decorin exclusively targeting a receptor tyrosine kinase to achieve a specific effect. The overall effects of soluble decorin on the tumor microenvironment would cause an immediately-early as well as a sustained antiangiogenic response

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

    PubMed Central

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

    2015-01-01

    Calcium ions are considered ubiquitous second messengers in eukaryotic signal transduction pathways. Intracellular Ca2+ concentration are modulated by various signals such as hormones and biotic and abiotic stresses. Modulation of Ca2+ ion leads to stimulation of calcium dependent protein kinase genes (CPKs), which results in regulation of gene expression and therefore mediates plant growth and development as well as biotic and abiotic stresses. Here, we reported the CPK gene family of 40 different plant species (950 CPK genes) and provided a unified nomenclature system for all of them. In addition, we analyzed their genomic, biochemical and structural conserved features. Multiple sequence alignment revealed that the kinase domain, auto-inhibitory domain and EF-hands regions of regulatory domains are highly conserved in nature. Additionally, the EF-hand domains of higher plants were found to contain four D-x-D and two D-E-L motifs, while lower eukaryotic plants had two D-x-D and one D-x-E motifs in their EF-hands. Phylogenetic analysis showed that CPK genes are clustered into four different groups. By studying the CPK gene family across the plant lineage, we provide the first evidence of the presence of D-x-D motif in the calcium binding EF-hand domain of CPK proteins. PMID:26734045

  6. A 2-mW 50-dB DR wideband hybrid AGC for a GNSS receiver in 65 nm CMOS

    NASA Astrophysics Data System (ADS)

    Yang, Xu; Baoyong, Chi; Yang, Xu; Nan, Qi; Zhihua, Wang

    2012-07-01

    A low-power wideband hybrid automatic gain control (AGC) loop for a GNSS receiver is presented. Single AGC in the I/Q path is composed of four-stage programmable gain amplifiers (PGAs), a differential peak detector, two comparators, a control algorithm logic, a decoder and the reference voltage source. Besides being controlled by an AGC loop, the gain of PGAs could alternatively be controlled by an off-chip digital baseband processor through the SPI interface. To obtain low power consumption and noise, an improved source degenerated amplifier is adopted, and the I/Q path phase mismatch within the ±5° range is calibrated with 0.2° accuracy. Implemented in 65 nm CMOS, the measured PGA total gains range from 9.8 to 59.5 dB with an average step of 0.95 dB and simulated bandwidth of more than 110 MHz. The settling time is about 180 μs with 80% AM input with measured signal power from -76.7 to -56.6 dBm from a radio-frequency amplifier (RFA) input port, and also reduces to 90 μs with clock frequency doubling. The single AGC consumes almost 0.8 mA current from the 2.5-V supply and occupies an area of 750 × 300 μm2.

  7. Design, synthesis and biological evaluation of pyrazol-furan carboxamide analogues as novel Akt kinase inhibitors.

    PubMed

    Zhan, Wenhu; Xu, Lei; Dong, Xiaowu; Dong, Jun; Yi, Xiao; Ma, Xiaodong; Qiu, Ni; Li, Jia; Yang, Bo; Zhou, Yubo; Hu, Yongzhou

    2016-07-19

    A series of novel pyrazol-furan carboxamide analogues were designed, synthesized and biologically evaluated for their Akt1 inhibitory activities, as well as anti-proliferative efficacies against HCT116 and OVCAR-8 cell lines. Most compounds exhibited moderate to excellent Akt1 inhibitory activities, together with favorable cytotoxicities. Further kinase selectivity assay of the most promising compound 25e illustrated that it was also potent against the structurally related AGC kinases, including Akt2, Akt3, ROCK1 and PKA, but was specific over kinases from other subfamilies. In addition, the Western blot analysis indicated that 25e could significantly suppress the phosphorylation level of Akt substrate GSK3β in PC-3 cell. Moreover, 25e demonstrated a concentration-dependent inhibition of phosphorylation of PRAS40 in LNCaP cell, with IC50 value of 30.4 nM. PMID:27089211

  8. Electron transport properties and spin modulation effect analysis for C60, Au@C60 and Ag@C60

    NASA Astrophysics Data System (ADS)

    Wang, Fangyuan; Li, Guiqin

    2016-04-01

    The electron transport properties of C60, Au@C60 and Ag@C60 junctions between Au electrodes are investigated with density functional theory and the first-principle nonequilibrium Greens function. Density of states and transmission possibility as functions of energy are examined, and current-voltage characteristics of all these models are calculated with and without spin correction. With spin correction, we find a spin-current difference in Au@C60 as large as an order of magnitude. It may be used to obtain high-purity arbitrary spin current. By comparing the calculation results with and without spin correction, we attribute this spin difference to the spin resonance between transmission electrons and the valence electron of endohedral Au atom.

  9. Range and AGC normalization in airborne discrete-return LiDAR intensity data for forest canopies

    NASA Astrophysics Data System (ADS)

    Korpela, Ilkka; Ørka, Hans Ole; Hyyppä, Juha; Heikkinen, Ville; Tokola, Timo

    Recently, the intensity characteristics of discrete-return LiDAR sensors were studied for vegetation classification. We examined two normalization procedures affecting LiDAR intensity through the scanning geometry and the system settings, namely, range normalization and the effects of the automatic gain control (AGC) in the Optech ALTM3100 and Leica ALS50-II sensors. Range normalization corresponds to weighting of the observed intensities with the term (, where R is the range, R is a mean reference range, and a∈[2,4] is the exponent that is, according to theory, dependent on the target geometry. LiDAR points belonging to individual tree crowns were extracted for 13 887 trees in southern Finland. The coefficient of variation (CV) of the intensity was analyzed for a range of values of exponent a. The tree species classification performance using 13 intensity variables was also used for sensitivity analysis of the effect of a. The results were in line with the established theory, since the optimal level of a was lower (a≈2) for trees with large or clumped leaves and higher (a≈3) for diffuse coniferous crowns. Different echo groups also showed varying responses. Single-return pulses that represented strong reflections had a lower optimal value of a than the first and all echoes in a pulse. The gain in classification accuracy from the optimal selection of the exponent was 2%-3%, and the optimum for classification was different from that obtained using the CV analysis. In the ALS50-II sensor, the combined and optimized AGC and R normalizations had a notably larger effect (6%-9%) on classification accuracy. Our study demonstrates the ambiguity of R normalization in vegetation canopies.

  10. Long Wavelength Monitoring of Protein Kinase Activity

    PubMed Central

    Oien, Nathan P.; Nguyen, Luong T.; Jernigan, Finith E.; Priestman, Melanie A.

    2014-01-01

    A family of long wavelength protein kinase fluorescent reporters is described in which the probing wavelength is pre-programmed using readily available fluorophores. These agents can assess protein kinase activity within the optical window of tissue, as exemplified by monitoring endogenous cAMP-dependent protein kinase activity (1) in erythrocyte lysates and (2) in intact erythrocytes using a light-activatable reporter. PMID:24604833

  11. Physical and functional association of the cbl protooncogen product with an src-family protein tyrosine kinase, p53/56lyn, in the B cell antigen receptor-mediated signaling.

    PubMed

    Tezuka, T; Umemori, H; Fusaki, N; Yagi, T; Takata, M; Kurosaki, T; Yamamoto, T

    1996-02-01

    To identify novel signal transducers involved in signaling mediated by the Src-family protein tyrosine kinases (PTKs), we used a yeast two-hybrid system with a probe corresponding to the regulatory region of p56lyn, a member of Src-family PTKs. One of the isolated clones contained the COOH-terminal 470 amino acid residues of p120c-cbl, the product of the cellular homologue of the v-cbl retroviral oncogene. p120c-cbl is a cytoplasmic protein with nuclear protein-like motifs. Here we show in vivo association of p120c-cbl with p53/56lyn. After stimulation of the B cell antigen receptor (BCR), p120c-cbl was rapidly tyrosine phosphorylated. Studies with lyn- or syk-negative chicken B cells demonstrated that p53/56lyn, but not p72syk, was crucial for tyrosine phosphorylation of p120c-cbl upon stimulation of the BCR. We also show the importance of p59fyn in tyrosine phosphorylation of p120c-cbl in the T-cell receptor-mediated signaling using fyn-overexpressing T cell hybridomas and splenic T cells from fyn-deficient mice. These results suggest that p120c-cbl is an important substrate of Src-family PTKs in the intracellular signaling mediated by the antigen receptors PMID:8627181

  12. Physical and functional association of the cbl protooncogen product with an src-family protein tyrosine kinase, p53/56lyn, in the B cell antigen receptor-mediated signaling

    PubMed Central

    1996-01-01

    To identify novel signal transducers involved in signaling mediated by the Src-family protein tyrosine kinases (PTKs), we used a yeast two- hybrid system with a probe corresponding to the regulatory region of p56lyn, a member of Src-family PTKs. One of the isolated clones contained the COOH-terminal 470 amino acid residues of p120c-cbl, the product of the cellular homologue of the v-cbl retroviral oncogene. p120c-cbl is a cytoplasmic protein with nuclear protein-like motifs. Here we show in vivo association of p120c-cbl with p53/56lyn. After stimulation of the B cell antigen receptor (BCR), p120c-cbl was rapidly tyrosine phosphorylated. Studies with lyn- or syk-negative chicken B cells demonstrated that p53/56lyn, but not p72syk, was crucial for tyrosine phosphorylation of p120c-cbl upon stimulation of the BCR. We also show the importance of p59fyn in tyrosine phosphorylation of p120c- cbl in the T-cell receptor-mediated signaling using fyn-overexpressing T cell hybridomas and splenic T cells from fyn-deficient mice. These results suggest that p120c-cbl is an important substrate of Src-family PTKs in the intracellular signaling mediated by the antigen receptors PMID:8627181

  13. Txk, a member of the non-receptor tyrosine kinase of the Tec family, forms a complex with poly(ADP-ribose) polymerase 1 and elongation factor 1α and regulates interferon-γ gene transcription in Th1 cells

    PubMed Central

    Maruyama, T; Nara, K; Yoshikawa, H; Suzuki, N

    2007-01-01

    We have found previously that Txk, a member of the Tec family tyrosine kinases, is involved importantly in T helper 1 (Th1) cytokine production. However, how Txk regulates interferon (IFN)-γ gene transcription in human T lymphocytes was not fully elucidated. In this study, we identified poly(ADP-ribose) polymerase 1 (PARP1) and elongation factor 1α (EF-1α) as Txk-associated molecules that bound to the Txk responsive element of the IFN-γ gene promoter. Txk phosphorylated EF-1α and PARP1 formed a complex with them, and bound to the IFN-γ gene promoter in vitro. In particular, the N terminal region containing the DNA binding domain of PARP1 was important for the trimolecular complex formation involving Txk, EF-1α and PARP1. Several mutant Txk which lacked kinase activity were unable to form the trimolecular complex. A PARP1 inhibitor, PJ34, suppressed IFN-γ but not interleukin (IL)-4 production by normal peripheral blood lymphocytes (PBL). Multi-colour confocal analysis revealed that Txk and EF-1α located in the cytoplasm in the resting condition. Upon activation, a complex involving Txk, EF-1α and PARP1 was formed and was located in the nucleus. Collectively, Txk in combination with EF-1α and PARP1 bound to the IFN-γ gene promoter, and exerted transcriptional activity on the IFN-γ gene. PMID:17177976

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

    SciTech Connect

    Schuster, V.; Seidenspinner, S.; Wolfgang Kreth, H.

    1996-05-03

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

  15. Role of melanoma inhibitor of apoptosis (ML-IAP) protein, a member of the baculoviral IAP repeat (BIR) domain family, in the regulation of C-RAF kinase and cell migration.

    PubMed

    Oberoi-Khanuja, Tripat Kaur; Karreman, Christiaan; Larisch, Sarit; Rapp, Ulf R; Rajalingam, Krishnaraj

    2012-08-17

    Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration. PMID:22711539

  16. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert . E-mail: biocrr@lsuhsc.edu

    2005-05-27

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.

  17. MST kinases in development and disease

    PubMed Central

    2015-01-01

    The mammalian MST kinase family, which is related to the Hippo kinase in Drosophila melanogaster, includes five related proteins: MST1 (also called STK4), MST2 (also called STK3), MST3 (also called STK24), MST4, and YSK1 (also called STK25 or SOK1). MST kinases are emerging as key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. Here we review the regulation and function of these kinases in normal physiology and pathologies, including cancer, endothelial malformations, and autoimmune disease. PMID:26370497

  18. 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.

  19. Aurora Kinase Inhibitors: Current Status and Outlook

    PubMed Central

    Bavetsias, Vassilios; Linardopoulos, Spiros

    2015-01-01

    The Aurora kinase family comprises of cell cycle-regulated serine/threonine kinases important for mitosis. Their activity and protein expression are cell cycle regulated, peaking during mitosis to orchestrate important mitotic processes including centrosome maturation, chromosome alignment, chromosome segregation, and cytokinesis. In humans, the Aurora kinase family consists of three members; Aurora-A, Aurora-B, and Aurora-C, which each share a conserved C-terminal catalytic domain but differ in their sub-cellular localization, substrate specificity, and function during mitosis. In addition, Aurora-A and Aurora-B have been found to be overexpressed in a wide variety of human tumors. These observations led to a number of programs among academic and pharmaceutical organizations to discovering small molecule Aurora kinase inhibitors as anti-cancer drugs. This review will summarize the known Aurora kinase inhibitors currently in the clinic, and discuss the current and future directions. PMID:26734566

  20. Effect of visible-light illumination on resistive switching characteristics in Ag/Ce2W3O12/FTO devices

    NASA Astrophysics Data System (ADS)

    Sun, Bai; Li, Xiaoping; Liang, Dandan; Chen, Peng

    2016-01-01

    The resistive switching device is a fascinating candidate for next generation nonvolatile memories. In this Letter, we report a simple hydrothermal way to prepare Ce2W3O12 powder. Furthermore, we fabricated a resistive switching memory device with Ag/Ce2W3O12/fluorine-doped tin oxide (FTO) structure. Moreover, we observed the effect of visible-light illumination on resistive switching memory behaviour in Ag/Ce2W3O12/FTO devices. This Letter is useful for exploring the new potential materials for resistive switching memory device, and provides the visible-light as a new control method for resistive switching random access memory (RRAM).

  1. Targeting Drug-Sensitive and -Resistant Strains of Mycobacterium tuberculosis by Inhibition of Src Family Kinases Lowers Disease Burden and Pathology

    PubMed Central

    Chandra, Pallavi; Rajmani, R. S.; Verma, Garima; Bhavesh, Neel Sarovar

    2016-01-01

    ABSTRACT In view of emerging drug resistance among bacterial pathogens, including Mycobacterium tuberculosis, the development of novel therapeutic strategies is increasingly being sought. A recent paradigm in antituberculosis (anti-TB) drug development is to target the host molecules that are crucial for intracellular survival of the pathogen. We previously showed the importance of Src tyrosine kinases in mycobacterial pathogenesis. Here, we report that inhibition of Src significantly reduced survival of H37Rv as well as multidrug-resistant (MDR) and extremely drug-resistant (XDR) strains of M. tuberculosis in THP-1 macrophages. Src inhibition was also effective in controlling M. tuberculosis infection in guinea pigs. In guinea pigs, reduced M. tuberculosis burden due to Src inhibition also led to a marked decline in the disease pathology. In agreement with the theoretical framework of host-directed approaches against the pathogen, Src inhibition was equally effective against an XDR strain in controlling infection in guinea pigs. We propose that Src inhibitors could be developed into effective host-directed anti-TB drugs, which could be indiscriminately used against both drug-sensitive and drug-resistant strains of M. tuberculosis. IMPORTANCE The existing treatment regimen for tuberculosis (TB) suffers from deficiencies like high doses of antibiotics, long treatment duration, and inability to kill persistent populations in an efficient manner. Together, these contribute to the emergence of drug-resistant tuberculosis. Recently, several host factors were identified which help intracellular survival of Mycobacterium tuberculosis within the macrophage. These factors serve as attractive targets for developing alternate therapeutic strategies against M. tuberculosis. This strategy promises to be effective against drug-resistant strains. The approach also has potential to considerably lower the risk of emergence of new drug-resistant strains. We explored tyrosine kinase

  2. Targeting Drug-Sensitive and -Resistant Strains of Mycobacterium tuberculosis by Inhibition of Src Family Kinases Lowers Disease Burden and Pathology.

