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1
Haj, Slimane Ammar Zeineb. "Dynamique Spatiotemporelle de la protéine kinase AMPc dépendante dans les myocytes cardiaques". Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00954406.
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La protéine kinase AMPc-dépendante (PKA) joue un rôle crucial dans la régulation neurohormonale de la fonction cardiaque. L'activation aiguë de la PKA est bénéfique car elle conduit à une augmentation de la contraction cardiaque en phosphorylant les acteurs clés du couplage excitation-contraction. En revanche, son activation chronique est délétère et ces effets semblent faire intervenir la régulation de protéines nucléaires pouvant conduire au remodelage hypertrophique et à l'insuffisance cardiaque. La localisation subcellulaire de la PKA, assurée par des protéines d'ancrage (AKAPs), est importante pour la rapidité et la spécificité d'action des hormones mettant en jeu la voie de l'AMPc. Les niveaux d'AMPc sont régulés par l'activité des adénylate cyclases et des phosphodiestérases (PDEs), et l'état de phosphorylation des protéines cibles de la PKA dépend de l'activité des Ser/Thr phosphatases (PPs). Dans le cœur, les PDEs les plus importantes dégradant l'AMPc sont les PDE3 et les PDE4. Les principales PPs cardiaques sont PP1, PP2A et PP2B. Dans une première partie de mon travail, j'ai mis au point, dans les cardiomyocytes de rats adultes, une mesure de l'activité de la PKA en temps réel dans les compartiments cytoplasmiques et nucléaires. J'ai utilisé pour cela des sondes de type AKAR (A-kinase activity reporters) basées sur le transfert d'énergie de fluorescence (FRET) et localisées spécifiquement dans le noyau ou dans le cytoplasme par des séquences d'adressage ou d'exclusion nucléaires. J'ai ainsi pu montrer qu'une stimulation maintenue des récepteurs β-adrénergiques active la PKA de façon plus importante dans le cytoplasme que dans le noyau, et que cette activation se développe lentement au niveau nucléaire que dans le cytoplasme. De ce fait, une stimulation brève des récepteurs β-adrénergiques active maximalement la PKA dans le cytoplasme, mais de façon marginale dans le noyau. Dans une seconde partie de l'étude, je me suis intéressée au rôle des PDE3 et PDE4 ainsi qu'à celui de PP1, PP2A et PP2B dans la régulation de l'activité PKA cytoplasmique et nucléaire, en réponse à une stimulation β-adrénergique. J'ai montré que la PDE4, mais pas la PDE3, régule l'activité de la PKA cytoplasmique et nucléaire. L'utilisation de souris invalidées pour les gènes Pde4b et Pde4d a révélé que l'isoforme PDE4B est prédominante pour la modulation de l'activité PKA cytoplasmique, alors que les deux isoformes PDE4B et PDE4D contribuent à la régulation de l'activité PKA nucléaire. Finalement, j'ai montré que la PP1 et la PP2A, mais pas la PP2B, participent à la terminaison des réponses β-adrénergiques dans le cytoplasme, alors qu'au niveau nucléaire, la PP1 semble jouer un rôle majeur. En conclusion, ce travail a mis en évidence le rôle des phosphodiestérases et des phosphatases dans l'intégration différentielle des réponses PKA à une stimulation β-adrénergique dans le cytoplasme et le noyau de cardiomyocytes adultes.
2
Galioot, Amandine. "Contribution à l'étude du rôle des SER/THR protéine-phosphatases PP1/PP2A dans les processus de mort cellulaire et de maturation du précurseur du peptide Beta-Amyloïde". Paris 7, 2013. http://www.theses.fr/2013PA077219.
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Notre laboratoire a précédemment proposé un concept appelé "Drug phosphatase Technology" (DPT) basé sur l'utilisation de séquences pénétrantes cationiques capables d'interagir avec les phosphatases PP1/PP2A pour déréguler spécifiquement des signaux intracellulaires. Les Ser. Thr protéine-phosphatases PP1 et PP2A sont des acteurs clé de la signalisation cellulaire et la dérégulation de leur activité via leur interaction avec des partenaires d'origine cellulaire ou virale conduit souvent à de graves dysfonctionnements. Par exemple, la protéine E4orf4 des adénovirus interagit avec PP2A1 et entraîne l'apoptose des cellules infectées ou transformées alors que les cellules saines ne sont pas affectées. Lors de cette étude nous avons identifié une séquence d'interaction entre E4orf4 et PP2A1 à partir de laquelle nous avons caractérisé le peptide DPT-E4orf44, qui provoque l'apoptose de certaines cellules tumorales humaines sans affecter les cellules saines de type fibroblastes. Les protéine-phosphatases de la famille PP1 et PP2A jouent également un rôle important dans la régulation physiologique des substrats neuronaux Tau et APP (précurseur de peptide amyloïde). La diminution de l'activité phosphatase dans les cellules nerveuses entraine une augmentation de l'état de phosphorylation de ces substrats chez les patients atteints de la maladie d'Alzheimer. La deuxième partie de ce travail de thèse a permis de mettre en évidence l'implication les protéine-phosphatases PP1 et PP2A dans la régulation de la phosphorylation du résidu T668 d'APP dont la modification post-traductionnelle est essentielle pour la maturation et le clivage de l'APPOuTlabhas previousry proposed a concept denominated "Drug Phosphatase Technology" (DFT) based on the use of cationic penetrating sequences capable of interacting with PP1/PP2A phosphatases in order to deregulate specific intracellular signais Ser/Thr proteins-phosphatases of PP1 and PP2A family are key factors of the cellular signalization and deregulation of their activity through interaction with cellular or viral protein often leads to severe dysfunctions. For exemple, E4orf4 protein of adenoviruses interacts with PP2A1 and leads to apoptotic death in infected or transformed cells while healthy cells remain unaffected. In a first part of this work, we have identified an interacting sequence between PP2A1 and E4orf4 from which we - have characterized the peptide DFT-F4orf44, which provokes the apoptosis of a subset of human tumoral cellswithout affecting healthy cells of fibroblastic type. Protein-phosphatases of PP1 and PP2A family also play a crucial role in the physiological regulation of neuronal substracts Tau and APP (Amylold Peptid Precursor). A diminution of the phosphatase activity in nerve cells leads to a drastically increasjng of phosphorylation state of these proteins for patients affected by Alzheimer's disease. I The second part of this work has allowed the identification of the phosphatase proteins responsible for the regulation of phosphorylation of APP T668 residue, which is an essential modification for APP maturation and processing
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Tan, Yves S. H. "Regulation of the type 1 protein phosphatase in saccharomyces cerevisiae". free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3013031.