    PubMed

    Chandra, Pallavi; Rajmani, R S; Verma, Garima; Bhavesh, Neel Sarovar; Kumar, Dhiraj

    2016-01-01

    In view of emerging drug resistance among bacterial pathogens, including Mycobacterium tuberculosis, the development of novel therapeutic strategies is increasingly being sought. A recent paradigm in antituberculosis (anti-TB) drug development is to target the host molecules that are crucial for intracellular survival of the pathogen. We previously showed the importance of Src tyrosine kinases in mycobacterial pathogenesis. Here, we report that inhibition of Src significantly reduced survival of H37Rv as well as multidrug-resistant (MDR) and extremely drug-resistant (XDR) strains of M. tuberculosis in THP-1 macrophages. Src inhibition was also effective in controlling M. tuberculosis infection in guinea pigs. In guinea pigs, reduced M. tuberculosis burden due to Src inhibition also led to a marked decline in the disease pathology. In agreement with the theoretical framework of host-directed approaches against the pathogen, Src inhibition was equally effective against an XDR strain in controlling infection in guinea pigs. We propose that Src inhibitors could be developed into effective host-directed anti-TB drugs, which could be indiscriminately used against both drug-sensitive and drug-resistant strains of M. tuberculosis. IMPORTANCE The existing treatment regimen for tuberculosis (TB) suffers from deficiencies like high doses of antibiotics, long treatment duration, and inability to kill persistent populations in an efficient manner. Together, these contribute to the emergence of drug-resistant tuberculosis. Recently, several host factors were identified which help intracellular survival of Mycobacterium tuberculosis within the macrophage. These factors serve as attractive targets for developing alternate therapeutic strategies against M. tuberculosis. This strategy promises to be effective against drug-resistant strains. The approach also has potential to considerably lower the risk of emergence of new drug-resistant strains. We explored tyrosine kinase Src as a

  3. Catalytic dechlorination of carbon tetrachloride in liquid phase with methanol as H-donor over Ag/C catalyst.

    PubMed

    Lu, Mohong; Li, Xuebing; Chen, Bo; Li, Mingshi; Xin, Hongchuan; Song, Liang

    2014-09-01

    Catalytic hydrodechlorination of carbon tetrachloride (CCl4) is an effective measure to remove CCl4 due to its pollutant character. The dechlorination of CCl4 to dichloromethane (CH2Cl2) and chloroform (CHCl3) with a molar ratio of 3:2 was catalyzed by carbon-supported silver (Ag/C) catalyst in methanol solution. It was proposed from the catalytic results and characterization (X-ray diffraction, transmission electron microscopy and X-ray photoelectron spectroscopy) data that, the chloride ion is abstracted from adsorbed CCl4 by Ag to form CCl3 and CCI2 radicals and silver chloride (AgCl), and meanwhile the dehydrogenation of methanol over Ag domains intrigues initial active Ag-H species and formaldehyde (HCHO); then the CCI3 and CCI, radicals are combined with Ag-H to generate reaction products (CHCl3 and CH2Cl2) and Ag, and the dehydrogenated product HCHO facilitates the regeneration of formed AgCl to Ag with formation of carbon monoxide and hydrogen chloride. The catalyst can be recovered and recycled, and there was no significant decrease in catalytic activity and selectivity after 4th recycling. PMID:25924408

  4. Variable intron/exon structure in the oligochaete lombricine kinase gene.

    PubMed

    Doumen, Chris

    2012-09-01

    Lombricine kinase is an annelid enzyme that belongs to the phosphagen kinase family of which creatine kinase and arginine kinase are the typical representatives. The enzymes play important roles in the cellular energy metabolism of animals. Biochemical, physiological and molecular information with respect to lombricine kinase is limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two smaller oligochaetes, Enchytraeus sp. and Stylaria sp. The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases. The intron/exon structure of the lombricine kinase gene was determined for these two species as well as two additional oligochaetes, Lumbriculus variegatus and Tubifex tubifex, and compared with available data for annelid phosphagen kinases. The data indicate the existence of a variable organization of the proposed 8-intron/9-exon gene structure. The results provide further insights in the evolution and position of these enzymes within the phosphagen kinase family. PMID:22705027

  5. Mitotic regulation by NIMA-related kinases

    PubMed Central

    O'Regan, Laura; Blot, Joelle; Fry, Andrew M

    2007-01-01

    The NIMA-related kinases represent a family of serine/threonine kinases implicated in cell cycle control. The founding member of this family, the NIMA kinase of Aspergillus nidulans, as well as the fission yeast homologue Fin1, contribute to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Mammals contain a large family of eleven NIMA-related kinases, named Nek1 to Nek11. Of these, there is now substantial evidence that Nek2, Nek6, Nek7 and Nek9 also regulate mitotic events. At least three of these kinases, as well as NIMA and Fin1, have been localized to the microtubule organizing centre of their respective species, namely the centrosome or spindle pole body. Here, they have important functions in microtubule organization and mitotic spindle assembly. Other Nek kinases have been proposed to play microtubule-dependent roles in non-dividing cells, most notably in regulating the axonemal microtubules of cilia and flagella. In this review, we discuss the evidence that NIMA-related kinases make a significant contribution to the orchestration of mitotic progression and thereby protect cells from chromosome instability. Furthermore, we highlight their potential as novel chemotherapeutic targets. PMID:17727698

  6. Bisgma Stimulates Prostaglandin E2 Production in Macrophages via Cyclooxygenase-2, Cytosolic Phospholipase A2, and Mitogen-Activated Protein Kinases Family

    PubMed Central

    Lee, Shiuan-Shinn; Li, Yi-Ching; Chang, Yu-Chao

    2013-01-01

    Background Bisphenol A-glycidyl-methacrylate (BisGMA) employs as a monomer in dental resins. The leakage of BisGMA from composite resins into the peripheral environment can result in inflammation via macrophage activation. Prostaglandin E2 (PGE2) is a key regulator of immunopathology in inflammatory reactions. Little is known about the mechanisms of BisGMA-induced PGE2 expression in macrophage. The aim of this study was to evaluate the signal transduction pathways of BisGMA-induced PGE2 production in murine RAW264.7 macrophages. Methodology/Principal Findings Herein, we demonstrate that BisGMA can exhibit cytotoxicity to RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). In addition, PGE2 production, COX-2 expression, and cPLA2 phosphorylation were induced by BisGMA on RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). Moreover, BisGMA could induce the phosphorylation of ERK1/2 pathway (MEK1/2, ERK1/2, and Elk), p38 pathway (MEK3/6, p38, and MAPKAPK2), and JNK pathway (MEK4, JNK, and c-Jun) in a dose- and time-dependent manner (p<0.05). Pretreatment with AACOCF3, U0126, SB203580, and SP600125 significantly diminished the phosphorylation of cPLA2, ERK1/2, p38, and JNK stimulated by BisGMA, respectively (p<0.05). BisGMA-induced cytotoxicity, cPLA2 phosphorylation, PGE2 generation, and caspases activation were reduced by AACOCF3, U0126, SB203580, and SP600125, respectively (p<0.05). Conclusions These results suggest that BisGMA induced-PGE2 production may be via COX-2 expression, cPLA2 phosphorylation, and the phosphorylation of MAPK family. Cytotoxicity mediated by BisGMA may be due to caspases activation through the phosphorylation of cPLA2 and MAPKs family. PMID:24376609

  7. Neuronal Apoptosis Induced by Selective Inhibition of Rac GTPase versus Global Suppression of Rho Family GTPases Is Mediated by Alterations in Distinct Mitogen-activated Protein Kinase Signaling Cascades*

    PubMed Central

    Stankiewicz, Trisha R.; Ramaswami, Sai Anandi; Bouchard, Ron J.; Aktories, Klaus; Linseman, Daniel A.

    2015-01-01

    Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad. PMID:25666619

  8. Neuronal apoptosis induced by selective inhibition of Rac GTPase versus global suppression of Rho family GTPases is mediated by alterations in distinct mitogen-activated protein kinase signaling cascades.

    PubMed

    Stankiewicz, Trisha R; Ramaswami, Sai Anandi; Bouchard, Ron J; Aktories, Klaus; Linseman, Daniel A

    2015-04-10

    Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad. PMID:25666619

  9. Ascofuranone stimulates expression of adiponectin and peroxisome proliferator activated receptor through the modulation of mitogen activated protein kinase family members in 3T3-L1, murine pre-adipocyte cell line

    SciTech Connect

    Chang, Young-Chae; Cho, Hyun-Ji

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Ascofuranone increases expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Inhibitors for MEK and JNK increased the expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Ascofuranone significantly suppressed phosho-ERK, while increasing phospho-p38. -- Abstract: Ascofuranone, an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of the phytopathogenic fungus, Ascochyta visiae. Adiponectin is mainly synthesized by adipocytes. It relieves insulin resistance by decreasing the plasma triglycerides and improving glucose uptake, and has anti-atherogenic properties. Here, we found that ascofuranone increases expression of adiponectin and PPAR{gamma}, a major transcription factor for adiponectin, in 3T3-L1, murine pre-adipocytes cell line, without promoting accumulation of lipid droplets. Ascofuranone induced expression of adiponectin, and increases the promoter activity of adiponectin and PPRE, PPAR response element, as comparably as a PPAR{gamma} agonist, rosiglitazone, that stimulates lipid accumulation in the preadipocyte cell line. Moreover, inhibitors for MEK and JNK, like ascofuranone, considerably increased the expression of adiponectin and PPAR{gamma}, while a p38 inhibitor significantly suppressed. Ascofuranone significantly suppressed ERK phosphorylation, while increasing p38 phosphorylation, during adipocyte differentiation program. These results suggest that ascofuranone regulates the expression of adiponectin and PPAR{gamma} through the modulation of MAP kinase family members.

  10. Leishmania Infection Engages Non-Receptor Protein Kinases Differentially to Persist in Infected Hosts

    PubMed Central

    Zhang, Naixin; Kima, Peter E.

    2016-01-01

    Protein kinases play important roles in the regulation of cellular activities. In cells infected by pathogens, there is an increasing appreciation that dysregulated expression of protein kinases promotes the success of intracellular infections. In Leishmania-infected cells, expression and activation of protein kinases, such as the mitogen-activated protein kinases, kinases in the PI3-kinase signaling pathway, and kinases in the NF-κB-signaling pathway, are modulated in some manner. Several recent reviews have discussed our current understanding of the roles of these kinases in Leishmania infections. Apart from the kinases in the pathways enumerated above, there are other host cell protein kinases that are activated during the Leishmania infection of mammalian cells whose roles also appear to be significant. This review discusses recent observations on the Abl family of protein kinases and the protein kinase regulated by RNA in Leishmania infections. PMID:27148265

  11. Assignment of the human aggrecan gene (AGC1) to 15q26 using fluorescence in situ hybridization analysis

    SciTech Connect

    Korenberg, J.R.; Chen, X.N.; Doege, K.; Grover, J.; Roughley, P.J.

    1993-05-01

    The large aggregating proteoglycan aggrecan is a major structural component of the extracellular matrix of articular cartilage. Recent cDNA cloning of the human aggrecan gene (AGC1) reveals a core protein of at least 2316 amino acids characterized by several distinct structural domains. Two globular domains, termed G1 and G2, are present at the amino terminus of the molecule and a third, termed G3, is present at the carboxy terminus. The G1 domain is homologous in structure to the cartilage link protein and accounts for the aggregating potential of aggrecan through its ability to interact with hyaluronic acid. The aggrecan gene is known to consist of 15 exons, with each exon encoding a distinct functional region of the mature protein. However, while the link protein gene is known to reside on chromosome 5 in the human, the location of the aggrecan gene is currently undetermined in any species. The probe (pAGG2) for the aggrecan gene was mapped on chromosome band 15q26, most likely in the subregion of 15q26.1, using fluorescence in situ hybridization. Clear signals were noted on both chromatids of chromosome band 15q26 in over 80% of the 300 metaphase cells examined in three independent experiments using pAGG2. No other sites of hybridization were noted on both chromatids of any other chromosome band. The precise band location was identified by using chromsomes of about 650 bands and employing fluorescence reverse banding with chromomycin A3 and distamycin. 14 refs., 1 fig.

  12. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

    PubMed

    Challa, Anil Kumar; Chatti, Kiranam

    2013-09-01

    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome. PMID:23234507

  13. Conservation and Early Expression of Zebrafish Tyrosine Kinases Support the Utility of Zebrafish as a Model for Tyrosine Kinase Biology

    PubMed Central

    Challa, Anil Kumar

    2013-01-01

    Abstract Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome. PMID:23234507

  14. 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.

  15. PRKX, a Novel cAMP-Dependent Protein Kinase Member, Plays an Important Role in Development.

    PubMed

    Huang, Sizhou; Li, Qian; Alberts, Ian; Li, Xiaohong

    2016-03-01

    The human protein kinase X gene (PRKX) and cAMP-dependent protein kinase (PKA) are both c-AMP-dependent serine/threonine protein kinases within the protein kinase AGC subgroup. Of all the protein kinases in this group, PRKX is the least studied. PRKX has been isolated from patients with chondrodysplasia punctate and is involved in numerous processes, including sexual differentiation and fertilization, normal kidney development and autosomal dominant polycystic kidney disease (ADPKD), blood maturation, neural development, and angiogenesis in vitro. Although the role of PRKX in development and disease has been reported recently, the underlying mechanism of PRKX activity is largely unknown. In addition, based on the expression pattern of PRKX and the extensive role of PKA in disease and development, PRKX might have additional crucial functions that have not been addressed in the literature. In this review, we summarize the characteristics and developmental functions of PRKX that have been reported by recent studies. In particular, we elucidate the structural and functional differences between PRKX and PKA, as well as the possible roles of PRKX in development and related diseases. Finally, we propose future studies that could lead to important discoveries of more PRKX functions and the underlying mechanisms involved. PMID:26252946

  16. Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

    SciTech Connect

    Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian; Huber, Jochen; Tesmer, John J.G.

    2012-07-11

    G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G{beta}{gamma} complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G{beta}{gamma} structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

  17. Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor

    PubMed Central

    Akhtar, Saghir; Chandrasekhar, Bindu; Attur, Sreeja; Dhaunsi, Gursev S.; Yousif, Mariam H. M.; Benter, Ibrahim F.

    2015-01-01

    Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1–7) [Ang-(1–7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1–7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1–7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1–7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1–7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro7-Ang-(1–7). Ang-(1–7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1–7) that could be prevented by D-Pro7-Ang-(1–7) in VSMC. These data suggest that Ang-(1–7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1–7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications. PMID:26536590

  18. Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor.

    PubMed

    Akhtar, Saghir; Chandrasekhar, Bindu; Attur, Sreeja; Dhaunsi, Gursev S; Yousif, Mariam H M; Benter, Ibrahim F

    2015-01-01

    Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1-7) [Ang-(1-7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1-7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1-7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1-7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1-7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro7-Ang-(1-7). Ang-(1-7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1-7) that could be prevented by D-Pro7-Ang-(1-7) in VSMC. These data suggest that Ang-(1-7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1-7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications. PMID:26536590

  19. A Fully Digital AGC System with 100MHz Bandwidth and 35dB Dynamic Range Power Detectors for DVB-S2 Application

    NASA Astrophysics Data System (ADS)

    Pu, Younggun; Lee, Kang-Yoon

    This paper presents a fully digital gain control system with a new high bandwidth and wide dynamic range power detector for DVB-S2 application. Because the peak-to-average power ratio (PAPR) of DVB-S2 system is so high and the settling time requirement is so stringent, the conventional closed-loop analog gain control scheme cannot be used. The digital gain control is necessary for the robust gain control and the direct digital interface with the baseband modem. Also, it has several advantages over the analog gain control in terms of the settling time and insensitivity to the process, voltage and temperature variation. In order to have a wide gain range with fine step resolution, a new AGC system is proposed. The system is composed of high-bandwidth digital VGAs, wide dynamic range power detectors with RMS detector, low power SAR type ADC, and a digital gain controller. To reduce the power consumption and chip area, only one SAR type ADC is used, and its input is time-interleaved based on four power detectors. Simulation and measurement results show that the new AGC system converges with gain error less than 0.25dB to the desired level within 10µs. It is implemented in a 0.18µm CMOS process. The measurement results of the proposed IF AGC system exhibit 80-dB gain range with 0.25-dB resolution, 8nV/\\\\!\\\\sqrt{Hz} input referred noise, and 5-dBm IIP3 at 60-mW power consumption. The power detector shows the 35dB dynamic range for 100MHz input.

  20. Diversity, classification and function of the plant protein kinase superfamily

    PubMed Central

    Lehti-Shiu, Melissa D.; Shiu, Shin-Han

    2012-01-01

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants. PMID:22889912

  1. 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

  2. The JAK kinases: not just another kinase drug discovery target.

    PubMed

    Wilks, Andrew F

    2008-08-01

    There are four members of the JAK family of protein tyrosine kinases (PTKs) in the human genome. Since their discovery in 1989, great strides have been made in the understanding of their role in normal intracellular signalling. Importantly, their roles in pathologies ranging from cancer to immune deficiencies have placed them front and centre as potential drug targets. The recent discovery of the role of activating mutations in the kinase-like domain (KLD) of JAK2 in the development of polycythemia rubra vera, and the elaboration of KLD mutation as a broader mechanism by which cells might become hyperproliferative has sparked enormous interest in the development of JAK selective drug candidates. I review herein the progress that has been made in the discovery of JAK-targeted inhibitors, and discuss the challenges that face the development of these drugs for use in the clinic. PMID:18721891

  3. Identification of four plastid-localized protein kinases.

    PubMed

    Richter, Andreas S; Gartmann, Hans; Fechler, Mona; Rödiger, Anja; Baginsky, Sacha; Grimm, Bernhard

    2016-06-01

    In chloroplasts, protein phosphorylation regulates important processes, including metabolism, photosynthesis, gene expression, and signaling. Because the hitherto known plastid protein kinases represent only a fraction of existing kinases, we aimed at the identification of novel plastid-localized protein kinases that potentially phosphorylate enzymes of the tetrapyrrole biosynthesis (TBS) pathway. We screened publicly available databases for proteins annotated as putative protein kinase family proteins with predicted chloroplast localization. Additionally, we analyzed chloroplast fractions which were separated by sucrose density gradient centrifugation by mass spectrometry. We identified four new candidates for protein kinases, which were confirmed to be plastid localized by expression of GFP-fusion proteins in tobacco leaves. A phosphorylation assay with the purified kinases confirmed the protein kinase activity for two of them. PMID:27214872

  4. Structural and functional diversity in the activity and regulation of DAPK-related protein kinases.

    PubMed

    Temmerman, Koen; Simon, Bertrand; Wilmanns, Matthias

    2013-11-01

    Within the large group of calcium/calmodulin-dependent protein kinases (CAMKs) of the human kinome, there is a distinct branch of highly related kinases that includes three families: death-associated protein-related kinases, myosin light-chain-related kinases and triple functional domain protein-related kinases. In this review, we refer to these collectively as DMT kinases. There are several functional features that span the three families, such as a broad involvement in apoptotic processes, cytoskeletal association and cellular plasticity. Other CAMKs contain a highly conserved HRD motif, which is a prerequisite for kinase regulation through activation-loop phosphorylation, but in all 16 members of the DMT branch, this is replaced by an HF/LD motif. This DMT kinase signature motif substitutes phosphorylation-dependent active-site interactions with a local hydrophobic core that maintains an active kinase conformation. Only about half of the DMT kinases have an additional autoregulatory domain, C-terminal to the kinase domain that binds calcium/calmodulin in order to regulate kinase activity. Protein substrates have been identified for some of the DMT kinases, but little is known about the mechanism of recognition. Substrate conformation could be an equally important parameter in substrate recognition as specific preferences in sequence position. Taking the data together, this kinase branch encapsulates a treasure trove of features that renders it distinct from many other protein kinases and calls for future research activities in this field. PMID:23745726

  5. Synthesis of single-crystal α-MnO2 nanotubes-loaded Ag@C core-shell matrix and their application for electrochemical sensing of nonenzymatic hydrogen peroxide.

    PubMed

    Zhang, Sai; Zheng, Jianbin

    2016-10-01

    A nonenzymatic hydrogen peroxide sensor was fabricated by combing the crystal α-MnO2 nanotubes and Ag@C core-shell matrix with their own superior characteristics. The morphology, size and electrochemical of the sensing interface materials and the relationship between the electrical catalytic properties and sensor response performance were also studied, established a new method for the detection of hydrogen peroxide (H2O2). The structure and morphology of hollow tubular-like MnO2 and MnO2-Ag@C film were characterized by scanning electron micrograph (SEM), transmission electron microscopy (TEM) and X-ray diffraction. The electrochemical properties of the sensor were explored by cyclic voltammetry and amperometry. The investigation showed that the MnO2-Ag@C at the sensor exhibited a high electrocatalytic activity towards electroreduction of hydrogen peroxide; and under the optimal conditions, the linear ranges of hydrogen peroxide were 0.5μM to 5.7mM with a low detection limit of 0.17μM (S/N=3) and high sensitivity of 127.2μAmM(-1)cm(-2). Compared with other nonenzymatic hydrogen peroxide sensor, the fabricated sensor own lower detection limit, demonstrating that MnO2-Ag@C nanocomposite film will be a new promising platform for the construction of hydrogen peroxide sensors. PMID:27474303

  6. Brassinosteroid regulated kinases (BRKs) that mediate brassinosteroid signal transduction and uses thereof

    DOEpatents

    Wang, Zhi-Yong; Tang, Wenqiang

    2013-09-24

    The present invention identifies a novel family of kinases regulated by brassinosteroids, referred to as BRKs (brassinosteroid regulated kinases) or BSKs (brassinosteroid signaling kinases). The present invention provides methods for modulating the response of a plant cell to a brassinosteroid using BRKs.

  7. A comprehensive update of the sequence and structure classification of kinases

    PubMed Central

    Cheek, Sara; Ginalski, Krzysztof; Zhang, Hong; Grishin, Nick V

    2005-01-01

    Background A comprehensive update of the classification of all available kinases was carried out. This survey presents a complete global picture of this large functional class of proteins and confirms the soundness of our initial kinase classification scheme. Results The new survey found the total number of kinase sequences in the protein database has increased more than three-fold (from 17,310 to 59,402), and the number of determined kinase structures increased two-fold (from 359 to 702) in the past three years. However, the framework of the original two-tier classification scheme (in families and fold groups) remains sufficient to describe all available kinases. Overall, the kinase sequences were classified into 25 families of homologous proteins, wherein 22 families (~98.8% of all sequences) for which three-dimensional structures are known fall into 10 fold groups. These fold groups not only include some of the most widely spread proteins folds, such as the Rossmann-like fold, ferredoxin-like fold, TIM-barrel fold, and antiparallel β-barrel fold, but also all major classes (all α, all β, α+β, α/β) of protein structures. Fold predictions are made for remaining kinase families without a close homolog with solved structure. We also highlight two novel kinase structural folds, riboflavin kinase and dihydroxyacetone kinase, which have recently been characterized. Two protein families previously annotated as kinases are removed from the classification based on new experimental data. Conclusion Structural annotations of all kinase families are now revealed, including fold descriptions for all globular kinases, making this the first large functional class of proteins with a comprehensive structural annotation. Potential uses for this classification include deduction of protein function, structural fold, or enzymatic mechanism of poorly studied or newly discovered kinases based on proteins in the same family. PMID:15771780

  8. Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

    PubMed

    Craige, Siobhan M; Reif, Michaella M; Kant, Shashi

    2016-09-01

    Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases. PMID:27259981

  9. 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

  10. A novel nonreceptor tyrosine kinase, Srm: cloning and targeted disruption.

    PubMed Central

    Kohmura, N; Yagi, T; Tomooka, Y; Oyanagi, M; Kominami, R; Takeda, N; Chiba, J; Ikawa, Y; Aizawa, S

    1994-01-01

    We have isolated a novel nonreceptor tyrosine kinase, Srm, that maps to the distal end of chromosome 2. It has SH2, SH2', and SH3 domains and a tyrosine residue for autophosphorylation in the kinase domain but lacks an N-terminal glycine for myristylation and a C-terminal tyrosine which, when phosphorylated, suppresses kinase activity. These are structural features of the recently identified Tec family of nonreceptor tyrosine kinases. The Srm N-terminal unique domain, however, lacks the structural characteristics of the Tec family kinases, and the sequence similarity is highest to Src in the SH region. The expression of two transcripts is rather ubiquitous and changes according to tissue and developmental stage. Mutant mice were generated by gene targeting in embryonic stem cells but displayed no apparent phenotype as in mutant mice expressing Src family kinases. These results suggest that Srm constitutes a new family of nonreceptor tyrosine kinases that may be redundant in function. Images PMID:7935409

  11. Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization

    PubMed Central

    Lavoie, Hugo; Thevakumaran, Neroshan; Gavory, Gwenaëlle; Li, John; Padeganeh, Abbas; Guiral, Sébastien; Duchaine, Jean; Mao, Daniel Y. L.; Bouvier, Michel; Sicheri, Frank; Therrien, Marc

    2016-01-01

    RAF kinases play a prominent role in cancer. Their mode of activation is complex, but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and as a result undesirably stimulate RAS/ERK-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe BRET-based biosensors for the extended RAF family enabling the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization as well as for probing structural determinants of RAF dimerization in vivo. Our findings, which appear generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain. PMID:23685672

  12. Salicylic acid activates a 48-kD MAP kinase in tobacco.

    PubMed Central

    Zhang, S; Klessig, D F

    1997-01-01

    The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors. Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate. This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase). Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not. Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation. The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells. The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity. Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene. Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses. PMID:9165755

  13. The selectivity of protein kinase inhibitors: a further update

    PubMed Central

    Bain, Jenny; Plater, Lorna; Elliott, Matt; Shpiro, Natalia; Hastie, C. James; Mclauchlan, Hilary; Klevernic, Iva; Arthur, J. Simon C.; Alessi, Dario R.; Cohen, Philip

    2007-01-01

    The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70–80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)–raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes. PMID:17850214

  14. Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses.

    PubMed

    Balzano, Deborah; Fawal, Mohamad-Ali; Velázquez, Jose V; Santiveri, Clara M; Yang, Joshua; Pastor, Joaquín; Campos-Olivas, Ramón; Djouder, Nabil; Lietha, Daniel

    2015-10-01

    Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects. PMID:26286748

  15. RAF protein-serine/threonine kinases: Structure and regulation

    SciTech Connect

    Roskoski, Robert

    2010-08-27

    Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.

  16. Mitogen activated protein kinase at the nuclear pore complex

    PubMed Central

    Faustino, Randolph S; Maddaford, Thane G; Pierce, Grant N

    2011-01-01

    Abstract Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function. PMID:20497490

  17. A conserved isoleucine maintains the inactive state of Bruton’s tyrosine kinase

    PubMed Central

    Boyken, Scott E.; Chopra, Nikita; Xie, Qian; Joseph, Raji E.; Wales, Thomas E.; Fulton, D. Bruce; Engen, John R.; Jernigan, Robert L.; Andreotti, Amy H.

    2014-01-01

    Despite high homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamic simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest this isoleucine side chain in the Tec family kinases acts as a ‘wedge’ that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain. PMID:25193673

  18. cDNA identification, comparison and phylogenetic aspects of lombricine kinase from two oligochaete species.

    PubMed

    Doumen, Chris

    2010-06-01

    Creatine kinase and arginine kinase are the typical representatives of an eight-member phosphagen kinase family, which play important roles in the cellular energy metabolism of animals. The phylum Annelida underwent a series of evolutionary processes that resulted in rapid divergence and radiation of these enzymes, producing the greatest diversity of the phosphagen kinases within this phylum. Lombricine kinase (EC 2.7.3.5) is one of such enzymes and sequence information is rather limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two oligochaete species, the California blackworm (Lumbriculus variegatus) and the sludge worm (Tubifex tubifex). The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases, including two additional lombricine kinase sequences extracted from DNA databases and provide further insights in the evolution and position of these enzymes within the phosphagen kinase family. The data confirms the presence of a deleted region within the flexible loop (the GS region) of all six examined lombricine kinases. A phylogenetic analysis of these six lombricine kinases clearly positions the enzymes together in a small subcluster within the larger creatine kinase (EC 2.7.3.2) clade. PMID:20230902

  19. From Phosphosites to Kinases.

    PubMed

    Munk, Stephanie; Refsgaard, Jan C; Olsen, Jesper V; Jensen, Lars J

    2016-01-01

    Kinases play a pivotal role in propagating the phosphorylation-mediated signaling networks in living cells. With the overwhelming quantities of phosphoproteomics data being generated, the number of identified phosphorylation sites (phosphosites) is ever increasing. Often, proteomics investigations aim to understand the global signaling modulation that takes place in different biological conditions investigated. For phosphoproteomics data, identifying the kinases central to mediating this response is key. This has prompted several efforts to catalogue the immense amounts of phosphorylation data and known or predicted kinases responsible for the modifications. However, barely 20 % of the known phosphosites are assigned to a kinase, initiating various bioinformatics efforts that attempt to predict the responsible kinases. These algorithms employ different approaches to predict kinase consensus sequence motifs, mostly based on large scale in vivo and in vitro experiments. The context of the kinase and the phosphorylated proteins in a biological system is equally important for predicting association between the enzymes and substrates, an aspect that is also being tackled with available bioinformatics tools. This chapter summarizes the use of the larger phosphorylation databases, and approaches that can be applied to predict kinases that phosphorylate individual sites or that are globally modulated in phosphoproteomics datasets. PMID:26584935

  20. Structure of the human dimeric ATM kinase.

    PubMed

    Lau, Wilson C Y; Li, Yinyin; Liu, Zhe; Gao, Yuanzhu; Zhang, Qinfen; Huen, Michael S Y

    2016-01-01

    DNA-double strand breaks activate the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) to initiate DNA damage signal transduction. This activation process involves autophosphorylation and dissociation of inert ATM dimers into monomers that are catalytically active. Using single-particle electron microscopy (EM), we determined the structure of dimeric ATM in its resting state. The EM map could accommodate the crystal structure of the N-terminal truncated mammalian target of rapamycin (mTOR), a closely related enzyme of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, allowing for the localization of the N- and the C-terminal regions of ATM. In the dimeric structure, the actives sites are buried, restricting the access of the substrates to these sites. The unanticipated domain organization of ATM provides a basis for understanding its mechanism of inhibition. PMID:27097373

  1. Structure of the human dimeric ATM kinase

    PubMed Central

    Lau, Wilson C. Y.; Li, Yinyin; Liu, Zhe; Gao, Yuanzhu; Zhang, Qinfen; Huen, Michael S. Y.

    2016-01-01

    ABSTRACT DNA-double strand breaks activate the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) to initiate DNA damage signal transduction. This activation process involves autophosphorylation and dissociation of inert ATM dimers into monomers that are catalytically active. Using single-particle electron microscopy (EM), we determined the structure of dimeric ATM in its resting state. The EM map could accommodate the crystal structure of the N-terminal truncated mammalian target of rapamycin (mTOR), a closely related enzyme of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, allowing for the localization of the N- and the C-terminal regions of ATM. In the dimeric structure, the actives sites are buried, restricting the access of the substrates to these sites. The unanticipated domain organization of ATM provides a basis for understanding its mechanism of inhibition. PMID:27097373

  2. Conservation of structural fluctuations in homologous protein kinases and its implications on functional sites.

    PubMed

    Kalaivani, Raju; de Brevern, Alexandre G; Srinivasan, Narayanaswamy

    2016-07-01

    Our aim is to explore the similarities in structural fluctuations of homologous kinases. Gaussian Network Model based Normal Mode Analysis was performed on 73 active conformation structures in Ser/Thr/Tyr kinase superfamily. Categories of kinases with progressive evolutionary divergence, viz. (i) Same kinase with many crystal structures, (ii) Within-Subfamily, (iii) Within-Family, (iv) Within-Group, and (v) Across-Group, were analyzed. We identified a flexibility signature conserved in all kinases involving residues in and around the catalytic loop with consistent low-magnitude fluctuations. However, the overall structural fluctuation profiles are conserved better in closely related kinases (Within-Subfamily and Within-family) than in distant ones (Within-Group and Across-Group). A substantial 65.4% of variation in flexibility was not accounted by variation in sequences or structures. Interestingly, we identified substructural residue-wise fluctuation patterns characteristic of kinases of different categories. Specifically, we recognized statistically significant fluctuations unique to families of protein kinase A, cyclin-dependent kinases, and nonreceptor tyrosine kinases. These fluctuation signatures localized to sites known to participate in protein-protein interactions typical of these kinase families. We report for the first time that residues characterized by fluctuations unique to the group/family are involved in interactions specific to the group/family. As highlighted for Src family, local regions with differential fluctuations are proposed as attractive targets for drug design. Overall, our study underscores the importance of consideration of fluctuations, over and above sequence and structural features, in understanding the roles of sites characteristic of kinases. Proteins 2016; 84:957-978. © 2016 Wiley Periodicals, Inc. PMID:27028938

  3. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials. PMID:25337443

  4. Family Preservation & Family Functioning.

    ERIC Educational Resources Information Center

    McCroskey, Jacquelyn; Meezan, William

    This book reports a study of the outcomes of home-based family preservation services for abusive and neglectful families in Los Angeles County. Using the Family Assessment Form, the research project evaluated services provided by two voluntary agencies, and focused on changes in family functioning between the opening and closing of services during…

  5. Characterizing land condition variability in Ferlo, Senegal (2001-2009) using multi-temporal 1-km Apparent Green Cover (AGC) SPOT Vegetation data

    NASA Astrophysics Data System (ADS)

    Martínez, B.; Gilabert, M. A.; García-Haro, F. J.; Faye, A.; Meliá, J.

    2011-04-01

    The ecosystem state or 'land condition' can be characterized by a set of attributes, which show variations at different temporal scales. A multi-resolution analysis (MRA) based on the wavelet transform (WT) has been implemented to examine the land condition of a dryland region in Ferlo (Senegal) over the period 2001-2009. This methodology has proven to be useful for smoothing time series while considering those temporal resolutions that incorporate information about the vegetation dynamics. For this purpose, time series of the 1-km Apparent Green Cover (AGC) from the 10-day composites SPOT Vegetation (VGT) data are analyzed. Two relevant outputs from the MRA, A1 (de-noised) and the A6 (inter-annual) components have served us for characterizing the annual vegetation production and assess the long-term variation, respectively. In a first stage, the vegetation seasonality (or intra-annual variation) over the area is described by using several metrics related to vegetation phenology derived from the de-noised time series ( A1). In a second phase, the temporal variability of the inter-annual component series ( A6) is accomplished to detect potential vegetation changes over the considered period. A Mann-Kendall test has been applied to confirm the significance of the observed inter-annual changes. A higher number of significant pixels (86%) are obtained when considering the inter-annual component in the trend analysis instead of the original time-series (47%). The results confirm a general greening up over the period 2001-2009, not fully explained by precipitation, as well as rather local negative trends. The Rain-Use Efficiency (RUE) ratio computed using the AGC as a proxy of vegetation production has been considered to further analyze the detected changes. Inter-annual changes in RUE provide a potential method of separating vegetation declines due to lack of rainfall from declines associated with degradation. Some spots of negative values in inter-annual RUE changes

  6. Molecular Physiology of SPAK and OSR1: Two Ste20-Related Protein Kinases Regulating Ion Transport

    PubMed Central

    Gagnon, Kenneth B.; Delpire, Eric

    2015-01-01

    SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress responsive kinase) are members of the germinal center kinase VI sub-family of the mammalian Ste20 (Sterile20)-related protein kinase family. Although there are 30 enzymes in this protein kinase family, their conservation across the fungi, plant and animal kingdom confirms their evolutionary importance. Already, a large volume of work has accumulated on the tissue distribution, binding partners, signaling cascades, and physiological roles of mammalian SPAK and OSR1 in multiple organ systems. After reviewing this basic information, we will examine newer studies that demonstrate the pathophysiological consequences to SPAK and/or OSR1 disruption, discuss the development and analysis of genetically-engineered mouse models, and address the possible role these serine/threonine kinases might have in cancer proliferation and migration. PMID:23073627

  7. Cytoskeletal protein kinases: titin and its relations in mechanosensing.

    PubMed

    Gautel, Mathias

    2011-07-01

    Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca(2+)-calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other "MLCKs", is not Ca(2+)-calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation. PMID:21416260

  8. A Novel Mode of Protein Kinase Inhibition Exploiting Hydrophobic Motifs of Autoinhibited Kinases

    SciTech Connect

    S Eathiraj; R Palma; M Hirschi; E Volckova; E Nakuci; J Castro; C Chen; T Chan; D France; M Ashwell

    2011-12-31

    Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.

  9. Chromosomal location of the Syk and ZAP-70 tyrosine kinase genes in mice and humans

    SciTech Connect

    Ku, G.; Malissen, B.; Mattei, M.G.

    1994-12-31

    Several protein tyrosine kinases (PTKs), which may be grouped into two structurally different families, have been implicated in antigen receptor proximal signaling. Blk, Fyn, Lck, Lyn, and Yes belong to the Src-family kinases, whereas the spleen tyrosine kinase (Syk) and the CD3-{zeta}-associated PTK (ZAP-70) define a new one, the Syk family. These kinases differ from the Src-family kinases in that they are non-myristylated cytoplasmic polypeptides composed of two N-terminal Src homology-2 (SH2) domains and a C-terminal catalytic domain. ZAP-70 appears to be expressed exclusively in T cells and NK cells, whereas Syk is preferentially expressed in B cells, T cells, and myeloid cells. 15 refs., 2 figs.

  10. Diversification of Lrk/Tak Kinase Gene Clusters is Associated with Subfunctionalization and Cultivar-specific Transcript Accumulation in Barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lrk (Lr10 receptor-like kinase) and Tak (Triticum aestivum kinase) belong to the receptor-like kinase (RLK) super-gene family in higher plants. Three Lrk/Tak gene regions spanning greater than 600 kb were identified via a genome-wide survey of barley gene-rich BAC (Bacterial Artificial Chromosome) ...

  11. A fast Monte Carlo EM algorithm for estimation in latent class model analysis with an application to assess diagnostic accuracy for cervical neoplasia in women with AGC

    PubMed Central

    Kang, Le; Carter, Randy; Darcy, Kathleen; Kauderer, James; Liao, Shu-Yuan

    2013-01-01

    In this article we use a latent class model (LCM) with prevalence modeled as a function of covariates to assess diagnostic test accuracy in situations where the true disease status is not observed, but observations on three or more conditionally independent diagnostic tests are available. A fast Monte Carlo EM (MCEM) algorithm with binary (disease) diagnostic data is implemented to estimate parameters of interest; namely, sensitivity, specificity, and prevalence of the disease as a function of covariates. To obtain standard errors for confidence interval construction of estimated parameters, the missing information principle is applied to adjust information matrix estimates. We compare the adjusted information matrix based standard error estimates with the bootstrap standard error estimates both obtained using the fast MCEM algorithm through an extensive Monte Carlo study. Simulation demonstrates that the adjusted information matrix approach estimates the standard error similarly with the bootstrap methods under certain scenarios. The bootstrap percentile intervals have satisfactory coverage probabilities. We then apply the LCM analysis to a real data set of 122 subjects from a Gynecologic Oncology Group (GOG) study of significant cervical lesion (S-CL) diagnosis in women with atypical glandular cells of undetermined significance (AGC) to compare the diagnostic accuracy of a histology-based evaluation, a CA-IX biomarker-based test and a human papillomavirus (HPV) DNA test. PMID:24163493

  12. Deviations from one-electron behavior in the Ag and Pd M4,5-VV Auger spectra of AgcPd1-c alloys

    NASA Astrophysics Data System (ADS)

    Mariot, J.-M.; Hague, C. F.; Dufour, G.

    1981-04-01

    A systematic investigation of the Ag and Pd M4,5-VV Auger spectra in the pure metals and in the AgcPd1-c(0.1<=c<=0.9) alloys is presented. The shape of the Auger spectra is discussed in relation to the values of the effective Coulomb interaction Ueff between the two holes present in the final state of the Auger transition and of the width W of the one-electron local densities of states as obtained from Lβ2,15 soft-x-ray emission bands. The Auger spectra of Ag in the metal and the alloys have a pronounced quasiatomic character, as can be expected from the Ueff2W ratio which is found to be close to unity. The Auger spectrum of pure Pd (Ueff2W~0.4) can be explained in terms of an atomic model in which strong band effects are present. For alloys with low-Pd content where Pd forms an impurity state, it is shown that final states other than the localized [4d2] two-hole state have to be invoked to explain the line shape.