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Rivera, Reyes Brenda Mariola. "Regulation of the TCR signaling pathway". Connect to text online, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1132588714.
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Mills, Elena Claire. "Characterisation of the trypanosomatid PPEF-like phosphatases : novel members of the RDGC/PP5-related protein phosphatase family". Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423156.
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Lee, Gui-in. "Structure and dynamics of the receptor kinase interacting FHA domain of kinase associated protein kinase from arabidopsis". Free to MU campus, others may purchase, 2003. http://wwwlib.umi.com/cr/mo/fullcit?p3100058.
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Conde, Rui Miguel Esteves Antunes Seabra. "Protein phosphatases acting on the replication checkpoint". Doctoral thesis, Universidade de Aveiro, 2010. http://hdl.handle.net/10773/3494.
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Doutoramento em BiologiaA génese de um cancro está dependente da acumulação de mutações genéticas que dão origem a instabilidade genómica, que por sua vez resulta na proliferação descontrolada. Para prevenir a acumulação destas mutações, as células têm mecanismos de controlo (checkpoints) que suspendem o ciclo celular e accionam as vias de reparação do ADN. Estes eventos são muitas vezes regulados por dinâmicas de (des)fosforilação de proteínas. As proteínas fosfatases (PPs), enzimas responsáveis pela remoção do grupo fosfato de resíduos fosforilados, desempenham funções cruciais na regulação de muitos mecanismos celulares. Enquanto que no início do projecto as cinases envolvidas no checkpoint da replicação estavam bem estabelecidas, as PPs envolvidas não eram conhecidas. A Chk1, um componente da maquinaria do checkpoint da replicação, é exemplo dessa regulação por (des)fosforilação, como sejam nos resíduos Ser317 e Ser345. Assim, como primeira abordagem para determinar quais os grupos de PPs envolvidos na regulação do checkpoint da replicação, decidimos investigar o seu papel na regulação da fosforilação da Chk1. A primeira conclusão é que a desfosforilação da Chk1 ao longo do tempo, tanto in vivo como in vitro, ocorre com uma dinâmica bi-fásica. Em segundo, a abordagem in vitro sugere que as famílias PP1, PP2A e PP2C estão envolvidas na desfosforilação da Chk1. Uma vez que a família PP2A foi a que mostrou a maior acção nesta reacção, decidimos investigar outros membros da família in vivo, primeiro com uma abordagem geral (tratando com OA ou sobreexpressando a PME-1), e depois com o knockdown específico da PP4 e PP6 (através de siRNA). Os resultados mostram que a inibição das PPs afectam tanto a desfosforilação como o estado de activação da Chk1 em resposta a tratamento com Hidroxiureia (HU). Todas as PPs testadas in vivo pareceram ser capazes de regular, a níveis diferentes, tanto a fosforilação como a desfosforilação da Chk1. A função das PPs foi também investigada ao nível: da regulação do disparo das origens de replicação, e da recuperação da suspensão da replicação, induzida pela HU. No último caso, os dados indicam que na situação simultânea de knockdown da PP4 com tratamento de HU, há um atraso do ciclo celular na resolução da transição de G2/M. No ensaio de replicação por pulse-chase, os resultamos mostram que tanto o tratamento com OA, como a sobre-expressão de I-2 ou PME-1, atrasam a cronologia do disparo programado das origens de replicação. No entanto, nenhum dos tratamentos efectuados parece desregular o início do checkpoint da replicação. Um rastreio de 2-híbrido de levedura com uma biblioteca de cDNA de testículo humano foi realizado, usando a Chk1 como isco, no sentido de descobrir novos interactores e definir novas possíveis funções para a Chk1 no contexto da meiose. Com base nos resultados do rastreio, duas novas funções são sugeridas: a interacção com a GAGE12 sugere uma função na recombinação genómica/vigilância do genoma durante a meiose, e as interacções com a EEF1α1 e a RPS5 sugerem uma função na regulação da síntese proteíca. Estas experiências fornecem um visão geral para a compreensão da diversidade de funções das proteínas fosfatases envolvidas no checkpoint da replicação, bem como, abre novos caminhos para o desenvolvimento de novas drogas para o tratamento do cancro.
The emergence of cancer is dependent on the accumulation of small numbers of genetic mutations that give rise to genomic instability, which in turn results in uncontrolled cell proliferation. To prevent the accumulation of such mutations and the evolution into a cancer state, cells operate checkpoints by arresting the cell cycle and triggering the DNA repair pathways. Often these events are regulated by protein (de)phosphorylation dynamics. Protein phosphatases (PPs), enzymes responsible for removing the phosphate from phosphorylated residues, play key roles in the regulation of many cellular mechanisms. While at the beginning of the project the kinases involved in the replication checkpoint were well established, PPs involved in the regulation of this pathway were not known. Chk1, a key component of the replication checkpoint machinery, is an example of regulation by (de)phosphorylation at several residues, such as Ser317 and Ser345. Hence, as a first approach to determine which PPs could be involved in the regulation of the replication checkpoint, we decided to investigate their role in the regulation of Chk1 phosphorylation state. The first finding from our study was that Chk1 dephosphorylation time course, both in vivo and in vitro, occurred with a bi-phasic dynamics. Secondly, a preliminary in vitro approach suggested that PP1, PP2A and PP2C families are involved in Chk1 dephosphorylation. Given that PP2A was shown to be the major contributor for this reaction, we investigated close family members in vivo, first by a general approach (either with OA treatment or PME-1 overexpression), and then by specific knockdown using siRNA for PP4 and PP6. Our data shows that besides affecting Chk1 dephosphorylation, the inhibition of PPs also affects Chk1 activation state in response to Hydroxyurea treatment. All the protein phosphatases tested in vivo seemed, to some extent, to be able to regulate either Chk1 phosphorylation or dephosphorylation. The PPs function was also investigated at the level of replication origin-firing regulation and the recovery from HU-induced replication arrest. In the latter study, our results indicate that PP4 knockdown triggers a HU-dependent cell cycle arrest in G2/M. In the pulse-chase replication assay, we show that OA treatment as well as I-2 or PME-1 overexpression, delay the timing of the normal replication origin-firing. None of the treatments performed seemed to abrogate the onset of the replication checkpoint. We have also performed a Yeast Two-hybrid screen in a human testis cDNA library using Chk1 as bait, in order to find novel interactors and identify new Chk1 putative functions in the context of meiosis. From this screen, two new Chk1 functions were defined due to the new putative interactions. Hence, the interaction with GAGE12 suggests a function in genomic recombination/ surveillance in meiosis and the interactions with both EEF1α1 and RPS5 suggest a function in protein synthesis regulation. These experiments provide a framework for understanding the diversity of the protein phosphatase functions involved in the replication checkpoint, as well as, for opening new avenues for the development of new drugs for cancer therapy.
FCT (POCI 2010) / FSE - SFRH/BD/11310/2002
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Luechapanichkul, Rinrada. "Determination of the Sequence Specificity and Protein Substrates of Protein Phosphatases". The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1398868380.
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Davis, Anthony John. "Characterization of the protein phosphatase 2A regulatory subunit PR70". Access to abstract only; dissertation is embargoed until after 12/19/2006, 2005. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=123.
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Bemporad, Francesco. "Folding and aggregation studies in the acylphosphatase-like family /". Firenze : Firenze University Press, 2009. http://digital.casalini.it/9788884539465.
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Bielinski, Vincent Anthony. "The role of protein phosphatases in regulation of Drosophila S6 by nutrient signaling pathways". Access to abstract only; dissertation is embargoed until after 5/15/2007, 2006. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=147.
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Esteves, Sara Luísa de Castro. "Characterization of human brain protein phosphatase 1a interacting proteins using the yeast two-hybrid system". Master's thesis, Universidade de Aveiro, 2008. http://hdl.handle.net/10773/917.
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Clark, O. R. "The role of protein tyrosine phosphatases in neuroblastoma". Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1335714/.
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Reversible phosphorylation of tyrosine residues on proteins is a cornerstone of cell to cell signalling, and is achieved through the concerted action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), which catalyse the addition and removal of phosphate moieties respectively. While the contribution of PTKs to oncogenesis and tumour maintenance has been well documented in a range of human cancers, knowledge of the specific role of PTPs has generally lagged behind. This thesis examines that role in neuroblastoma, a common and deadly sympathoadrenal tumour of infancy. We provide evidence that vanadium-‐based PTP inhibitors cause synergistic hyperactivation of Akt and Erk, which drives an irreversible differentiation and senescence program that is independent of p53, p16INK4A and PTEN. It is then shown that in another group of tumour cell lines, such compounds used alone can induce selective caspase-dependent, p53-independent apoptosis that is associated with activation of Akt and altered redox homeostasis. Lastly we examine the expression profile of the PTPome in a wide panel of neuroblastoma cell lines, providing preliminary evidence for candidate PTP oncogenes and tumour suppressors, as well as mediators of the effects of vanadium compounds.
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Wood, Steven Leslie. "The protein phosphatases acting on hormone-sensitive lipase". Thesis, University of Newcastle Upon Tyne, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282920.
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Ng, Wai Sun. "Multi-functions of the PP2A domain of axin /". View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?BICH%202004%20NGW.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004.Includes bibliographical references (leaves 82-89). Also available in electronic version. Access restricted to campus users.
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Dahche, Hanan Mohamad. "Dual-specific protein phosphatases in the Archaea". Diss., Virginia Tech, 2010. http://hdl.handle.net/10919/37625.