  13. Diacylglycerol kinases in membrane trafficking

    PubMed Central

    Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    Diacylglycerol kinases (DGKs) belong to a family of cytosolic kinases that regulate the phosphorylation of diacylglycerol (DAG), converting it into phosphatidic acid (PA). There are 10 known mammalian DGK isoforms, each with a different tissue distribution and substrate specificity. These differences allow regulation of cellular responses by fine-tuning the delicate balance of cellular DAG and PA. DGK isoforms are best characterized as mediators of signal transduction and immune function. However, since recent studies reveal that DAG and PA are also involved in the regulation of endocytic trafficking, it is therefore anticipated that DGKs also plays an important role in membrane trafficking. In this review, we summarize the literature discussing the role of DGK isoforms at different stages of endocytic trafficking, including endocytosis, exocytosis, endocytic recycling, and transport from/to the Golgi apparatus. Overall, these studies contribute to our understanding of the involvement of PA and DAG in endocytic trafficking, an area of research that is drawing increasing attention in recent years. PMID:27057419

  14. Rho Kinases and Cardiac Remodeling.

    PubMed

    Shimizu, Toru; Liao, James K

    2016-06-24

    Hypertensive cardiac remodeling is characterized by left ventricular hypertrophy and interstitial fibrosis, which can lead to heart failure with preserved ejection fraction. The Rho-associated coiled-coil containing kinases (ROCKs) are members of the serine/threonine protein kinase family, which mediates the downstream effects of the small GTP-binding protein RhoA. There are 2 isoforms: ROCK1 and ROCK2. They have different functions in different types of cells and tissues. There is growing evidence that ROCKs contribute to the development of cardiovascular diseases, including cardiac fibrosis, hypertrophy, and subsequent heart failure. Recent experimental studies using ROCK inhibitors, such as fasudil, have shown the benefits of ROCK inhibition in cardiac remodeling. Mice lacking each ROCK isoform also exhibit reduced myocardial fibrosis in a variety of pathological models of cardiac remodeling. Indeed, clinical studies with fasudil have suggested that ROCKs could be potential novel therapeutic targets for cardiovascular diseases. In this review, we summarize the current understanding of the roles of ROCKs in the development of cardiac fibrosis and hypertrophy and discuss their therapeutic potential for deleterious cardiac remodeling. (Circ J 2016; 80: 1491-1498). PMID:27251065

  15. Pyruvate kinase blood test

    MedlinePlus

    ... break down faster than normal, a condition called hemolytic anemia . This test helps diagnose pyruvate kinase deficiency (PKD) . ... Pa: Elsevier Saunders; 2011:chap 32. Gallagher PG. Hemolytic anemias: red cell membrane and metabolic defects In: Goldman ...

  16. Indolinones as promising scaffold as kinase inhibitors: a review.

    PubMed

    Prakash, C R; Raja, S

    2012-02-01

    Kinases are probably the most important signaling enzymes, which represent about 20% of the druggable genome. Currently, more than 150 kinases are known. So, kinase inhibition therapy has become a very important area of drug research since most of our diseases are related to intra or intercellular signaling by kinases. Indole alkaloids are extensively studied for their biological activities in several pharmaceutical areas, including, for example, antitumor. Among this chemical family, indolinone displays very promising antitumor properties by inhibiting various kinase families. These small molecules have a low molecular weight and most of them bind to protein kinases competing with ATP for the ATP-binding site. This review focuses on the indolinone based drugs approved for the treatment of cancer, drugs under clinical trial and then chemical diversity of various synthetic analogues of indolinone and their metabolites as various kinase inhibitors. This review also focused on structural activity relationship (SAR), mechanisms of action and biological targets through which indolinone and its derivatives display their antitumor activity. PMID:22372601

  17. Mechanism of substrate specificity of phosphatidylinositol phosphate kinases.

    PubMed

    Muftuoglu, Yagmur; Xue, Yi; Gao, Xiang; Wu, Dianqing; Ha, Ya

    2016-08-01

    The phosphatidylinositol phosphate kinase (PIPK) family of enzymes is primarily responsible for converting singly phosphorylated phosphatidylinositol derivatives to phosphatidylinositol bisphosphates. As such, these kinases are central to many signaling and membrane trafficking processes in the eukaryotic cell. The three types of phosphatidylinositol phosphate kinases are homologous in sequence but differ in catalytic activities and biological functions. Type I and type II kinases generate phosphatidylinositol 4,5-bisphosphate from phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate, respectively, whereas the type III kinase produces phosphatidylinositol 3,5-bisphosphate from phosphatidylinositol 3-phosphate. Based on crystallographic analysis of the zebrafish type I kinase PIP5Kα, we identified a structural motif unique to the kinase family that serves to recognize the monophosphate on the substrate. Our data indicate that the complex pattern of substrate recognition and phosphorylation results from the interplay between the monophosphate binding site and the specificity loop: the specificity loop functions to recognize different orientations of the inositol ring, whereas residues flanking the phosphate binding Arg244 determine whether phosphatidylinositol 3-phosphate is exclusively bound and phosphorylated at the 5-position. This work provides a thorough picture of how PIPKs achieve their exquisite substrate specificity. PMID:27439870

  18. Mini-review: bmx kinase inhibitors for cancer therapy.

    PubMed

    Jarboe, John S; Dutta, Shilpa; Velu, Sadanandan E; Willey, Christopher D

    2013-09-01

    Kinase inhibitors are among the fastest growing class of anti-cancer therapies. One family of kinases that has recently gained attention as a target for treating malignant disorders is the Tec kinase family. Evidence has been published that one member of this family; the Bmx kinase, may play a role in the pathogenesis of glioblastoma, prostate, breast and lung cancer. Bmx has also shown potential as an anti-vascular therapy in combination with radiation or as a sensitizer to chemotherapeutic agents. Therefore, several companies such as Pharmacyclics, Avila Therapeutics, Merck and Co., Metaproteomics, IRM, and Moerae Matrix have developed compounds or peptides that function as Bmx kinase inhibitors. These companies have subsequently been issued patents for these inhibitors. Additionally, it has been shown that current clinical stage EGFR inhibitors can irreversibly inhibit Bmx, suggesting these compounds might be rapidly moved to clinical trials for other malignancies. This review will discuss current patents issued since 2009 that contain data specifically on inhibition of the Bmx kinase, and will also discuss the scientific literature that suggests their potential application as therapeutics in the treatment of the aforementioned malignancies. PMID:23198769

  19. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.

    PubMed

    González-Fernández, Lauro; Macías-García, Beatriz; Loux, Shavahn C; Varner, Dickson D; Hinrichs, Katrin

    2013-06-01

    Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways. PMID:23595906

  20. Activity-based kinase profiling of approved tyrosine kinase inhibitors.

    PubMed

    Kitagawa, Daisuke; Yokota, Koichi; Gouda, Masaki; Narumi, Yugo; Ohmoto, Hiroshi; Nishiwaki, Eiji; Akita, Kensaku; Kirii, Yasuyuki

    2013-02-01

    The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K(m) for ATP. In addition, profiling at a physiological ATP concentration (1 mm) was carried out, and the IC(50) values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C(trough) and C(max) values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K(m) and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors. PMID:23279183

  1. Structural and Functional Analysis of G Protein–Coupled Receptor Kinase Inhibition by Paroxetine and a Rationally Designed Analog

    PubMed Central

    Homan, Kristoff T.; Wu, Emily; Wilson, Michael W.; Singh, Puja; Larsen, Scott D.

    2014-01-01

    Recently we identified the serotonin reuptake inhibitor paroxetine as an inhibitor of G protein–coupled receptor kinase 2 (GRK2) that improves cardiac performance in live animals. Paroxetine exhibits up to 50-fold selectivity for GRK2 versus other GRKs. A better understanding of the molecular basis of this selectivity is important for the development of even more selective and potent small molecule therapeutics and chemical genetic probes. We first sought to understand the molecular mechanisms underlying paroxetine selectivity among GRKs. We directly measured the KD for paroxetine and assessed its mechanism of inhibition for each of the GRK subfamilies and then determined the atomic structure of its complex with GRK1, the most weakly inhibited GRK tested. Our results suggest that the selectivity of paroxetine for GRK2 largely reflects its lower affinity for adenine nucleotides. Thus, stabilization of off-pathway conformational states unique to GRK2 will likely be key for the development of even more selective inhibitors. Next, we designed a benzolactam derivative of paroxetine that has optimized interactions with the hinge of the GRK2 kinase domain. The crystal structure of this compound in complex with GRK2 confirmed the predicted interactions. Although the benzolactam derivative did not significantly alter potency of inhibition among GRKs, it exhibited 20-fold lower inhibition of serotonin reuptake. However, there was an associated increase in the potency for inhibition of other AGC kinases, suggesting that the unconventional hydrogen bond formed by the benzodioxole ring of paroxetine is better accommodated by GRKs. PMID:24220010

  2. Molecular physiology of SPAK and OSR1: two Ste20-related protein kinases regulating ion transport.

    PubMed

    Gagnon, Kenneth B; Delpire, Eric

    2012-10-01

    SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress responsive kinase) are members of the germinal center kinase VI subfamily of the mammalian Ste20 (Sterile20)-related protein kinase family. Although there are 30 enzymes in this protein kinase family, their conservation across the fungi, plant, and animal kingdom confirms their evolutionary importance. Already, a large volume of work has accumulated on the tissue distribution, binding partners, signaling cascades, and physiological roles of mammalian SPAK and OSR1 in multiple organ systems. After reviewing this basic information, we will examine newer studies that demonstrate the pathophysiological consequences to SPAK and/or OSR1 disruption, discuss the development and analysis of genetically engineered mouse models, and address the possible role these serine/threonine kinases might have in cancer proliferation and migration. PMID:23073627

  3. Bruton tyrosine kinase (Btk) suppresses osteoblastic differentiation.

    PubMed

    Kaneshiro, Shoichi; Ebina, Kosuke; Shi, Kenrin; Yoshida, Kiyoshi; Otsuki, Dai; Yoshikawa, Hideki; Higuchi, Chikahisa

    2015-09-01

    The Tec family of nonreceptor tyrosine kinases has been shown to play a key role in inflammation and bone destruction. Bruton tyrosine kinase (Btk) has been the most widely studied because of its critical role in B cells. Furthermore, recent evidence has demonstrated that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. The role of Btk in osteoblastic differentiation has not been well elucidated. In this study, we demonstrated the role of Btk in osteoblastic differentiation and investigated the effects of a Btk inhibitor on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells, primary calvarial osteoblasts, and bone marrow stromal ST2 cells. Btk expression was detected in all three cell lines. Btk inhibition stimulated mRNA expression of osteoblastic markers (alkaline phosphatase, osteocalcin, and osterix) and promoted mineralization of the extracellular matrix. In addition, Btk knockdown caused increased mRNA expression of osteoblastic markers. Furthermore, Btk inhibition suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NFκB), and protein kinase Cα (PKCα). Our results indicate that Btk may regulate osteoblastic differentiation through the MAPK, NFκB, and PKCα signaling pathways. PMID:25230818

  4. Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons.

    PubMed

    Llorente-Folch, Irene; Rueda, Carlos B; Amigo, Ignacio; del Arco, Araceli; Saheki, Takeyori; Pardo, Beatriz; Satrústegui, Jorgina

    2013-08-28

    Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads. PMID:23986233

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

    SciTech Connect

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

    2008-11-01

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

  6. Human UMP-CMP kinase 2, a novel nucleoside monophosphate kinase localized in mitochondria.

    PubMed

    Xu, Yunjian; Johansson, Magnus; Karlsson, Anna

    2008-01-18

    Enzyme deficiency in the salvage pathway of deoxyribonucleotide synthesis in mitochondria can cause mtDNA depletion syndromes. We have identified a human mitochondrial UMP-CMP kinase (UMP-CMPK, cytidylate kinase; EC 2.7.4.14), designated as UMP-CMP kinase 2 (UMP-CMPK2). The C-terminal domain of this 449-amino acid protein contains all consensus motifs of a nucleoside monophosphate kinase. Phylogenetic analysis showed that UMP-CMPK2 belonged to a novel nucleoside monophosphate kinase family, which was closer to thymidylate kinase than to cytosolic UMP-CMP kinase. Subcellular localization with green fluorescent protein fusion proteins illustrated that UMP-CMPK2 was localized in the mitochondria of HeLa cells and that the mitochondrial targeting signal was included in the N-terminal 22 amino acids. The enzyme was able to phosphorylate dUMP, dCMP, CMP, and UMP with ATP as phosphate donor, but the kinetic properties were different compared with the cytosolic UMP-CMPK. Its efficacy to convert dUMP was highest, followed by dCMP, whereas CMP and UMP were the poorest substrates. It also phosphorylated the monophosphate forms of the nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd, which suggests that UMP-CMPK2 may be involved in mtDNA depletion caused by long term treatment with ddC or other pyrimidine analogs. UMP-CMPK2 mRNA expression was exclusively detected in chronic myelogenous leukemia K-562 and lymphoblastic leukemia MOLT-4 among eight studied cancer cell lines. Particular high expression in leukemia cells, dominant expression in bone marrow, and tight correlation with macrophage activation and inflammatory response suggest that UMP-CMPK2 may have other functions in addition to the supply of substrates for mtDNA synthesis. PMID:17999954

  7. Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1(BmLsd)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The structurally-related members of the PAT family of proteins, which are so name based on similarity amongst perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47), are cytoplasmic lipid droplet (LD)-associated proteins charac...

  8. Phosphatidylinositol kinase from rabbit reticulocytes

    SciTech Connect

    Tuazon, P.T.; Heng, A.B.W.; Traugh, J.A.

    1986-05-01

    Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of (/sup 32/P)ATP and Mg/sup 2 +/. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements.

  9. Kinases as targets for chemical modulators: Structural aspects and their role in spermatogenesis

    PubMed Central

    Jenardhanan, Pranitha; Mathur, Premendu P

    2014-01-01

    Protein phosphorylation and de-phosphorylation events are crucial in deciding the fate of cells. They regulate cellular growth, differentiation and cell death, and kinases are the key players of these events. The members of ser/thr kinases and tyrosine kinases form the majority of protein kinase family, exerting their regulatory mechanism in almost all cells. In testis, they impact signal transduction events, regulate all stages of sperm development from mitosis through fertilization. Understanding the function of these kinases at the structural level and studying their interactions with inhibitors can help in understanding the machinery of spermatogenesis. In view of this, we have reviewed some of the prominent kinases that are known to play a role in spermatogenesis. A better understanding of the impacts of kinase inhibition on spermatogenesis should aid in the interpretation of lesions and hopefully further the development of more efficient and potent drug candidates. PMID:26413395

  10. Structural basis for the regulation of maternal embryonic leucine zipper kinase.

    PubMed

    Cao, Lu-Sha; Wang, Jue; Chen, Yuling; Deng, Haiteng; Wang, Zhi-Xin; Wu, Jia-Wei

    2013-01-01

    MELK (maternal embryonic leucine zipper kinase), which is a member of the AMPK (AMP-activated protein kinase)-related kinase family, plays important roles in diverse cellular processes and has become a promising drug target for certain cancers. However, the regulatory mechanism of MELK remains elusive. Here, we report the crystal structure of a fragment of human MELK that contains the kinase domain and ubiquitin-associated (UBA) domain. The UBA domain tightly binds to the back of the kinase domain, which may contribute to the proper conformation and activity of the kinase domain. Interestingly, the activation segment in the kinase domain displays a unique conformation that contains an intramolecular disulfide bond. The structural and biochemical analyses unravel the molecular mechanisms for the autophosphorylation/activation of MELK and the dependence of its catalytic activity on reducing agents. Thus, our results may provide the basis for designing specific MELK inhibitors for cancer treatment. PMID:23922895

  11. The Predikin webserver: improved prediction of protein kinase peptide specificity using structural information

    PubMed Central

    Saunders, Neil F. W.

    2008-01-01

    The Predikin webserver allows users to predict substrates of protein kinases. The Predikin system is built from three components: a database of protein kinase substrates that links phosphorylation sites with specific protein kinase sequences; a perl module to analyse query protein kinases and a web interface through which users can submit protein kinases for analysis. The Predikin perl module provides methods to (i) locate protein kinase catalytic domains in a sequence, (ii) classify them by type or family, (iii) identify substrate-determining residues, (iv) generate weighted scoring matrices using three different methods, (v) extract putative phosphorylation sites in query substrate sequences and (vi) score phosphorylation sites for a given kinase, using optional filters. The web interface provides user-friendly access to each of these functions and allows users to obtain rapidly a set of predictions that they can export for further analysis. The server is available at http://predikin.biosci.uq.edu.au. PMID:18477637

  12. Functional mutations in 5′UTR of the BMPR2 gene identified in Chinese families with pulmonary arterial hypertension

    PubMed Central

    Zhang, Chenting; Liu, Chunli; Wang, Wei; Zhang, Nuofu; Hadadi, Cyrus; Huang, Junyi; Zhong, Nanshan; Lu, Wenju

    2016-01-01

    Abstract Pulmonary arterial hypertension (PAH) is a progressive pulmonary vasculopathy with significant morbidity and mortality. Bone morphogenetic protein receptor type 2 (BMPR2) has been well recognized as the principal gene responsible for heritable and sporadic PAH. Four unrelated Chinese patients with PAH and their family members, both symptomatic and asymptomatic, were genetically evaluated by sequencing all exons and the flanking regions of BMPR2. Functionality of the aberrant mutations at the 5′ untranslated region (UTR) of BMPR2 in the families with PAH was determined by site mutation, transient transfection, and promoter-reporter assays. Four individual mutations in the BMPR2 gene were identified in the 4 families, respectively: 10-GGC repeats, 13-GGC repeats, 4-AGC repeats in 5′UTR, and a novel missense mutation in exon 7 (c.961C>T; p.Arg321X). Moreover, we demonstrated that (1) these 5′UTR mutations decreased the transcription of BMPR2 and (2) the GGC repeats and AGC repeats in BMPR2 5′UTR bore functional binding sites of EGR-1 and MYF5, respectively. This is the first report demonstrating the presence of functional BMPR2 5′UTR mutations in familial patients with PAH and further indicating that EGR-1 and MYF5 are potential targets for correcting these genetic abnormalities for PAH therapy. PMID:27162618

  13. The molecular basis of targeting protein kinases in cancer therapeutics.

    PubMed

    Tsai, Chung-Jung; Nussinov, Ruth

    2013-08-01

    In this paper, we provide an overview of targeted anticancer therapies with small molecule kinase inhibitors. First, we discuss why a single constitutively active kinase emanating from a variety of aberrant genetic alterations is capable of transforming a normal cell, leading it to acquire the hallmarks of a cancer cell. To draw attention to the fact that kinase inhibition in targeted cancer therapeutics differs from conventional cytotoxic chemotherapy, we exploit a conceptual framework explaining why suppressed kinase activity will selectively kill only the so-called oncogene 'addicted' cancer cell, while sparing the healthy cell. Second, we introduce the protein kinase superfamily in light of its common active conformation with precisely positioned structural elements, and the diversified auto-inhibitory conformations among the kinase families. Understanding the detailed activation mechanism of individual kinases is essential to relate the observed oncogenic alterations to the elevated constitutively active state, to identify the mechanism of consequent drug resistance, and to guide the development of the next-generation inhibitors. To clarify the vital importance of structural guidelines in studies of oncogenesis, we explain how somatic mutations in EGFR result in kinase constitutive activation. Third, in addition to the common theme of secondary (acquired) mutations that prevent drug binding from blocking a signaling pathway which is hijacked by the aberrant activated kinase, we discuss scenarios of drug resistance and relapse by compensating lesions that bypass the inactivated pathway in a vertical or horizontal fashion. Collectively, these suggest that the future challenge of cancer therapy with small molecule kinase inhibitors will rely on the discovery of distinct combinations of optimized drugs to target individual subtypes of different cancers. PMID:23651790

  14. The molecular regulation of Janus kinase (JAK) activation.

    PubMed

    Babon, Jeffrey J; Lucet, Isabelle S; Murphy, James M; Nicola, Nicos A; Varghese, Leila N

    2014-08-15

    The JAK (Janus kinase) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2), was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haemopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organization of the component FERM (4.1/ezrin/radixin/moesin)-SH2 (Src homology 2), pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain-containing proteins, SOCS (suppressors of cytokine signalling) proteins and LNK. These recent studies highlight the diversity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors. PMID:25057888

  15. WNK Kinases, Renal Ion Transport and Hypertension

    PubMed Central

    San-Cristobal, Pedro; de los Heros, Paola; Ponce-Coria, José; Moreno, Erika; Gamba, Gerardo

    2008-01-01

    Two members of a recently discovered family of protein kinases are the cause of an inherited disease known as pseudohypoaldosteronism type II (PHAII). These patients exhibit arterial hypertension together with hyperkalemia and metabolic acidosis. This is a mirror image of Gitelman disease that is due to inactivating mutations of the SLC12A3 gene that encodes the thiazide-sensitive Na+: Cl− cotransporter. The uncovered genes causing PHAII encode for serine/threonine kinases known as WNK1 and WNK4. Physiological and biochemical studies have revealed that WNK1 and WNK4 modulate the activity of several transport pathways of the aldosterone-sensitive distal nephron, thus increasing our understanding of how diverse renal ion transport proteins are coordinated to regulate normal blood pressure levels. Observations discussed in the present work place WNK1 and WNK4 as genes involved in the genesis of essential hypertension and as potential targets for the development of antihypertensive drugs. PMID:18547946

  16. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    SciTech Connect

    Boura, Evzen Nencka, Radim

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  17. Osmotic stress signaling via protein kinases.

    PubMed

    Fujii, Hiroaki; Zhu, Jian-Kang

    2012-10-01

    Plants face various kinds of environmental stresses, including drought, salinity, and low temperature, which cause osmotic stress. An understanding of the plant signaling pathways that respond to osmotic stress is important for both basic biology and agriculture. In this review, we summarize recent investigations concerning the SNF1-related protein kinase (SnRK) 2 kinase family, which play central roles in osmotic stress responses. SnRK2s are activated by osmotic stress, and a mutant lacking SnRK2s is hypersensitive to osmotic stress. Many questions remain about the signaling pathway upstream and downstream of SnRK2s. Because some SnRK2s also functions in the abscisic acid (ABA) signaling pathway, which has recently been well clarified, study of SnRK2s in ABA signaling can provide clues regarding their roles in osmotic stress signaling. PMID:22828864

  18. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    1996-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK.

  19. Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

    PubMed

    Altman, Amnon; Kong, Kok-Fai

    2016-05-20

    The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system. PMID:27168244

  20. Structural dynamic analysis of apo and ATP-bound IRAK4 kinase

    NASA Astrophysics Data System (ADS)

    Gosu, Vijayakumar; Choi, Sangdun

    2014-07-01

    Interleukin-1 receptor-associated kinases (IRAKs) are Ser/Thr protein kinases that play an important role as signaling mediators in the signal transduction facilitated by the Toll-like receptor (TLR) and interleukin-1 receptor families. Among IRAK family members, IRAK4 is one of the drug targets for diseases related to the TLR and IL-1R signaling pathways. Experimental evidence suggests that the IRAK4 kinase domain is phosphorylated in its activation loop at T342, T345, and S346 in the fully activated state. However, the molecular interactions of subdomains within the active and inactive IRAK4 kinase domain are poorly understood. Hence, we employed a long-range molecular dynamics (MD) simulation to compare apo IRAK4 kinase domains (phosphorylated and unphosphorylated) and ATP-bound phosphorylated IRAK4 kinase domains. The MD results strongly suggested that lobe uncoupling occurs in apo unphosphorylated IRAK4 kinase via the disruption of the R334/T345 and R310/T345 interaction. In addition, apo unphosphorylated trajectory result in high mobility, particularly in the N lobe, activation segment, helix αG, and its adjoining loops. The Asp-Phe-Gly (DFG) and His-Arg-Asp (HRD) conserved kinase motif analysis showed the importance of these motifs in IRAK4 kinase activation. This study provides important information on the structural dynamics of IRAK4 kinase, which will aid in inhibitor development.

  1. Clinical experience with aurora kinase inhibitors: a review.

    PubMed

    Boss, David S; Beijnen, Jos H; Schellens, Jan H M

    2009-08-01

    The aurora kinase family of serine/threonine kinases comprises three members, designated auroras A, B, and C. Auroras A and B are essential components of the mitotic pathway, ensuring proper chromosome assembly, formation of the mitotic spindle, and cytokinesis. The role of aurora C is less clear. Overexpression of aurora A and B has been observed in several tumor types, and has been linked with a poor prognosis of cancer patients. Several small molecules targeting aurora kinases A and B or both have been evaluated preclinically and in early phase I trials. In this review we aim to summarize the most recent advances in the development of aurora kinase inhibitors, with a focus on the clinical data. PMID:19684075

  2. LRRK2 and ubiquitination: implications for kinase inhibitor therapy

    PubMed Central

    Melrose, Heather L.

    2015-01-01

    Pathogenic mutations and risk variants in LRRK2 (leucine-rich repeat kinase 2) represent the most common genetic cause of familial and sporadic PD (Parkinson's disease). LRRK2 protein is widely expressed throughout the brain and the periphery. Structurally, LRRK2 contains several functional domains, including a dual enzymatic core consisting of a kinase and GTPase domain. Disease-linked variants are found in both these enzymatic domains as well as in the COR [C-terminal of ROC (Ras of complex proteins)] and WD40 protein–protein binding domain. The kinase domain is widely believed to be linked to toxicity, and thus the thrust of pharmaceutical effort has focused on developing LRRK2 kinase inhibitors. However, recent data have suggested that inhibition of LRRK2 activity results in reduced LRRK2 levels and peripheral side effects, which are similar to those observed in homozygous LRRK2-knockout and LRRK2 kinase-dead rodent models. In a recent issue of the Biochemical Journal, a study led by Nichols reveals that dephosphorylation of LRRK2 cellular phosphorylation sites (Ser910/Ser935/Ser955/Ser973) triggers its ubiquitination and subsequent degradation and thus may account for the loss of function phenotypes observed in peripheral tissues in LRRK2-knockout/kinase-dead or inhibitor-treated rodents and primates. Albeit negative from a kinase inhibitor standpoint, the data open new avenues for LRRK2 biology and therapeutic approaches to counteract LRRK2 toxicity. PMID:26341487

  3. Targeting kinases with anilinopyrimidines: discovery of N-phenyl-N’-[4-(pyrimidin-4-ylamino)phenyl]urea derivatives as selective inhibitors of class III receptor tyrosine kinase subfamily

    PubMed Central

    Gandin, Valentina; Ferrarese, Alessandro; Dalla Via, Martina; Marzano, Cristina; Chilin, Adriana; Marzaro, Giovanni

    2015-01-01

    Kinase inhibitors are attractive drugs/drug candidates for the treatment of cancer. The most recent literature has highlighted the importance of multi target kinase inhibitors, although a correct balance between specificity and non-specificity is required. In this view, the discovery of multi-tyrosine kinase inhibitors with subfamily selectivity is a challenging goal. Herein we present the synthesis and the preliminary kinase profiling of a set of novel 4-anilinopyrimidines. Among the synthesized compounds, the N-phenyl-N’-[4-(pyrimidin-4-ylamino)phenyl]urea derivatives selectively targeted some members of class III receptor tyrosine kinase family. Starting from the structure of hit compound 19 we synthesized a further compound with an improved affinity toward the class III receptor tyrosine kinase members and endowed with a promising antitumor activity both in vitro and in vivo in a murine solid tumor model. Molecular modeling simulations were used in order to rationalize the behavior of the title compounds. PMID:26568452

  4. Efficacy of the Irreversible ErbB Family Blocker Afatinib in Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI)–Pretreated Non–Small-Cell Lung Cancer Patients with Brain Metastases or Leptomeningeal Disease

    PubMed Central

    Tufman, Amanda; Wehler, Thomas; Pelzer, Theo; Wiewrodt, Rainer; Schütz, Martin; Serke, Monika; Stöhlmacher-Williams, Jan; Märten, Angela; Maria Huber, Rudolf; Dickgreber, Nicolas J.

    2015-01-01

    Introduction: Afatinib is an effective first-line treatment in patients with epidermal growth factor receptor (EGFR)-mutated non–small-cell lung cancer (NSCLC) and has shown activity in patients progressing on EGFR-tyrosine kinase inhibitors (TKIs). First-line afatinib is also effective in patients with central nervous system (CNS) metastasis. Here we report on outcomes of pretreated NSCLC patients with CNS metastasis who received afatinib within a compassionate use program. Methods: Patients with NSCLC progressing after at least one line of chemotherapy and one line of EGFR-TKI treatment received afatinib. Medical history, patient demographics, EGFR mutational status, and adverse events including tumor progression were documented. Results: From 2010 to 2013, 573 patients were enrolled and 541 treated with afatinib. One hundred patients (66% female; median age, 60 years) had brain metastases and/or leptomeningeal disease with 74% having documented EGFR mutation. Median time to treatment failure for patients with CNS metastasis was 3.6 months, and did not differ from a matched group of 100 patients without CNS metastasis. Thirty-five percent (11 of 31) of evaluable patients had a cerebral response, five (16%) responded exclusively in brain. Response duration (range) was 120 (21–395) days. Sixty-six percent (21 of 32) of patients had cerebral disease control on afatinib. Data from one patient with an impressive response showed an afatinib concentration in the cerebrospinal fluid of nearly 1 nMol. Conclusion: Afatinib appears to penetrate into the CNS with concentrations high enough to have clinical effect on CNS metastases. Afatinib may therefore be an effective treatment for heavily pretreated patients with EGFR-mutated or EGFR–TKI-sensitive NSCLC and CNS metastasis. PMID:25247337

  5. Functional screen identifies kinases driving prostate cancer visceral and bone metastasis

    PubMed Central

    Faltermeier, Claire M.; Drake, Justin M.; Clark, Peter M.; Smith, Bryan A.; Zong, Yang; Volpe, Carmen; Mathis, Colleen; Morrissey, Colm; Castor, Brandon; Huang, Jiaoti; Witte, Owen N.

    2016-01-01

    Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention. PMID:26621741

  6. Functional screen identifies kinases driving prostate cancer visceral and bone metastasis.

    PubMed

    Faltermeier, Claire M; Drake, Justin M; Clark, Peter M; Smith, Bryan A; Zong, Yang; Volpe, Carmen; Mathis, Colleen; Morrissey, Colm; Castor, Brandon; Huang, Jiaoti; Witte, Owen N

    2016-01-12

    Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention. PMID:26621741

  7. Targeting cancer with kinase inhibitors

    PubMed Central

    Gross, Stefan; Rahal, Rami; Stransky, Nicolas; Lengauer, Christoph; Hoeflich, Klaus P.

    2015-01-01

    Kinase inhibitors have played an increasingly prominent role in the treatment of cancer and other diseases. Currently, more than 25 oncology drugs that target kinases have been approved, and numerous additional therapeutics are in various stages of clinical evaluation. In this Review, we provide an in-depth analysis of activation mechanisms for kinases in cancer, highlight recent successes in drug discovery, and demonstrate the clinical impact of selective kinase inhibitors. We also describe the substantial progress that has been made in designing next-generation inhibitors to circumvent on-target resistance mechanisms, as well as ongoing strategies for combining kinase inhibitors in the clinic. Last, there are numerous prospects for the discovery of novel kinase targets, and we explore cancer immunotherapy as a new and promising research area for studying kinase biology. PMID:25932675

  8. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2005-03-08

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine 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.

  9. Oncoprotein protein kinase

    DOEpatents

    Davis, Roger; Derijard, Benoit; Karin, Michael; Hibi, Masahiko; Lin, Anning

    2005-01-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine 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. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

    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.

  11. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine 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.

  12. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine 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.

  13. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine 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.

  14. Oncoprotein protein kinase

    DOEpatents

    Karin, M.; Hibi, M.; Lin, A.

    1997-02-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

  15. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine 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.

  16. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

    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.

  17. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine 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.

  18. KINATEST-ID: a pipeline to develop phosphorylation-dependent terbium sensitizing kinase assays.

    PubMed

    Lipchik, Andrew M; Perez, Minervo; Bolton, Scott; Dumrongprechachan, Vasin; Ouellette, Steven B; Cui, Wei; Parker, Laurie L

    2015-02-25

    Nonreceptor protein tyrosine kinases (NRTKs) are essential for cellular homeostasis and thus are a major focus of current drug discovery efforts. Peptide substrates that can enhance lanthanide ion luminescence upon tyrosine phosphorylation enable rapid, sensitive screening of kinase activity, however design of suitable substrates that can distinguish between tyrosine kinase families is a huge challenge. Despite their different substrate preferences, many NRTKs are structurally similar even between families. Furthermore, the development of lanthanide-based kinase assays is hampered by incomplete understanding of how to integrate sequence selectivity with metal ion binding, necessitating laborious iterative substrate optimization. We used curated proteomic data from endogenous kinase substrates and known Tb(3+)-binding sequences to build a generalizable in silico pipeline with tools to generate, screen, align, and select potential phosphorylation-dependent Tb(3+)-sensitizing substrates that are most likely to be kinase specific. We demonstrated the approach by developing several substrates that are selective within kinase families and amenable to high-throughput screening (HTS) applications. Overall, this strategy represents a pipeline for developing efficient and specific assays for virtually any tyrosine kinase that use HTS-compatible lanthanide-based detection. The tools provided in the pipeline also have the potential to be adapted to identify peptides for other purposes, including other enzyme assays or protein-binding ligands. PMID:25689372

  19. Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1.

    PubMed Central

    Hernández, M; Bayón, Y; Sánchez Crespo, M; Nieto, M L

    1997-01-01

    The release of [3H]arachidonic acid was studied in the 1321N1 astrocytoma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simultaneously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A2 (cPLA2) takes part in thrombin-induced arachidonate release was provided by the finding that thrombin induced retardation of the mobility of cPLA2 in SDS/polyacrylamide gels, which is a feature of the activation of cPLA2 by mitogen-activated protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPLA2, namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA2 more clearly than the activation of p42-MAK kinase did. Both cPLA2 and c-Jun kinase activation were not affected by PD-98059, a specific inhibitor of MAP kinase kinases, which indeed completely blocked p42-MAP kinase shift. Heat shock, a well-known activator of c-Jun kinase, also phosphorylated cPLA2 but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin receptor stimulation that activates a kinase cascade acting on the Pro-Leu-Ser-Pro consensus primary sequence, activates cPLA2, and associates the release of arachidonate with nuclear signalling pathways. PMID:9359863

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

    PubMed

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

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. PMID:27226575

  1. A High-Throughput Radiometric Kinase Assay.

    PubMed

    Duong-Ly, Krisna C; Peterson, Jeffrey R

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening of libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small-molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

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

    SciTech Connect

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

    2005-01-01

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

  3. Some implications of receptor kinase signaling pathway for development of multitargeted kinase inhibitors.

    PubMed

    Mitrasinovic, Petar M

    2013-03-01

    Epidermal growth factor receptors (EGFRs) belong to the ErbB family of receptor tyrosine kinases (TKs). Based on the role of EGFR signaling pathway in malignant progression of various types of tumors, a growing interest in the use of EGFR-TK inhibitors as probes for molecular imaging of EGFR-overexpressing tumors via positron emission tomography (PET) and single photon emission computed tomography (SPECT) is being notable. On one side, such noninvasive and repetitive monitoring of the activity of EGFR at the kinase level is intended to provide a direct measure of EGFR occupancy and inhibition by EGFR-targeting drugs. On the other side, all oncologic imaging tracers are molecularly targeted radiopharmaceuticals, which are strongly dependent on the tumor biochemistry including increased metabolism, hyperproliferation, angiogenesis, hypoxia, apoptosis, and specific tumor biomarkers (tumor specific antigens and tumor-specific receptors). The present article is an attempt to reconcile these two vital standpoints influencing the choice of appropriate radiolabeled agents for PET and SPECT imaging aimed to support the development of a new generation of multi-targeted kinase inhibitors in the time ahead, because the routine accomplishment of drug selectivity for particular protein kinases is a substantial challenge. PMID:23278847

  4. Glycogen Synthase Kinase 3β Interaction 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 kinase 3β interaction 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

  5. The regulation and function of the NUAK family.

    PubMed

    Sun, Xianglan; Gao, Ling; Chien, Hung-Yu; Li, Wan-Chun; Zhao, Jiajun

    2013-10-01

    AMP-activated protein kinase (AMPK) is a critical regulator of cellular and whole-body energy homeostasis. Twelve AMPK-related kinases (ARKs; BRSK1, BRSK2, NUAK1, NUAK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4, and MELK) have been identified recently. These kinases show a similar structural organization, including an N-terminal catalytic domain, followed by a ubiquitin-associated domain and a C-terminal spacer sequence, which in some cases also contains a kinase-associated domain 1. Eleven of the ARKs are phosphorylated and activated by the master upstream kinase liver kinase B1. However, most of these ARKs are largely unknown, and the NUAK family seems to have different regulations and functions. This review contains a brief discussion of the NUAK family including the specific characteristics of NUAK1 and NUAK2. PMID:23873311

  6. Diacylglycerol Kinase Inhibition and Vascular Function.

    PubMed

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

    2009-01-01

    Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction. PMID:21547002

  7. Structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae

    SciTech Connect

    Michalska, Karolina; Cuff, Marianne E.; Tesar, Christine; Feldmann, Brian; Joachimiak, Andrzej

    2011-08-01

    The crystal structure of 2-oxo-3-deoxygalactonate kinase from the De Ley–Doudoroff pathway of galactose metabolism has been determined at 2.1 Å resolution. In most organisms, efficient d-galactose utilization requires the highly conserved Leloir pathway that converts d-galactose to d-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of d-galactose assimilation have been identified. In the so-called De Ley–Doudoroff pathway, d-galactose is metabolized into pyruvate and d-glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-oxo-3-deoxygalactonate kinase, with ATP serving as a phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley–Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein.