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Three distinct families of PTPs, the conventional (cPTPs), low molecular weight (LMW PTPs), and Cdc25 PTPs, have converged upon a common catalytic mechanism and active site sequence, mainly, the phosphate-binding loop encompassing the PTP signature motif (H/V)C(X)₅R(S/T) and an essential Asp residue on a surface loop. There is little sequence similarity among the three families of phosphatases. All known LMW PTP remove phosphoryl groups esterified to the hydroxyl amino acid: tyrosine, whereas all members of the Cdc25 family are dual-specificity protein phosphatases that dephosphorylate all the hydroxyl amino acids: tyrosine, serine and threonine. The cPTP family primarily functions as tyrosine phosphatases, but it also includes dual-specific members.
ORFs encoding potential cPTPs have been identified in five archaeal species: Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Thermococcus kodakaraensis, Pyrococcus horikoshii, and S. solfataricus. Only one has been partially characterized, Tk-PTP from T. kodakaraensis. Hence, our current body of knowledge concerning the functional properties and physiological roles of these enzymes remains fragmented.
The genome of S. solfataricus encodes a single conventional protein tyrosine phosphatase, SsoPTP. SsoPTP is the smallest known archaeal PTP (18.3 kDa) with a primary amino acid sequence that conforms to the cPTP protein tyrosine phosphatase paradigm, HCX₅R(S/T).
Relatively little is known about its mode of action " whether it follows the conventional PTP mechanism or employs a different route for catalysis " or its physiological role.
ORF sso2453 from the genome of Sulfolobus solfataricus, encoding a protein tyrosine phosphatase, was cloned and its recombinant protein product, SsoPTP, was expressed in E. coli and purified by immobilized metal affinity chromatography. SsoPTP displayed the ability to dephosphorylate protein-bound phosphotyrosine as well as protein-bound phosphoserine/phosphothreonine. SsoPTP hydrolyzed both isomers of naphthyl phosphate, an indication of dual specificity. The four conserved residues within the presumed active site sequence: Asp⁶⁹, His⁹⁵, and Arg¹⁰², and the invariant Gln¹³⁹ residue were essential for catalysis, as it was predicted for the established members of the PTP family in both bacteria and eukaryotes. A substrate trapping protein variant, SsoPTP-C96S/D69A, was constructed to isolate possible SsoPTP substrates present in S. solfataricus cell lysates. Several potential substrates were isolated and identified by mass spectroscopy.Ph. D.
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McLaren, Lorna J. "The mouse protein phosphatase inhibitor-1 gene". Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/24958.
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Intracellular proteins are phosphorylated by kinases and dephosphorylated by phosphatases. Protein phosphatase inhibitor-1 (I-1) inhibits protein phosphatase-1 (PP-1), thereby increasing the phosphorylated state of proteins in the cell. The initial aim of the work reported here was to determine whether I-1 is a kidney 'stem' cell marker (Svennilson et al., 1995); if so, it would be the first unique marker for these cells. The project involved characterizing the protein coding region of the mouse I-1 gene, determining the I-1 protein expression pattern in the developing mouse embryo and analysing potential promoter elements of the I-1 gene. A mouse genomic library was screened using a mouse cDNA clone homologous to the published rat I-1 mRNA and two types of clones with positive sequence homology to the rat mRNA sequence were isolated. These overlapping clones were sequenced, and analysis showed that the predicted mRNA and protein sequences contain 513 nucleotides and 171 amino acids, respectively, with both sharing over 95% homology with the known rat sequences. Further comparison of the predicted mouse protein I-1 sequence to those of rabbit, rat and human showed that there is strong homology across species, and that all share motifs which are important for inhibiting PP-1 (a KIQF sequence at amino acid positions 9-12 and a threonine at position 35). The mouse I-1 gene contains seven protein-coding exons which lie in a 7 kb region of DNA on chromosome 15, band F. Svennilson et al. (1995) detected I-1 expression in peripheral metanephric mesenchyme (nephron progenitor) cells in rate sections using RNA in situ hibridization. With this in mind, the mouse protein expression pattern was determined using wholemount tissue, a commercially available anti-I-1 antibody and fluorescent confocal microscopy. The results showed that I-1 is not a 'stem' cell marker in the developing kidney, but is restricted to the peripheral epithelial layer of cells of the kidney i.e. the mesothelium.
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Conn, Olga. "Functional studies on receptor-type protein tyrosine phosphatases of the R3 subgroup". Thesis, University of Westminster, 2017. https://westminsterresearch.westminster.ac.uk/item/q2w27/functional-studies-on-receptor-type-protein-tyrosine-phosphatases-of-the-r3-subgroup.