  8. Familial pituitary tumors.

    PubMed

    Alband, Neda; Korbonits, Márta

    2014-01-01

    Pituitary adenomas are benign intracranial neoplasms that present a major clinical concern due to hormone overproduction and/or tumor mass effects. The majority of pituitary adenomas occur sporadically; however, familial cases are increasingly being recognized, such as multiple endocrine neoplasia type 1 (MEN1), Carney complex (CNC), and familial isolated pituitary adenoma (FIPA). Familial pituitary tumors appear to differ from their sporadic counterparts both in their genetic basis and in clinical characteristics. Evidence suggests that, especially in MEN1 and FIPA, tumors are more aggressive and affect patients at a younger age, therefore justifying the importance of early diagnosis, while in Carney complex pituitary hyperplasia is common. The genetic alterations responsible for the formation of familial pituitary syndromes include the MEN1 gene, responsible for about 80% of MEN1 cases, the regulatory subunit of the protein kinase A, PRKAR1A, responsible for about 70% of Carney complex cases, and AIP, the gene coding the aryl hydrocarbon receptor interacting protein, responsible for about 20% of FIPA cases. Rarely other genes have also been found responsible for familial pituitary adenoma cases. McCune-Albright syndrome (MAS) also has a genetic origin due to mosaic mutations in the G protein-coupled α subunit coded by the GNAS1 gene. In this chapter, we summarize the genetic and clinical characteristics of these familial pituitary syndromes and MAS. PMID:25248598

  9. PI3 kinase is indispensable for oncogenic transformation by the V560D mutant of c-Kit in a kinase-independent manner.

    PubMed

    Lindblad, Oscar; Kazi, Julhash U; Rönnstrand, Lars; Sun, Jianmin

    2015-11-01

    Oncogenic mutants of c-Kit are often found in mastocytosis, gastrointestinal stromal tumors and acute myeloid leukemia. The activation mechanism of the most commonly occurring mutation, D816V in exon 17 of c-Kit, has been well-studied while other mutations remain fairly uncharacterized in this respect. In this study, we show that the constitutive activity of the exon 11 mutant V560D is weaker than the D816V mutant. Phosphorylation of downstream signaling proteins induced by the ligand for c-Kit, stem cell factor, was stronger in c-Kit/V560D expressing cells than in cells expressing c-kit/D816V. Although cells expressing c-Kit/V560D showed increased ligand-independent proliferation and survival compared to wild-type c-Kit-expressing cells, these biological effects were weaker than in c-Kit/D816V-expressing cells. In contrast to cells expressing wild-type c-Kit, cells expressing c-Kit/V560D were independent of Src family kinases for downstream signaling. However, the independence of Src family kinases was not due to a Src-like kinase activity that c-Kit/D816V displayed. Point mutations that selectively block the association of PI3 kinase with c-Kit/V560D inhibited ligand-independent activation of the receptor, while inhibition of the kinase activity of PI3 kinase with pharmacological inhibitors did not affect the kinase activity of the receptor. This suggests a lipid kinase-independent key role of PI3 kinase in c-Kit/V560D-mediated oncogenic signal transduction. Thus, PI3 kinase is an attractive therapeutic target in malignancies induced by c-Kit mutations independent of its lipid kinase activity. PMID:26040420

  10. Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2

    PubMed Central

    Konopatskaya, Olga; Matthews, Sharon A.; Harper, Matthew T.; Gilio, Karen; Cosemans, Judith M. E. M.; Williams, Christopher M.; Navarro, Maria N.; Carter, Deborah A.; Heemskerk, Johan W. M.; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W.

    2016-01-01

    Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion. PMID:21527521

  11. Biochemical and Structural Insights into Doublecortin-like Kinase Domain 1.

    PubMed

    Patel, Onisha; Dai, Weiwen; Mentzel, Mareike; Griffin, Michael D W; Serindoux, Juliette; Gay, Yoann; Fischer, Stefanie; Sterle, Shoukat; Kropp, Ashleigh; Burns, Christopher J; Ernst, Matthias; Buchert, Michael; Lucet, Isabelle S

    2016-09-01

    Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that belongs to the family of microtubule-associated proteins. Originally identified for its role in neurogenesis, DCLK1 has recently been shown to regulate biological processes outside of the CNS. DCLK1 is among the 15 most common putative driver genes for gastric cancers and is highly mutated across various other human cancers. However, our present understanding of how DCLK1 dysfunction leads to tumorigenesis is limited. Here, we provide evidence that DCLK1 kinase activity negatively regulates microtubule polymerization. We present the crystal structure of the DCLK1 kinase domain at 1.7 Å resolution, providing detailed insight into the ATP-binding site that will serve as a framework for future drug design. This structure also allowed for the mapping of cancer-causing mutations within the kinase domain, suggesting that a loss of kinase function may contribute to tumorigenesis. PMID:27545623

  12. Structural insight into the mechanism of synergistic autoinhibition of SAD kinases

    PubMed Central

    Wu, Jing-Xiang; Cheng, Yun-Sheng; Wang, Jue; Chen, Lei; Ding, Mei; Wu, Jia-Wei

    2015-01-01

    The SAD/BRSK kinases participate in various important life processes, including neural development, cell cycle and energy metabolism. Like other members of the AMPK family, SAD contains an N-terminal kinase domain followed by the characteristic UBA and KA1 domains. Here we identify a unique autoinhibitory sequence (AIS) in SAD kinases, which exerts autoregulation in cooperation with UBA. Structural studies of mouse SAD-A revealed that UBA binds to the kinase domain in a distinct mode and, more importantly, AIS nestles specifically into the KD-UBA junction. The cooperative action of AIS and UBA results in an ‘αC-out' inactive kinase, which is conserved across species and essential for presynaptic vesicle clustering in C. elegans. In addition, the AIS, along with the KA1 domain, is indispensable for phospholipid binding. Taken together, these data suggest a model for synergistic autoinhibition and membrane activation of SAD kinases. PMID:26626945

  13. The extended protein kinase C superfamily.

    PubMed Central

    Mellor, H; Parker, P J

    1998-01-01

    Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in a multitude of cellular processes, ranging from control of fundamental cell autonomous activities, such as proliferation, to more organismal functions, such as memory. However, understanding of mammalian PKC signalling systems is complicated by the large number of family members. Significant progress has been made through studies based on comparative analysis, which have defined a number of regulatory elements in PKCs which confer specific location and activation signals to each isotype. Further studies on simple organisms have shown that PKC signalling paradigms are conserved through evolution from yeast to humans, underscoring the importance of this family in cellular signalling and giving novel insights into PKC function in complex mammalian systems. PMID:9601053

  14. Characterization of the Mechanisms Controlling Greatwall Activity ▿

    PubMed Central

    Vigneron, Suzanne; Gharbi-Ayachi, Aicha; Raymond, Anne-Aurélie; Burgess, Andrew; Labbé, Jean-Claude; Labesse, Gilles; Monsarrat, Bernard; Lorca, Thierry; Castro, Anna

    2011-01-01

    Here we investigate the mechanisms regulating Greatwall (Gwl), a serine/threonine kinase essential for promoting the correct timing of mitosis. We identify Gwl as a unique AGC kinase that, unlike most AGC members, appears to be devoid of a hydrophobic motif despite the presence of a functional hydrophobic pocket. Our results suggest that Gwl activation could be mediated by the binding of its hydrophobic pocket to the hydrophobic motif of another AGC kinase. Our molecular modeling and mutagenic analysis also indicate that Gwl displays a conserved tail/linker site whose phosphorylation mediates kinase activation by promoting the interaction of this phosphorylated residue with two lysines at the N terminus. This interaction could stabilize the αC-helix and maintain kinase activity. Finally, the different phosphorylation sites on Gwl are identified, and the role of each one in the regulation of Gwl kinase activity is determined. Our data suggest that only the phosphorylation of the tail/linker site, located outside the putative T loop, appears to be essential for Gwl activation. In summary, our results identify Gwl as a member of the AGC family of kinases that appears to be regulated by unique mechanisms and that differs from the other members of this family. PMID:21444715

  15. 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

  16. The molecular regulation of Janus kinase (JAK) activation

    PubMed Central

    2014-01-01

    The Janus Kinase (JAK) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2, was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haematopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organisation of the component FERM-SH2, pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain containing proteins, SOCS (Suppressors of Cytokine Signalling) proteins and Lnk. These recent advances highlight the diversity of regulatory mechanisms utilised by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors. PMID:25057888

  17. Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2.

    PubMed

    Saruhashi, Masashi; Kumar Ghosh, Totan; Arai, Kenta; Ishizaki, Yumiko; Hagiwara, Kazuya; Komatsu, Kenji; Shiwa, Yuh; Izumikawa, Keiichi; Yoshikawa, Harunori; Umezawa, Taishi; Sakata, Yoichi; Takezawa, Daisuke

    2015-11-17

    Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms. PMID:26540727

  18. Reevaluation of Phosphorylation Sites in the Parkinson Disease-associated Leucine-rich Repeat Kinase 2*

    PubMed Central

    Li, Xiaojie; Moore, Darren J.; Xiong, Yulan; Dawson, Ted M.; Dawson, Valina L.

    2010-01-01

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson disease and contribute to sporadic Parkinson disease. LRRK2 is a large complex protein with multiple functional domains, including a Roc-GTPase, protein kinase, and multiple protein-protein interaction domains. Previous studies have suggested an important role for kinase activity in LRRK2-induced neuronal toxicity and inclusion body formation. Disease-associated mutations in LRRK2 also tend to increase kinase activity. Thus, enhanced kinase activity may therefore underlie LRRK2-linked disease. Similar to the closely related mixed-lineage kinases, LRRK2 can undergo autophosphorylation in vitro. Three putative autophosphorylation sites (Thr-2031, Ser-2032, and Thr-2035) have been identified within the activation segment of the LRRK2 kinase domain based on sequence homology to mixed-lineage kinases. Phosphorylation at one or more of these sites is critical for the kinase activity of LRRK2. Sensitive phopho-specific antibodies to each of these three sites have been developed and validated by ELISA, dot-blot, and Western blot analysis. Using these antibodies, we have found that all three putative sites are phosphorylated in LRRK2, and Ser-2032 and Thr-2035 are the two important sites that regulate LRRK2 kinase activity. PMID:20595391

  19. Biochemical and molecular analysis of a transmembrane protein kinase from Arabidopsis thaliana. Progress report, January 1993

    SciTech Connect

    Bleecker, A.B.

    1993-06-01

    We have isolated genomic and cDNA clones encoding a novel receptor-like protein kinase from the higher plant Arabidopsis thaliana. This kinase is being studied by combining biochemical, molecular, and genetic approaches. Domain-specific antibodies immunodecorate a polypeptide with a molecular mass of 120,000 daltons in extracts of Arabidopsis, where it has been found in all portions of the plant examined including root, stem, leaf, flower, and silique. Cytochemical analysis and initial studies using the kinase promoter with the GUS reporter gene system also indicate that the kinase is present throughout the plant. The kinase is glycosylated, like animal receptor kinases, and has been partially purified from Arabidopsis by using lectin columns. The kinase has been expressed in E coli, purified, and found to autophosphorylate on serine and threonine residues, but not on tyrosine residues. As such, it belongs to the small family of receptor-like kinases with serine/threonine specificity. Transgenic plants are now being produced that either overexpress or carry altered forms of the protein kinase gene. These experiments will help determine the natural role the kinase plays in a pathway of signal transduction.

  20. Biochemical and molecular analysis of a transmembrane protein kinase from Arabidopsis thaliana

    SciTech Connect

    Bleecker, A.B.

    1993-01-01

    We have isolated genomic and cDNA clones encoding a novel receptor-like protein kinase from the higher plant Arabidopsis thaliana. This kinase is being studied by combining biochemical, molecular, and genetic approaches. Domain-specific antibodies immunodecorate a polypeptide with a molecular mass of 120,000 daltons in extracts of Arabidopsis, where it has been found in all portions of the plant examined including root, stem, leaf, flower, and silique. Cytochemical analysis and initial studies using the kinase promoter with the GUS reporter gene system also indicate that the kinase is present throughout the plant. The kinase is glycosylated, like animal receptor kinases, and has been partially purified from Arabidopsis by using lectin columns. The kinase has been expressed in E coli, purified, and found to autophosphorylate on serine and threonine residues, but not on tyrosine residues. As such, it belongs to the small family of receptor-like kinases with serine/threonine specificity. Transgenic plants are now being produced that either overexpress or carry altered forms of the protein kinase gene. These experiments will help determine the natural role the kinase plays in a pathway of signal transduction.

  1. Phosphatidylinositol 3-kinase in myogenesis.

    PubMed

    Kaliman, P; Zorzano, A

    1997-08-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been cloned and characterized in a wide range of organisms. PI 3-kinases are activated by a diversity of extracellular stimuli and are involved in multiple cell processes such as cell proliferation, protein trafficking, cell motility, differentiation, regulation of cytoskeletal structure, and apoptosis. It has recently been shown that PI 3-kinase is a crucial second messenger in the signaling of myogenesis. Two structurally unrelated highly specific inhibitors of PI 3-kinase-wortmannin and LY294002-block the morphological and biochemical differentiation program of different skeletal-muscle cell models. Moreover, L6E9 myoblasts overexpressing a dominant-negative mutant of PI 3-kinase p85 regulatory subunit (Δp85) are unable to differentiate. Furthermore, PI 3-kinase is specifically involved in the insulinlike growth factor (IGF)-dependent myogenic pathway. Indeed, the ability of IGF-I, des-1,3-IGF-I, and IGF-II to promote cell fusion and muscle-specific protein expression is impaired after treatment with PI 3-kinase inhibitors or in cells overexpressing Δp85. The identification of additional key downstream elements of the IGF/PI 3-kinase myogenic cascade is crucial to a detailed understanding of the process of muscle differentiation and may generate new tools for skeletal and cardiac muscle regeneration therapies. (Trends Cardiovasc Med 1997;7:198-202). © 1997, Elsevier Science Inc. PMID:21235885

  2. [Kinase inhibitors and their resistance].

    PubMed

    Togashi, Yosuke; Nishio, Kazuto

    2015-08-01

    Kinase cascades are involved in all stages of tumorigenesis through modulation of transformation and differentiation, cell-cycle progression, and motility. Advances in molecular targeted drug development allow the design and synthesis of inhibitors targeting cancer-associated signal transduction pathways. Potent selective inhibitors with low toxicity can benefit patients especially with several malignancies harboring an oncogenic driver addictive signal. This article evaluates information on solid tumor-related kinase signals and inhibitors, including receptor tyrosine kinase or serine/threonine kinase signals that lead to successful application in clinical settings. In addition, the resistant mechanisms to the inhibitors is summarized. PMID:26281685

  3. Family Meals

    MedlinePlus

    ... Story" 5 Things to Know About Zika & Pregnancy Family Meals KidsHealth > For Parents > Family Meals Print A ... even more important as kids get older. Making Family Meals Happen It can be a big challenge ...

  4. Family History

    MedlinePlus

    Your family history includes health information about you and your close relatives. Families have many factors in common, including their genes, ... as heart disease, stroke, and cancer. Having a family member with a disease raises your risk, but ...

  5. Family Arguments

    MedlinePlus

    ... Spread the Word Shop AAP Find a Pediatrician Family Life Medical Home Family Dynamics Adoption & Foster Care ... Life Listen Español Text Size Email Print Share Family Arguments Page Content Article Body We seem to ...

  6. Family History

    MedlinePlus

    ... CDC Cancel Submit Search The CDC Family Health History Note: Javascript is disabled or is not supported ... visit this page: About CDC.gov . Family Health History The Basics Family Health History & Chronic Disease Planning ...

  7. Family Folklore

    ERIC Educational Resources Information Center

    Kotkin, Amy J.; Baker, Holly C.

    1977-01-01

    Discusses the Family Folklore Program of the Smithsonian Institution's annual Festival of American Folklife, in which the whole family can be involved in tracing family history through story telling, photographs, etc. (MS)

  8. Venus Kinase Receptors: Prospects in Signaling and Biological Functions of These Invertebrate Kinases

    PubMed Central

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. VKR gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in S. mansoni. VKRs are activated by amino acids and are highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates or in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens, and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel. PMID:24860549

  9. Venus kinase receptors: prospects in signaling and biological functions of these invertebrate kinases.

    PubMed

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. VKR gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in S. mansoni. VKRs are activated by amino acids and are highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates or in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens, and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel. PMID:24860549

  10. Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

    PubMed Central

    Lu, Q; Inouye, M

    1996-01-01

    Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8650159

  11. The early evolution of the phosphagen kinases--insights from choanoflagellate and poriferan arginine kinases.

    PubMed

    Conejo, Maria; Bertin, Matt; Pomponi, Shirley A; Ellington, W Ross

    2008-01-01

    Arginine kinase (AK) is a member of a large family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases. AKs are present in certain protozoans, sponges, cnidarians, and both lophotrochozoan and ecdysozoan protostomes. Another phosphagen kinase, creatine kinase (CK), is found in sponges, cnidarians, and both deuterostome and protostome groups but does not appear to be present in protozoans. To probe the early evolution of phosphagen kinases, we have amplified the cDNAs for AKs from three choanoflagellates and from the hexactinellid sponge Aphrocallistes beatrix and the demosponges Suberites fuscus and Microciona prolifera. Phylogenetic analysis using maximum likelihood of these choanoflagellate and sponge AKs with other AK sequences revealed that the AK from the choanoflagellate Monosiga brevicollis clusters with the AK from the glass sponge Aphrocallistes and is part of a larger cluster containing AKs from the demosponges Suberites and Microciona as well as basal and protostome invertebrates. In contrast, AKs from Codonosiga gracilis and Monosiga ovata form a distinct cluster apart from all other AK sequences. tBLASTn searches of the recently released M. brevicollis genome database showed that this species has three unique AK genes-one virtually identical to the M. brevicollis cDNA and the other two showing great similarity to C. gracilis and M. ovata AKs. Three distinct AK genes are likely present in choanoflagellates. Two of these AKs display extensive similarity to both CKs and an AK from sponges. Previous work has shown CK evolved from an AK-like ancestor prior to the divergence of sponges. The present results provide evidence suggesting that the initial gene duplication event(s) leading to the CK lineage may have occurred before the divergence of the choanoflagellate and animal lineages. PMID:18064398

  12. Cdc42 Regulation of Kinase Activity and Signaling by the Yeast p21-Activated Kinase Ste20

    PubMed Central

    Lamson, Rachel E.; Winters, Matthew J.; Pryciak, Peter M.

    2002-01-01

    The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated Gβγ complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway. PMID:11940652

  13. Preclinical validation of a novel compound targeting p70S6 kinase in breast cancer

    PubMed Central

    Segatto, Ilenia; Massarut, Samuele; Boyle, Robert; Baldassarre, Gustavo; Walker, David; Belletti, Barbara

    2016-01-01

    Breast cancer is a frequent and treatable disease. However, when recurrent, breast cancer often becomes refractory to therapy and progresses into metastatic forms that are typically incurable. Thus, understanding and targeting the critical pathways underlying breast cancer recurrence is urgently needed to eradicate primary disease and achieve better prognosis. Recently, we have demonstrated that the ribosomal protein p70S6K is activated in residual breast cancer cells as a result of post-surgical inflammation and that interfering with its activity in the peri-operative setting strongly suppresses recurrence in a mouse model. In order to develop clinically-exploitable treatments targeting p70S6K, we have tested a newly generated compound, called FS-115. FS-115 potently inhibited p70S6K1 (IC50 35nM) with high selectivity over other AGC kinases or PI3K pathway kinases. In vitro, treatment with FS-115 efficiently blocked p70S6K activity in breast cancer cell lines and impaired colony formation and anchorage independent growth. Pharmacokinetic profiling showed that FS-115 exhibited high oral bioavailability, optimal plasma distribution and high brain penetrance. In nude mice, FS-115 strongly suppressed tumor take-rate and primary tumor growth. Oral dosing with FS-115 in a peri-operative schedule was effective in decreasing local recurrence of breast cancer and a long-term treatment schedule was well tolerated and efficiently suppressed distant metastasis formation. Altogether, we propose that FS-115 might be a good candidate for the treatment of breast cancer patients at high risk to relapse. Summary Statement Our results confirm that inhibition of p70S6K represents a valuable opportunity for restraining loco-regional relapse and metastasis in breast cancer and identify in FS-115 a promising candidate-inhibitor to move from preclinical to clinical treatments. PMID:27155197

  14. Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana

    PubMed Central

    Xie, Yuan; Li, Mei; Chang, Wenrui

    2016-01-01

    The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. However, their exact physiological function and real substrates remain unknown. Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. Using mutation and activity analysis, we further verified the key residues important for its catalytic activity. Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process. Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1. PMID:27223615

  15. Identification and characterization of the role of c-terminal Src kinase in dengue virus replication

    PubMed Central

    Kumar, Rinki; Agrawal, Tanvi; Khan, Naseem Ahmed; Nakayama, Yuji; Medigeshi, Guruprasad R.

    2016-01-01

    We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication. PMID:27457684

  16. The minimal kinome of Giardia lamblia illuminates early kinase evolution and unique parasite biology

    PubMed Central

    2011-01-01

    Background The major human intestinal pathogen Giardia lamblia is a very early branching eukaryote with a minimal genome of broad evolutionary and biological interest. Results To explore early kinase evolution and regulation of Giardia biology, we cataloged the kinomes of three sequenced strains. Comparison with published kinomes and those of the excavates Trichomonas vaginalis and Leishmania major shows that Giardia's 80 core kinases constitute the smallest known core kinome of any eukaryote that can be grown in pure culture, reflecting both its early origin and secondary gene loss. Kinase losses in DNA repair, mitochondrial function, transcription, splicing, and stress response reflect this reduced genome, while the presence of other kinases helps define the kinome of the last common eukaryotic ancestor. Immunofluorescence analysis shows abundant phospho-staining in trophozoites, with phosphotyrosine abundant in the nuclei and phosphothreonine and phosphoserine in distinct cytoskeletal organelles. The Nek kinase family has been massively expanded, accounting for 198 of the 278 protein kinases in Giardia. Most Neks are catalytically inactive, have very divergent sequences and undergo extensive duplication and loss between strains. Many Neks are highly induced during development. We localized four catalytically active Neks to distinct parts of the cytoskeleton and one inactive Nek to the cytoplasm. Conclusions The reduced kinome of Giardia sheds new light on early kinase evolution, and its highly divergent sequences add to the definition of individual kinase families as well as offering specific drug targets. Giardia's massive Nek expansion may reflect its distinctive lifestyle, biphasic life cycle and complex cytoskeleton. PMID:21787419

  17. Identification and characterization of the role of c-terminal Src kinase in dengue virus replication.

    PubMed

    Kumar, Rinki; Agrawal, Tanvi; Khan, Naseem Ahmed; Nakayama, Yuji; Medigeshi, Guruprasad R

    2016-01-01

    We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication. PMID:27457684

  18. G-protein coupled receptor kinases in inflammation and disease

    PubMed Central

    Packiriswamy, Nandakumar; Parameswaran, Narayanan

    2015-01-01

    G-protein coupled receptor kinases (GRKs) are serine/threonine protein kinases originally discovered for their role in G-protein coupled receptor (GPCR) phosphorylation. Recent studies have demonstrated a much broader function for this kinase family including phosphorylation of cytosolic substrates involved in cell signaling pathways stimulated by GPCRs as well as non-GPCRs. In addition, GRKs modulate signaling via phosphorylation-independent functions. Because of these various biochemical functions, GRKs have been shown to affect critical physiological and pathophysiological processes and thus are considered as drug targets in diseases such as heart failure. Role of GRKs in inflammation and inflammatory diseases is an evolving area of research and several studies including work from our lab in the recent years have demonstrated critical role of GRKs in the immune system. In this review we discuss the classical and the newly emerging functions of GRKs in the immune system and their role in inflammation and disease processes. PMID:26226012

  19. Family Privilege

    ERIC Educational Resources Information Center

    Seita, John R.

    2014-01-01

    Family privilege is defined as "strengths and supports gained through primary caring relationships." A generation ago, the typical family included two parents and a bevy of kids living under one roof. Now, every variation of blended caregiving qualifies as family. But over the long arc of human history, a real family was a…

  20. Pharmacological modulation of protein kinases as a new approach to treat addiction to cocaine and opiates.

    PubMed

    García-Pardo, María Pilar; Roger-Sanchez, Concepción; Rodríguez-Arias, Marta; Miñarro, Jose; Aguilar, María Asunción

    2016-06-15

    Drug addiction shares brain mechanisms and molecular substrates with learning and memory processes, such as the stimulation of glutamate receptors and their downstream signalling pathways. In the present work we provide an up-to-date review of studies that have demonstrated the implication of the main memory-related calcium-dependent protein kinases in opiate and cocaine addiction. The effects of these drugs of abuse in different animal models of drug reward, dependence and addiction are altered by manipulation of the mitogen-activated protein kinase (MAPK) family, particularly extracellular signal regulated kinase (ERK), calcium/calmodulin-dependent kinase II (CaMKII), the protein kinase C (PKC) family (including PKMζ), cAMP-dependent protein kinase A (PKA), cGMP-dependent protein kinase G (PKG), the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream target mammalian target of Rapamycin (mTOR), cyclin-dependent kinase 5 (Cdk5), heat-shock proteins (Hsp) and other enzymes and proteins. Research suggests that drugs of abuse induce dependence and addiction by modifying the signalling pathways that involve these memory-related protein kinases, and supports the idea that drug addiction is an excessive aberrant learning disorder in which the maladaptive memory of drug-associated cues maintains compulsive drug use and contributes to relapse. Moreover, the studies we review offer new pharmacological strategies to treat opiate and cocaine dependence based on the manipulation of these protein kinases. In particular, disruption of reconsolidation of drug-related memories may have a high therapeutic value in the treatment of drug addiction. PMID:27056740

  1. Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38.

    PubMed

    Yustein, Jason T; Xia, Liang; Kahlenburg, J Michelle; Robinson, Dan; Templeton, Dennis; Kung, Hsing-Jien

    2003-09-18

    The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence. PMID:13679851

  2. Crystal structure of domain-swapped STE20 OSR1 kinase domain

    SciTech Connect

    Lee, Seung-Jae; Cobb, Melanie H.; Goldsmith, Elizabeth J.

    2009-09-15

    OSR1 (oxidative stress-responsive-1) and SPAK (Ste20/Sps1-related proline/alanine-rich kinase) belong to the GCK-VI subfamily of Ste20 group kinases. OSR1 and SPAK are key regulators of NKCCs (Na{sup +}/K{sup +}/2Cl{sup -} cotransporters) and activated by WNK family members (with-no-lysine kinase), mutations of which are known to cause Gordon syndrome, an autosomal dominant form of inherited hypertension. The crystal structure of OSR1 kinase domain has been solved at 2.25 {angstrom}. OSR1 forms a domain-swapped dimer in an inactive conformation, in which P+1 loop and {alpha}EF helix are swapped between dimer-related monomers. Structural alignment with nonswapped Ste20 TAO2 kinase indicates that the integrity of chemical interactions in the kinase domain is well preserved in the domain-swapped interfaces. The OSR1 kinase domain has now been added to a growing list of domain-swapped protein kinases recently reported, suggesting that the domain-swapping event provides an additional layer of complexity in regulating protein kinase activity.

  3. c-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution

    PubMed Central

    2016-01-01

    Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity. PMID:27166638

  4. Structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae

    PubMed Central

    Michalska, Karolina; Cuff, Marianne E.; Tesar, Christine; Feldmann, Brian; Joachimiak, Andrzej

    2011-01-01

    In most organisms, efficient d-galactose utilization requires the highly conserved Leloir pathway that converts d-galactose to d-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of d-galactose assimilation have been identified. In the so-called De Ley–Doudoroff pathway, d-galactose is metabolized into pyruvate and d-­glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-­oxo-3-deoxygalactonate kinase, with ATP serving as a phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley–Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein. PMID:21795809

  5. Structural Comparison of Human Mammalian Ste20-Like Kinases

    PubMed Central

    Record, Christopher J.; Chaikuad, Apirat; Rellos, Peter; Das, Sanjan; Pike, Ashley C. W.; Fedorov, Oleg; Marsden, Brian D.; Knapp, Stefan; Lee, Wen Hwa

    2010-01-01

    Background The serine/threonine mammalian Ste-20 like kinases (MSTs) are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains. Principal Findings We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family. Significance The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. PMID:20730082

  6. Cancer, Families, and Family Counselors.

    ERIC Educational Resources Information Center

    Duffy, Maureen; Gillig, Scott

    2003-01-01

    Examines the role of the family counselor in working with cancer patients and their families. Suggests ways in which the family counselor can work proactively with families in the area of cancer prevention and helping them cope more effectively with its impact on their lives. Uses a clinical case example to illustrate intervention with cancer…

  7. Family therapy by family doctors

    PubMed Central

    Neighbour, R.

    1982-01-01

    The experiences of a group of general practitioners learning and attempting family therapy are described. Three principles for working with whole families — facilitation, formulation and focussing — are illustrated by case histories. Family therapy in general practice can be effective for patients and worthwhile for family doctors. PMID:7153974

  8. Creating Order from Chaos: Cellular Regulation by Kinase Anchoring

    PubMed Central

    Scott, John D.; Dessauer, Carmen W.; Tasken, Kjetil

    2012-01-01

    Second messenger responses rely on where and when the enzymes that propagate these signals become active. Spatial and temporal organization of certain signaling enzymes is controlled in part by A-kinase anchoring proteins (AKAPs). This family of regulatory proteins was originally classified on the basis of their ability to compartmentalize the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (also known as protein kinase A, or PKA). However, it is now recognized that AKAPs position G protein–coupled receptors, adenylyl cyclases, G proteins, and their effector proteins in relation to protein kinases and signal termination enzymes such as phosphodiesterases and protein phosphatases. This arrangement offers a simple and efficient means to limit the scope, duration, and directional flow of information to sites deep within the cell. This review focuses on the pros and cons of reagents that define the biological role of kinase anchoring inside cells and discusses recent advances in our understanding of anchored second messenger signaling in the cardiovascular and immune systems. PMID:23043438

  9. Functions of Aurora kinase C in meiosis and cancer

    PubMed Central

    Quartuccio, Suzanne M.; Schindler, Karen

    2015-01-01

    The mammalian genome encodes three Aurora kinase protein family members: A, B, and C. While Aurora kinase A (AURKA) and B (AURKB) are found in cells throughout the body, significant protein levels of Aurora kinase C (AURKC) are limited to cells that undergo meiosis (sperm and oocyte). Despite its discovery nearly 20 years ago, we know little about the function of AURKC compared to that of the other 2 Aurora kinases. This lack of understanding can be attributed to the high sequence homology between AURKB and AURKC preventing the use of standard approaches to understand non-overlapping and meiosis I (MI)-specific functions of the two kinases. Recent evidence has revealed distinct functions of AURKC in meiosis and may aid in our understanding of why chromosome segregation during MI often goes awry in oocytes. Many cancers aberrantly express AURKC, but because we do not fully understand AURKC function in its normal cellular context, it is difficult to predict the biological significance of this expression on the disease. Here, we consolidate and update what is known about AURKC signaling in meiotic cells to better understand why it has oncogenic potential. PMID:26347867

  10. Maternal embryonic leucine zipper kinase: key kinase for stem cell phenotype in glioma and other cancers.

    PubMed

    Ganguly, Ranjit; Hong, Christopher S; Smith, Luke G F; Kornblum, Harley I; Nakano, Ichiro

    2014-06-01

    Maternal embryonic leucine zipper kinase (MELK) is a member of the snf1/AMPK family of protein serine/threonine kinases that has recently gained significant attention in the stem cell and cancer biology field. Recent studies suggest that activation of this kinase is tightly associated with extended survival and accelerated proliferation of cancer stem cells (CSC) in various organs. Overexpression of MELK has been noted in various cancers, including colon, breast, ovaries, pancreas, prostate, and brain, making the inhibition of MELK an attractive therapeutic strategy for a variety of cancers. In the experimental cancer models, depletion of MELK by RNA interference or small molecule inhibitors induces apoptotic cell death of CSCs derived from glioblastoma multiforme and breast cancer, both in vitro and in vivo. Mechanism of action of MELK includes, yet may not be restricted to, direct binding and activation of the oncogenic transcription factors c-JUN and FOXM1 in cancer cells but not in the normal counterparts. Following these preclinical studies, the phase I clinical trial for advanced cancers with OTSSP167 started in 2013, as the first-in-class MELK inhibitor. This review summarizes the current molecular understanding of MELK and the recent preclinical studies about MELK as a cancer therapeutic target. PMID:24795222

  11. 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

  12. Familial cutaneous melanoma.

    PubMed

    Hansson, Johan

    2010-01-01

    Approximately 5-10 % of all cutaneous melanomas occur in families with hereditary melanoma predisposition. Worldwide, approximately 20-40% of kindreds with familial elanoma harbor germline mutations in the CDKN2A gene, located on chromosome 9p21, which encodes two different proteins, p16INK4 and p14ARF, both involved in regulation of cell cycle progression and induction of senescence. In different populations several recurring CDKN2A founder mutations have been described. The risk of melanoma in CDKN2A mutations carriers varies between populations and is higher in regions with high sun exposure and high incidence of melanoma in the general population. Some CDKN2A mutations have been associated not only with melanoma but also with increased risk of other malignancies--most notably pancreatic carcinoma. A much smaller number of families have germline mutations in the CDK4 gene on chromosome 12q14, encoding a cyclin dependent kinase which normally interacts with p16INK4A. The management of families with hereditary melanoma is discussed. PMID:20687502

  13. FAMILIAL SUICIDE

    PubMed Central

    Unni, K.E. Sadanaandan

    1996-01-01

    Seven completed suicides in a family of lower socioeconomic status and suburban domicile in Pondicherry are reported. The presence of bipolar affective disorder in the family members and the absence of exogenous factors are illustrated by utilising both family history method and family study method. The details collected formed the basis for the terminology ‘familial suicide’. The management of the index case, one of the only three surviving male members of the family, who presented with suicidal ruminations and depressive features, is described. PMID:21584122

  14. Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2

    PubMed Central

    Saruhashi, Masashi; Kumar Ghosh, Totan; Arai, Kenta; Ishizaki, Yumiko; Hagiwara, Kazuya; Komatsu, Kenji; Shiwa, Yuh; Izumikawa, Keiichi; Yoshikawa, Harunori; Umezawa, Taishi; Sakata, Yoichi; Takezawa, Daisuke

    2015-01-01

    Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated “ARK” (for “ABA and abiotic stress-responsive Raf-like kinase”) plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms. PMID:26540727

  15. Synthesis, protein kinase inhibitory potencies, and in vitro antiproliferative activities of meridianin derivatives.

    PubMed

    Giraud, Francis; Alves, Georges; Debiton, Eric; Nauton, Lionel; Théry, Vincent; Durieu, Emilie; Ferandin, Yoan; Lozach, Olivier; Meijer, Laurent; Anizon, Fabrice; Pereira, Elisabeth; Moreau, Pascale

    2011-07-14

    The synthesis of new meridianin derivatives is described. The indolic ring system was substituted at the C-4 to C-7 positions either by a bromine atom or by nitro or amino groups. Additionally, an iodine atom or various aryl groups were introduced at the C-5 position of the 2-aminopyrimidine ring. These compounds as well as some of their synthetic intermediates were tested for their kinase inhibitory potencies and for their in vitro antiproliferative activities. We found that this series of compounds is particularly interesting in the development of new inhibitors of DYRK1A and CLK1 kinases. The most effective compounds toward these two kinase families are the 6- and 7-bromo derivatives 30, 33, and 34 that showed more than 45-fold selectivity toward DYRK1A/CLK1 kinases over the other kinases tested. Meridianin derivatives could thus be developed toward potent and selective inhibitors of key RNA splicing regulators and potential therapeutic agents. PMID:21623630

  16. Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.)

    PubMed Central

    Sun, Yun; Wang, Chen; Yang, Bo; Jiang, Yuan-Qing

    2014-01-01

    Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A–C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription–PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola. PMID:24604738

  17. Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.).

    PubMed

    Sun, Yun; Wang, Chen; Yang, Bo; Wu, Feifei; Hao, Xueyu; Liang, Wanwan; Niu, Fangfang; Yan, Jingli; Zhang, Hanfeng; Wang, Boya; Deyholos, Michael K; Jiang, Yuan-Qing

    2014-05-01

    Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A-C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription-PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola. PMID:24604738

  18. Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

    PubMed Central

    Li, John J.; Cao, Chune; Fixsen, Sarah M.; Young, Janet M.; Bando, Hisanori; Elde, Nels C.; Katsuma, Susumu; Dever, Thomas E.; Sicheri, Frank

    2015-01-01

    Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase function remains an enigma. Here, we probed the mechanism by which PK2 inhibits the model eIF2α kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2α kinases. Although PK2 structurally mimics the C-lobe of a protein kinase domain and possesses the required docking infrastructure to bind eIF2α, we show that PK2 directly binds the kinase domain of PKR (PKRKD) but not eIF2α. The PKRKD–PK2 interaction requires a 22-residue N-terminal extension preceding the globular PK2 body that we term the “eIF2α kinase C-lobe mimic” (EKCM) domain. The functional insufficiency of the N-terminal extension of PK2 implicates a role for the adjacent EKCM domain in binding and inhibiting PKR. Using a genetic screen in yeast, we isolated PK2-activating mutations that cluster to a surface of the EKCM domain that in bona fide protein kinases forms the catalytic cleft through sandwiching interactions with a kinase N-lobe. Interaction assays revealed that PK2 associates with the N- but not the C-lobe of PKRKD. We propose an inhibitory model whereby PK2 engages the N-lobe of an eIF2α kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. Finally, we show that PK2 enhances baculovirus fitness in insect hosts by targeting the endogenous insect heme-regulated inhibitor (HRI)–like eIF2α kinase. PMID:26216977

  19. Genome-wide analysis of lectin receptor-like kinases in Populus

    DOE PAGESBeta

    Yang, Yongil; Labbé, Jessy; Muchero, Wellington; Yang, Xiaohan; Jawdy, Sara S.; Kennedy, Megan; Johnson, Jenifer; Sreedasyam, Avinash; Schmutz, Jeremy; Tuskan, Gerald A.; et al

    2016-09-01

    Receptor-like kinases (RLKs) belong to a large protein family with over 600 members in Arabidopsis and over 1000 in rice. Among RLKs, the lectin receptor-like kinases (LecRLKs) possess a characteristic extracellular carbohydrate-binding lectin domain and play important roles in plant development and innate immunity. In addition, there are 75 and 173 LecRLKs in Arabidopsis and rice, respectively. However, little is known about LecRLKs in perennial woody plants.