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The receptor-type protein tyrosine phosphatases (RPTPs) of the R3subgroup play key roles in the immune, vascular and nervous system. They are characterised by an extracellular domain (ECD), comprised of multiple FNIII-like repeats, a transmembrane domain and a single intracellular phosphatase domain. Although their phosphatase domains have been Fstudied in detail the functional roles of their extracellular regions have not been clearly defined. Potential roles in ligand interaction, dimerisation and cell-cell contacts have been reported. Here I used a bimolecular fluorescence complementation (BiFC) assay in live cells to examine the molecular basis for the interaction of one of the R3 RPTP members, VE-PTP, with VE-cadherin, and explored the potential of others to interact with this protein. The potential of R3 RPTPs to homo-dimerise via extracellular domains in live cells was also addressed. Quantitative BiFC analysis using sialophorin (SPN), an unrelated membrane protein, and a membrane anchored C-terminal Venus-YFP (Myr-VC) fragment as controls revealed a specific interaction between VE-PTP and VE-cadherin using constructs expressing only the extracellular and transmembrane domains. Use of a deletion mutant indicated that, in contrast to previous studies, removal of the 17th FNIII-like domain of VE-PTP is not sufficient to disrupt this interaction. Other members of the R3 RPTP family (DEP-1, GLEPP1 and SAP-1) also exhibited the potential to interact with VE-cadherin suggesting that specificity of this protein-protein interaction is not determined by the ECD alone. The direct interaction of DEP-1 with VE-cadherin is likely to be of physiological relevance since both proteins are expressed in endothelial cells. GLEPP1 and SAP-1 exhibited homo-dimerisation, whereas DEP-1 and VE-PTP did not form dimers via their extracellular and/or transmembrane domains. SPN was identified as a possible bona fide ligand for DEP-1 and their interaction is likely to be of physiological relevance since they were both shown to regulate T cell receptor activation. The interactions identified in the present study suggest a role for both the extracellular domain and transmembrane domain of R3-PTPs in interaction with VE-cadherin. The study also highlights the importance of using multiple controls in BiFC experiments and quantitative analysis of results.
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Luo, Jiexin 1968. "Regulation of the CFTR CI- channel by protein phosphatases". Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=30119.
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Activation of the cystic fibrosis transmembrane regulator (CFTR) chloride channel by protein kinases has been studied intensively. The mechanism of deactivation by protein phosphatases has received less attention, although it is equally important for physiological regulation of the channel. Characterization and identification of the phosphatases that regulate CFTR has become a priority in CF research because they are potential targets for pharmacotherapies.The first goal of this project was to characterize the effects of purified phosphatases on single CFTR channels. I found that PP2A, PP2C and alkaline phosphatase all reduced channel activity in excised patches. PP1 and PP2B did not deactivate CFTR, despite having comparable phosphatase activity when assayed biochemically using a standard substrate. Deactivation by exogenous PP2C closely resembled the spontaneous rundown induced by endogenous phosphatase in CHO, BHK, and T84 cells.
Genistein and bromotetramisole (Br-t) have been proposed to activate CFTR by inhibiting phosphatases. The second goal of this project was to assess the role of phosphatases in CFTR activation by these drugs. Genistein did not affect phosphatase activities. By contrast Br-t inhibited all four types phosphatases (PP1, PP2A, PP2B and PP2C). Thus Br-t may activate the channel by inhibiting its dephosphorylation whereas genistein probably acts directly on CFTR.
These studies provide functional evidence that PP2C is the predominant phosphatase regulating CFTR, and clarify the role of phosphatases in CFTR activation by genistein and bromotetramisole.
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Luo, Jiexin. "Regulation of the CFTR CI¢channel by protein phosphatases". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0017/MQ55077.pdf.
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Selner, Nicholas. "PROFILING THE INTRINSIC SEQUENCE SPECIFICITY OF PROTEIN TYROSINE PHOSPHATASES". The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1384269733.
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Mahmoud, Ali Ibrahim Hamouda Shima. "Protein kinases and phosphatases regulating the yeast proton pump". Doctoral thesis, Universitat Politècnica de València, 2015. http://hdl.handle.net/10251/54131.
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[EN] The plasma membrane H+-ATPase (Pma1) is essential for yeast growth and is activated by glucose metabolism by an unknown mechanism involving double phosphorylation of a regulatory site at the C-terminus (Ser911 Thr912). In this thesis we have investigated in Saccharomyces cerevisiae the role of two protein phosphatases, type 1 Glc7 and type 2A Sit4, and of an essential atypical protein kinase, TORC1, in the activation of Pma1 by glucose. The regulatory site of activated Pma1 can be dephosphorylated "in vitro" by recombinant Glc7 and Sit4, but inhibition "in vivo" of these phosphatases does not activate Pma1. Inhibition of Glc7 by regulated expression of a dominant-negative truncated form (the null mutant is not viable) had no effect on Pma1 activity while deletion of SIT4 gene decreased both Pma1 activity and double phosphorylation of the regulatory site. Inhibition of TORC1 protein kinase by treatment of yeast cells with the drug rapamycin or by exposure to non-permissive temperature of a temperature-sensitive mutant (tor1¿ tor2ts) inhibited Pma1 and decreased double phosphorylation of the regulatory site. We conclude that Sit4 and TORC1 are required for full activation of Pma1 by glucose while Glc7 either does not participate or is redundant with other phosphatases.[ES] La H+-ATPasa de la membrana plasmática (Pma1) es esencial para el crecimiento de la levadura y se activa por metabolismo de glucosa por un mecanismo desconocido que lleva consigo la doble fosforilación de un sitio regulador en el extremo C-terminal (Ser911 Thr912). En la presente tesis hemos investigado en Saccharomyces cerevisiae la participación de dos proteína fosfatasas, Glc7 de tipo 1 y Sit4 de tipo 2A, y de una proteína kinasa atípica esencial, TORC1, en la activación de Pma1 por glucosa. El sitio regulador de Pma1 en su estado activo puede defosforilarse "in vitro" por Glc7 y Sit4 recombinantes pero la inhibición "in vivo" de estas fosfatasas no activa Pma1. La inhibición de Glc7 mediante la expresión regulada de una forma truncada que actúa como dominante-negativa (el mutante nulo no es viable) no tiene efecto en la actividad de Pma1 mientras que la deleción del gen SIT4 disminuye tanto la actividad de Pma1 como la doble fosforilación del sitio regulador. Inhibición de la proteína kinasa TORC1 mediante tratamiento de las células de levadura con el fármaco rapamicina o exponiéndolas a temperatura no permisiva en el caso de un mutante termosensible (tor1¿ tor2ts) resulta en inhibición de Pma1 y disminución de la doble fosforilación del sitio regulador. Estos resultados indican que Sit4 y TORC1 son necesarias para la máxima activación de Pma1 por glucosa mientras que Glc7 podría no participar o hacerlo de forma redundante con otras fosfatasas.