  20. Targeting Axl and Mer kinases in cancer.

    PubMed

    Verma, Anupam; Warner, Steven L; Vankayalapati, Hariprasad; Bearss, David J; Sharma, Sunil

    2011-10-01

    Receptor tyrosine kinases (RTK) are cell-surface transmembrane receptors that contain regulated kinase activity within their cytoplasmic domain and play an important role in signal transduction in both normal and malignant cells. The mammalian TAM RTK family includes 3 closely related members: Tyro-3, Axl, and Mer. Overexpression or ectopic expression of the TAM receptors has been detected in a wide array of human cancers. Growth arrest-specific gene 6 has been identified as the major ligand for these TAM RTKs, and its binding to the receptors has been shown to promote proliferation and survival of cancer cells in vitro. Abnormal expression and activation of Axl or Mer can provide a survival advantage for certain cancer cells. Inhibition of Axl and Mer may enhance the sensitivity of cancer cells to cytotoxic agents and would potentially be a therapeutic strategy to target cancer cells. This review elucidates the role of Axl and Mer in normal cellular function and their role in oncogenesis. In addition, we review the potential to inhibit these RTKs for the development of therapeutic targets in treatment of cancer. PMID:21933973

  1. Targeting the Pim kinases in multiple myeloma

    PubMed Central

    Keane, N A; Reidy, M; Natoni, A; Raab, M S; O'Dwyer, M

    2015-01-01

    Multiple myeloma (MM) is a plasma cell malignancy that remains incurable. Novel treatment strategies to improve survival are urgently required. The Pims are a small family of serine/threonine kinases with increased expression across the hematological malignancies. Pim-2 shows highest expression in MM and constitutes a promising therapeutic target. It is upregulated by the bone marrow microenvironment to mediate proliferation and promote MM survival. Pim-2 also has a key role in the bone destruction typically seen in MM. Additional putative roles of the Pim kinases in MM include trafficking of malignant cells, promoting oncogenic signaling in the hypoxic bone marrow microenvironment and mediating resistance to therapy. A number of Pim inhibitors are now under development with lead compounds entering the clinic. The ATP-competitive Pim inhibitor LGH447 has recently been reported to have single agent activity in MM. It is anticipated that Pim inhibition will be of clinical benefit in combination with standard treatments and/or with novel drugs targeting other survival pathways in MM. PMID:26186558

  2. Neuronal migration and protein kinases

    PubMed Central

    Ohshima, Toshio

    2015-01-01

    The formation of the six-layered structure of the mammalian cortex via the inside-out pattern of neuronal migration is fundamental to neocortical functions. Extracellular cues such as Reelin induce intracellular signaling cascades through the protein phosphorylation. Migrating neurons also have intrinsic machineries to regulate cytoskeletal proteins and adhesion properties. Protein phosphorylation regulates these processes. Moreover, the balance between phosphorylation and dephosphorylation is modified by extracellular cues. Multipolar-bipolar transition, radial glia-guided locomotion and terminal translocation are critical steps of radial migration of cortical pyramidal neurons. Protein kinases such as Cyclin-dependent kinase 5 (Cdk5) and c-Jun N-terminal kinases (JNKs) involve these steps. In this review, I shall give an overview the roles of protein kinases in neuronal migration. PMID:25628530

  3. Protein Crystals of Raf Kinase

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This image shows crystals of the protein raf kinase grown on Earth (photo a) and on USML-2 (photo b). The space-grown crystals are an order of magnitude larger. Principal Investigator: Dan Carter of New Century Pharmaceuticals

  4. Isolation of chloroplastic phosphoglycerate kinase

    SciTech Connect

    Macioszek, J.; Anderson, L.E. ); Anderson, J.B. )

    1990-09-01

    We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts. Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast.

  5. The crystal structure of the catalytic domain of the ser/thr kinase PknA from M. tuberculosis shows an Src-like autoinhibited conformation.

    PubMed

    Wagner, Tristan; Alexandre, Matthieu; Duran, Rosario; Barilone, Nathalie; Wehenkel, Annemarie; Alzari, Pedro M; Bellinzoni, Marco

    2015-05-01

    Signal transduction mediated by Ser/Thr phosphorylation in Mycobacterium tuberculosis has been intensively studied in the last years, as its genome harbors eleven genes coding for eukaryotic-like Ser/Thr kinases. Here we describe the crystal structure and the autophosphorylation sites of the catalytic domain of PknA, one of two protein kinases essential for pathogen's survival. The structure of the ligand-free kinase domain shows an auto-inhibited conformation similar to that observed in human Tyr kinases of the Src-family. These results reinforce the high conservation of structural hallmarks and regulation mechanisms between prokaryotic and eukaryotic protein kinases. PMID:25586004

  6. Stimulus-Specific Distinctions in Spatial and Temporal Dynamics of Stress-Activated Protein Kinase Kinase Kinases Revealed by a Fluorescence Resonance Energy Transfer Biosensor▿

    PubMed Central

    Tomida, Taichiro; Takekawa, Mutsuhiro; O'Grady, Pauline; Saito, Haruo

    2009-01-01

    The stress-activated protein kinases (SAPKs), namely, p38 and JNK, are members of the mitogen-activated protein kinase family and are important determinants of cell fate when cells are exposed to environmental stresses such as UV and osmostress. SAPKs are activated by SAPK kinases (SAP2Ks), which are in turn activated by various SAP2K kinases (SAP3Ks). Because conventional methods, such as immunoblotting using phospho-specific antibodies, measure the average activity of SAP3Ks in a cell population, the intracellular dynamics of SAP3K activity are largely unknown. Here, we developed a reporter of SAP3K activity toward the MKK6 SAP2K, based on fluorescence resonance energy transfer, that can uncover the dynamic behavior of SAP3K activation in cells. Using this reporter, we demonstrated that SAP3K activation occurs either synchronously or asynchronously among a cell population and in different cellular compartments in single cells, depending on the type of stress applied. In particular, SAP3Ks are activated by epidermal growth factor and osmostress on the plasma membrane, by anisomycin and UV in the cytoplasm, and by etoposide in the nucleus. These observations revealed previously unknown heterogeneity in SAPK responses and supplied answers to the question of the cellular location in which various stresses induce stimulus-specific SAPK responses. PMID:19737916

  7. Family Support.

    ERIC Educational Resources Information Center

    Wieck, Colleen, Ed.; McBride, Marijo, Ed.

    1990-01-01

    This "Feature Issue" of the quarterly journal "Impact" presents 19 brief articles on family support systems in the United States for persons with developmental disabilities and their families. Emphasis is on provisions of Public Law 99-457. Articles include: "Family Support in the United States: Setting a Course for the 1990s" (James Knoll);…

  8. Family Matters.

    ERIC Educational Resources Information Center

    Mainor, Peggy

    2001-01-01

    Describes a Kellogg Family Collaborative project that involves the University of Montana and four tribal colleges in a family-strengths approach to improving student retention and achievement. States that the project is grounded in social work theory and research that recognize and reinforce family and student resilience through promotion of…

  9. Rural Families.

    ERIC Educational Resources Information Center

    Goetz, Kathy, Ed.

    1992-01-01

    This "special focus" journal issue consists of 13 individual articles on the theme of rural family programs relating to school, health services, church, and other institutions. It includes: (1) "Towards a Rural Family Policy" (Judith K. Chynoweth and Michael D. Campbell); (2) "Montana: Council for Families Collaborates for Prevention (Jean…

  10. Tyrosine kinase gene rearrangements in epithelial malignancies

    PubMed Central

    Shaw, Alice T.; Hsu, Peggy P.; Awad, Mark M.; Engelman, Jeffrey A.

    2014-01-01

    Chromosomal rearrangements that lead to oncogenic kinase activation are observed in many epithelial cancers. These cancers express activated fusion kinases that drive the initiation and progression of malignancy, and often have a considerable response to small-molecule kinase inhibitors, which validates these fusion kinases as ‘druggable’ targets. In this Review, we examine the aetiologic, pathogenic and clinical features that are associated with cancers harbouring oncogenic fusion kinases, including anaplastic lymphoma kinase (ALK), ROS1 and RET. We discuss the clinical outcomes with targeted therapies and explore strategies to discover additional kinases that are activated by chromosomal rearrangements in solid tumours. PMID:24132104

  11. Identifying Human Kinase-Specific Protein Phosphorylation Sites by Integrating Heterogeneous Information from Various Sources

    PubMed Central

    Li, Tingting; Du, Pufeng; Xu, Nanfang

    2010-01-01

    Phosphorylation is an important type of protein post-translational modification. Identification of possible phosphorylation sites of a protein is important for understanding its functions. Unbiased screening for phosphorylation sites by in vitro or in vivo experiments is time consuming and expensive; in silico prediction can provide functional candidates and help narrow down the experimental efforts. Most of the existing prediction algorithms take only the polypeptide sequence around the phosphorylation sites into consideration. However, protein phosphorylation is a very complex biological process in vivo. The polypeptide sequences around the potential sites are not sufficient to determine the phosphorylation status of those residues. In the current work, we integrated various data sources such as protein functional domains, protein subcellular location and protein-protein interactions, along with the polypeptide sequences to predict protein phosphorylation sites. The heterogeneous information significantly boosted the prediction accuracy for some kinase families. To demonstrate potential application of our method, we scanned a set of human proteins and predicted putative phosphorylation sites for Cyclin-dependent kinases, Casein kinase 2, Glycogen synthase kinase 3, Mitogen-activated protein kinases, protein kinase A, and protein kinase C families (avaiable at http://cmbi.bjmu.edu.cn/huphospho). The predicted phosphorylation sites can serve as candidates for further experimental validation. Our strategy may also be applicable for the in silico identification of other post-translational modification substrates. PMID:21085571

  12. Acetylation of cyclin-dependent kinase 5 is mediated by GCN5

    SciTech Connect

    Lee, Juhyung; Yun, Nuri; Kim, Chiho; Song, Min-Young; Park, Kang-Sik; Oh, Young J.

    2014-04-25

    Highlights: • Cyclin-dependent kinase 5 (CDK5) is present as an acetylated form. • CDK5 is acetylated by GCN5. • CDK5’s acetylation site is mapped at Lys33. • Its acetylation may affect CDK5’s kinase activity. - Abstract: Cyclin-dependent kinase 5 (CDK5), a member of atypical serine/threonine cyclin-dependent kinase family, plays a crucial role in pathophysiology of neurodegenerative disorders. Its kinase activity and substrate specificity are regulated by several independent pathways including binding with its activator, phosphorylation and S-nitrosylation. In the present study, we report that acetylation of CDK5 comprises an additional posttranslational modification within the cells. Among many candidates, we confirmed that its acetylation is enhanced by GCN5, a member of the GCN5-related N-acetyl-transferase family of histone acetyltransferase. Co-immunoprecipitation assay and fluorescent localization study indicated that GCN5 physically interacts with CDK5 and they are co-localized at the specific nuclear foci. Furthermore, liquid chromatography in conjunction with a mass spectrometry indicated that CDK5 is acetylated at Lys33 residue of ATP binding domain. Considering this lysine site is conserved among a wide range of species and other related cyclin-dependent kinases, therefore, we speculate that acetylation may alter the kinase activity of CDK5 via affecting efficacy of ATP coordination.

  13. Differential regulation of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic dual-specificity phosphatase.

    PubMed Central

    Groom, L A; Sneddon, A A; Alessi, D R; Dowd, S; Keyse, S M

    1996-01-01

    The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location. Images PMID:8670865

  14. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    NASA Astrophysics Data System (ADS)

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-03-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx.

  15. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates.

    PubMed

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-01-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx. PMID:24681538

  16. Abl Kinases Regulate HGF/Met Signaling Required for Epithelial Cell Scattering, Tubulogenesis and Motility

    PubMed Central

    Li, Ran; Knight, Jennifer F.; Park, Morag; Pendergast, Ann Marie

    2015-01-01

    Tight regulation of receptor tyrosine kinases (RTKs) is crucial for normal development and homeostasis. Dysregulation of RTKs signaling is associated with diverse pathological conditions including cancer. The Met RTK is the receptor for hepatocyte growth factor (HGF) and is dysregulated in numerous human tumors. Here we show that Abl family of non-receptor tyrosine kinases, comprised of Abl (ABL1) and Arg (ABL2), are activated downstream of the Met receptor, and that inhibition of Abl kinases dramatically suppresses HGF-induced cell scattering and tubulogenesis. We uncover a critical role for Abl kinases in the regulation of HGF/Met-dependent RhoA activation and RhoA-mediated actomyosin contractility and actin cytoskeleton remodeling in epithelial cells. Moreover, treatment of breast cancer cells with Abl inhibitors markedly decreases Met-driven cell migration and invasion. Notably, expression of a transforming mutant of the Met receptor in the mouse mammary epithelium results in hyper-activation of both Abl and Arg kinases. Together these data demonstrate that Abl kinases link Met activation to Rho signaling and Abl kinases are required for Met-dependent cell scattering, tubulogenesis, migration, and invasion. Thus, inhibition of Abl kinases might be exploited for the treatment of cancers driven by hyperactivation of HGF/Met signaling. PMID:25946048

  17. Citron kinase enhances ubiquitination of HIV-1 Gag protein and intracellular HIV-1 budding.

    PubMed

    Ding, Jiwei; Zhao, Jianyuan; Sun, Lei; Mi, Zeyun; Cen, Shan

    2016-09-01

    Assembly and budding of human immunodeficiency virus type 1 (HIV-1) particles is a complex process involving a number of host proteins. We have previously reported that the RhoA effector citron kinase enhances HIV-1 production. However, the underlying mechanism is not clear. In this study, we found that citron kinase interacted with HIV-1 Gag protein via its zinc finger and leucine zipper domains. Electron microscopy analysis revealed that citron kinase induced viral particle assembly in multivesicular bodies (MVBs). Citron kinase enhanced ubiquitination of HIV-1 Gag protein. Knockdown of Nedd4L, a member of the HECT ubiquitin E3 ligase family, partly decreased the ability of citron kinase to enhance HIV-1 production and reduced ubiquitination of HIV-1 Gag. Interestingly, the function of citron kinase to promote HIV-1 budding was severely impaired when endogenous ALIX was knocked down. Overexpression of the AAA-type ATPase VPS4 eliminated citron-kinase-mediated enhancement of HIV-1 production. Our results suggest that citron kinase interacts with HIV-1 Gag and enhances HIV-1 production by promoting Gag ubiquitination and inducing viral release via the MVB pathway. PMID:27339686

  18. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    PubMed Central

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-01-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx. PMID:24681538

  19. TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo

    PubMed Central

    Prchal-Murphy, Michaela; Semper, Christian; Lassnig, Caroline; Wallner, Barbara; Gausterer, Christian; Teppner-Klymiuk, Ingeborg; Kobolak, Julianna; Müller, Simone; Kolbe, Thomas; Karaghiosoff, Marina; Dinnyés, Andras; Rülicke, Thomas; Leitner, Nicole R.; Strobl, Birgit; Müller, Mathias

    2012-01-01

    Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2K923E) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein’s stability. An inhibitory function was only observed upon over-expression of TYK2K923E in vitro. Tyk2K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors. PMID:22723949

  20. Purification, crystallization and preliminary X-ray diffraction analysis of the kinase domain of human tousled-like kinase 2

    SciTech Connect

    Garrote, Ana M.; Redondo, Pilar; Montoya, Guillermo; Muñoz, Inés G.

    2014-02-19

    The C-terminal kinase domain of TLK2 (a human tousled-like kinase) has been cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4{sub 1}22 and cubic P2{sub 1}3. Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in chromatin dynamics, including DNA replication and repair, transcription and chromosome segregation. The two members of the family reported in humans, namely TLK1 and TLK2, localize to the cell nucleus and are capable of forming homo- or hetero-oligomers by themselves. To characterize the role of TLK2, its C-terminal kinase domain was cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4{sub 1}22 and cubic P2{sub 1}3. The latter produced the best diffracting crystal (3.4 Å resolution using synchrotron radiation), with unit-cell parameters a = b = c = 126.05 Å, α = β = γ = 90°. The asymmetric unit contained one protein molecule, with a Matthews coefficient of 4.59 Å{sup 3} Da{sup −1} and a solvent content of 73.23%.

  1. Protein kinases in vascular smooth muscle tone--role in the pulmonary vasculature and hypoxic pulmonary vasoconstriction.

    PubMed

    Ward, Jeremy P T; Knock, Greg A; Snetkov, Vladimir A; Aaronson, Philip I

    2004-12-01

    Hypoxic pulmonary vasoconstriction (HPV) is an adaptive mechanism that in the normal animal diverts blood away from poorly ventilated areas of the lung, thereby maintaining optimal ventilation-perfusion matching. In global hypoxia however, such as in respiratory disease or at altitude, it causes detrimental increases in pulmonary vascular resistance and pulmonary artery (PA) pressure. The precise intracellular pathways and mechanisms underlying HPV remain unclear, although it is now recognised that both an elevation in smooth muscle intracellular [Ca2+] and a concomitant increase in Ca2+ sensitivity are involved. Several key intracellular protein kinases have been proposed as components of the signal transduction pathways leading to development of HPV, specifically Rho kinase, non-receptor tyrosine kinases (NRTK), p38 mitogen activated protein (MAP) kinase, and protein kinase C (PKC). All of these have been implicated to a greater or lesser extent in pathways leading to Ca2+ sensitisation, and in some cases regulation of intracellular [Ca2+] as well. In this article, we review the role of these key protein kinases in the regulation of vascular smooth muscle (VSM) constriction, applying what is known in the systemic circulation to the pulmonary circulation and HPV. We conclude that the strongest evidence for direct involvement of protein kinases in the mechanisms of HPV concerns a central role for Rho kinase in Ca2+ sensitisation, and a potential role for Src-family kinases in both modulation of Ca2+ entry via capacitative Ca2+ entry (CCE) and activation of Rho kinase, though others are likely to have indirect or modulatory influences. In addition, we speculate that Src family kinases may provide a central interface between the proposed hypoxia-induced generation of reactive oxygen species by mitochondria and both the elevation in intracellular [Ca2+] and Rho kinase mediated Ca2+ sensitisation. PMID:15556675

  2. Discovering the first tyrosine kinase

    PubMed Central

    Hunter, Tony

    2015-01-01

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month. PMID:26130799

  3. 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

  4. On the molecular mechanisms of mitotic kinase activation.

    PubMed

    Bayliss, Richard; Fry, Andrew; Haq, Tamanna; Yeoh, Sharon

    2012-11-01

    During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein-protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients. PMID:23226601

  5. On the molecular mechanisms of mitotic kinase activation

    PubMed Central

    Bayliss, Richard; Fry, Andrew; Haq, Tamanna; Yeoh, Sharon

    2012-01-01

    During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein–protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differen