[CAT] L'H+-ATPasa de la membrana plasmàtica (Pma1) és essencial per al creixement dels llevats i s'activa gràcies al metabolisme de glucosa per un mecanisme desconegut que porta associat la doble fosforilació d'una regió reguladora a l'extrem C-terminal (Ser911 Thr912). En aquesta tesi hem investigat en Saccharomyces cerevisiae la participació de dos proteïnes fosfatases, Glc7 de tipus 1 i Sit4 de tipus 2A, i d'una proteïna quinasa essencial atípica, TORC1, en l'activació de Pma1 per glucosa. La regió reguladora de Pma1, en seu estat activat, pot desfosforar-se "in vitro" per Glc7 i Sit4 recombinants, però la inhibició "in vivo" d'aquestes fosfatases no activa Pma1. La inhibició de Glc7 mitjançant l'expressió regulada d'una forma truncada que actua com a dominant-negativa (el mutant nul no és viable) no té cap efecte en l'activitat de Pma1 mentre que la deleció del gen SIT4 disminueix tant l'activitat de Pma1 com la doble fosforilació de la regió reguladora. La inhibició de la proteïna quinasa TORC mitjançant un tractament de cèl·lules de llevat amb el fàrmac rapamicina o la seua exposició a temperatures no permissives en el cas d'un mutant termosensible (tor1¿ tor2ts) resulta en la inhibició de Pma1 i la disminució de la doble fosforilació de la regió reguladora. Aquests resultats indiquen que Sit4 i TORC1 són necessàries per a l'activació màxima de Pma1 per glucosa, mentre que Glc7 podria no participar o fer-ho d'una forma redundant amb altres fosfatases.
Mahmoud Ali Ibrahim Hamouda, S. (2015). Protein kinases and phosphatases regulating the yeast proton pump [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/54131
TESIS
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Wadham, Carol. "Protein Tyrosine Phosphatase Pez : its role in the regulation of cell-cell adhesions". Title page, abstract and table of contents only, 2003. http://web4.library.adelaide.edu.au/theses/09PH/09phw122.pdf.
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"March 2003" Bibliography: leaves 206-233. The experimental data presented in this thesis provide evidence that PTP-Pez is an active phosphatase that interacts with and dephosphorylates the adherens junction protein ℓ-catenin. PTP-Pez also associates with proteins that form part of the tight junction complex, the scaffolding protein ZO-1 and the transmembrane protein occludin.
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Reetoo, Namrata. "Purification of protein phosphatases and 14-3-3-binding proteins and their regulation by the cAMP–PKA pathway". Thesis, University of Dundee, 2012. https://discovery.dundee.ac.uk/en/studentTheses/b725a974-2bcf-4af1-998e-69cb4dfe7d61.
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Currin, Andrew. "A study of protein phosphatases from the genomes of trypanosomatid parasites". Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/a-study-of-protein-phosphatases-from-the-genomes-of-trypanosomatid-parasites(6afd7092-b7e6-4816-903b-647d77942e95).html.
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Trypanosomiases and leishmaniases are amongst the world’s most neglected infectious diseases. Trypanosoma brucei, Trypanosoma cruzi and Leishmania major are the primary human pathogens of the trypanosomatidae family and the causative agents of African sleeping sickness, Chagas’ disease and cutaneous leishmaniasis, respectively. The molecular mechanism controlling each parasite’s life cycle and virulence is poorly understood however protein phosphatases are expected to play a critical role. This study presents the biochemical characterisation data of two groups of phosphatases from the trypanosomatids: the LRR-DSP and LM phosphatases. The LRR-DSPs are a group of twelve proteins, characterised by a unique domain architecture: a leucine-rich repeat (LRR) domain together with a dual- specificity phosphatase (DSP) catalytic domain. In this study, the recombinant expression of a representative LRR-DSP orthologue from T. brucei (TbLRR-DSP), proved to be highly problematic in E. coli. Most full-length and catalytic domain constructs were expressed at very low levels or as insoluble proteins. Soluble protein was obtained by denaturation, treatment with detergents, non-denaturing extraction from inclusion bodies and fusion to solubility-enhancing proteins. However, no method yielded protein with catalytic activity or detectable secondary structure. Soluble expression of TbLRR-DSP was achieved using baculovirus-infected insect cells, but the protein co-purified with endogenous chaperones and exhibited no catalytic activity thus implying a lack of correct folding. In the second part of this study, two phosphatases specific to Leishmania major, LM1 and LM2, were characterised and structural studies were initiated. LM2 was shown to readily hydrolyse phospho-tyrosine substrates in vitro, but not phosphoinositides like its homologue, LM1. Both proteins therefore have a differentiated catalytic profile and are likely to have different functions in vivo. Purification protocols for both proteins were established and crystallisation screenings set up. Preliminary hits were obtained for LM2 and a mutagenesis strategy was developed to improve chances of obtaining diffraction quality crystals. Recombinant LM1 samples exhibited heterogeneity and therefore will require additional engineering to improve chances of crystallization. Promising pilot NMR data was also obtained for both phosphatases. In conclusion, this study demonstrates that the recombinant expression of multi- domain trypanosomatid proteins (like the LRR-DSPs) can be highly problematic and may pose a challenge for their biochemical characterisation and functional elucidation. Future work into trypanosomatid phosphatases, however challenging, will improve our understanding of their cell biology and potentially identify therapeutic targets.
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Booth, Carmen Jane. "The role of protein tyrosine phosphatase receptor Q in development and disease /". Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/6330.
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Cho, Uhn-Soo. "Structural studies of protein phosphatase 2A, simian virus 40 small t antigen, and the PhoQ sensor domain /". Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/5677.
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Meiries, Sebastien. "Towards the synthesis of novel protein phosphatase inhibitors". Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/641/.
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Protein phosphatases (PPases) are important enzymes which mediate dephosphorylation of proteins in eukaryotic cells. These enzymes are involved in a variety of cellular processes, and disruption of their activity has been shown to be involved in the development of diseases such as cancers and Alzheimer’s. Protein phosphatase inhibitors (PPIs) have been used to regulate the activity of these enzymes, and hence, control the development of the cellular processes related to them. However, the lack of potent and selective readily accessible PPIs is a major obstacle to the understanding of these complex biological pathways, and the development of reliable synthetic routes towards new PPIs has therefore generated a significant amount of interest. Several naturally occurring PPIs possess the “ADDA” residue , which has been shown to be crucial for their inhibitory activity. We would like here to report the synthesis of “ADDA”-isoforms such as enantio-ADDA 1 and enantio-iso-ADDA 2 through a convergent approach taking advantage of cross-metathesis methodology, non-aldol aldol and aza-Claisen rearrangements, as well as β-lactam chemistry. We envisage using our synthetic approaches as a platform to generate a wide range of novel “ADDA”-containing analogues, which might lead to the discovery of potent and selective PPIs, which could be generated in multi-gram scale.
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Chen, Chun-Ti. "Regulation of the Cdc14-like Phosphatase CLP1 in Schizosaccharomyces pombe and Identification of SID2 Kinase Substrates: A Dissertation". eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/449.
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Coordination of mitosis and cytokinesis is crucial to generate healthy daughter cells with equal amounts of genetic and cytoplasmic materials. In the fission yeast Schizosaccharomyces pombe, an evolutionarily conserved Cdc14-like phosphatase (Clp1) functions to couple mitosis and cytokinesis by antagonizing CDK activity. The activity of Clp1 is thought to be regulated in part by its subcellular localization. It is sequestered in the nucleolus and the spindle pole body (SPB) during interphase. Upon mitotic entry, it is released into the cytoplasm and localized to the kinetochores, the actomyosin ring, and the mitotic spindle to carry out distinct functions. It is not clear how Clp1 is released from the nucleolus, however, once released, a conserved signaling pathway termed Septation Initiation Network (SIN) functions to retain Clp1 in the cytoplasm until completion of cytokinesis. The SIN and Clp1 function together in a positive feedback loop to promote each other’s activity. That is, the SIN promotes cytoplasmic retention of Clp1, and cytoplasmic Clp1 antagonizes CDK activity and reverses CDK inhibition on the SIN pathway to promote its function and activity. However, at the start of this thesis, the mechanism by which the SIN regulated Clp1 was unknown. The SIN pathway is also required to promote constriction of the actomyosin ring, and the septum formation. However, its downstream targets were still uncharacterized. In two separate studies, we studied how Clp1 is released from the nucleolus at mitotic entry and how the SIN kinase Sid2 acts to retain Clp1 in the cytoplasm. We identified several Sid2 candidate substrates, and revealed other functions of the SIN pathway in coordinating mitotic events.
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Roca, Suarez Armando Andres. "Expression of human protein phosphatases during chronic HCV infection and the development of hepatocellular carcinoma". Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ095.
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L'infection chronique par le virus de l'hépatite C (VHC) est un facteur étiologique majeur menant au développement de maladies hépatiques. Ce processus est favorisé par le VHC via l'altération des voies de signalisation impliquées dans l'inflammation chronique du foie. Etant donné que les voies de signalisation cellulaires sont notamment régulées par les protéines phosphatases, tout déséquilibre de leur activité peut entraîner des conséquences désastreuses pour la cellule. Dans ce contexte, les résultats obtenus lors de mon travail de doctorat ont démontré que l'infection par le VHC induit la diminution de la protéine phosphatase récepteur type delta (PTPRD), un suppresseur de tumeur impliqué dans le développement de plusieurs cancers humains. La fonction perturbée du PTPRD favorise l'activité du facteur de transcription STAT3 dans le foie des patients, entraînant la progression de la maladie et conduisant finalement au développement du carcinome hépatocellulaire (CHC). Mes résultats suggèrent qu'une évaluation plus approfondie des inhibiteurs de STAT3 pourrait conduire à de nouvelles stratégies chimio-préventives ciblant la formation du CHC chez les patients à risqueChronic hepatitis C virus (HCV) infection is a major etiological factor leading to liver disease development. This process is favored by HCV through the alteration of signaling pathways mediating chronic liver inflammation. Since signal transduction is tightly regulated by protein phosphatases, any imbalance in their activity can elicit dire consequences for the cell. In this context, the results obtained during my PhD studies demonstrated how HCV infection induces the downregulation of protein tyrosine phosphatase receptor type delta (PTPRD), a tumor suppressor implicated in the development several human cancers. This perturbed PTPRD function promotes STAT3 transcriptional activity in the liver of patients, driving disease progression and ultimately leading to the development of hepatocellular carcinoma (HCC). My results suggest that further evaluation of STAT3-inhibitors could lead to novel chemo-preventive strategies targeting HCC formation in patients at risk
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Pouliot, Philippe. "Implication of intracellular signalling pathways in allergic asthma pathogenesis". Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115896.
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The regulation of systemic immune responses is dependent on individual cell responses that will concur to induce a coherent response against a stimulus. In turn, cell response is dependent on the processing of intracellular signals generated at the cell membrane and transmitted through successive protein modifications to the nucleus in order to activate gene transcription. This is referred to as intracellular signalling. Tight control of these mechanisms is required to generate an appropriate cell response to environmental stimulations and globally to establish an appropriate immune response. Among protein modifications used to transmit a signal to the nucleus, protein tyrosine phosphorylation represents a pivotal method used by immune cells to rapidly induce signalling. While protein tyrosine kinases (PTKs) phosphorylate proteins, protein tyrosine phosphatases (PTPs) regulate the signalling by removing the phosphate group. The goal of this study was to better characterize intracellular signalling events involved in allergic asthma, a chronic inflammatory disease involving a Th2 immune response. In a first time, we investigated the role of PTPs in the development of asthma. We show that inhibition of global PTP activity in mice, during either the allergen sensitization or the allergen challenge phase, reduces asthma development and is linked to an increased Th1 response in the spleen and lung. Secondly, we revealed that TC-PTP inhibition reduces asthma development, while PTP-1B inhibition exacerbates inflammatory cells recruitment to the lung. Inhibition of either SHP-1 or PTP-PEST activity did not significantly modulate asthma development in our model. In a third set of experiments, we got interested in the signalling pathways triggered by the pro-inflammatory molecules myeloid-related proteins (MRPs) 8 and 14. MRPs are small cytosolic proteins recently described to have extracellular functions. MRP8 expression is resistant to corticosteroid treatment, and potentially promotes inflammation in corticosteroid-treated patients. We identified that MRPs induce signal through the action of TLR-4 and trigger the activation of MEK/ERK and JNK pathways that lead to NF-kappaB translocation. Collectively, our data provide a new characterization of signalling pathways engaged in allergic asthma. This should be helpful in the elaboration of new therapeutic approaches targeting precise pathways to inhibit mechanisms of inflammation.
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Qian, Yueping. "The roles of protein tyrosine phosphatases in the development of the neuromuscular junction /". View abstract or full-text, 2008. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202008%20QIAN.
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Chilton, John K. "The role of receptor protein tyrosine phosphatases in axon guidance". Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365814.
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Johnson, K. G. "Receptor protein tyrosine phosphatases in the developing Xenopus visual system". Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.605647.
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The aim of my research was to understand how RPTPs are involved in retinal ganglion cell (RGC) axon outgrowth and guidance in the development of the Xenopus visual system. I first describe the cloning and expression patterns of a variety of RPTPs in the developing Xenopus embryo, focusing on the retinotectal system. All three members of Type IIa RPTPs; LAR, PTP-δ, and CRYP-α, are expressed in RGCs during periods of differentiation, axonogenesis, and axon guidance from the retina to the tectum. These three RPTPs, as well as PTP-p, are expressed in overlapping but distinct patterns in the developing Xenopus embryo. Expression patterns of putative ligands for CRYP-α and LAR were examined using receptor affinity probe in situ hybridisation in the developing retina and brain, and putative ligands for CRYP-α were found along the optic pathway and in the tectum. These results demonstrate that Type IIa RPTPs and their putative ligands are expressed in a spatial and temporal pattern consistent with their involvement in RGC axon guidance and outgrowth from the retina to the tectum. I also describe functional studies examining the role of RPTPs in the developing Xenopus visual system both in vitro and in vivo. In vitro analysis of RGCs expressing dominant negative RPTPs demonstrates that dominant negative PTP-δ inhibits RGC axon outgrowth, while dominant negative CRYP-α promotes outgrowth, in a substrate-dependent manner. In vivo analysis of RGC axon outgrowth and guidance demonstrates that although dominant negative PTP-δ and LAR reduce the rate of axon outgrowth, none of these RPTPs appear to be involved in axon guidance.
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Fan, Wen Jun. "The role of protein phosphatases in myocardial ischaemia and reperfusion". Thesis, Stellenbosch : Stellenbosch University, 2008. http://hdl.handle.net/10019.1/21615.
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Thesis (MScMed)--Stellenbosch University, 2008.ENGLISH ABSTRACT: Protein kinases and phosphatases play important roles in the phosphorylation state of intracellular proteins under both physiologic and pathophysiologic conditions. Compared to the large number of studies investigating the significance of kinases, in particular the mitogen-activated protein kinases (MAPKs) in myocardial ischaemia/reperfusion and ischaemic preconditioning, relatively few studies have been done on the protein phosphatases in this scenario. Although several role players in the signal transduction cascade of ischaemia/reperfusion and ischaemic preconditioning have been identified thus far, the exact mechanism of cardioprotection still remains unclear. Previous studies from our laboratory have shown that the stress kinase, p38 MAPK, has a dual role in preconditioning: it acts as trigger of the process, while attenuation of its activation during sustained ischaemia and reperfusion is required for cardioprotection. Since the activation of p38 MAPK is dependent on both the upstream kinases for phosphorylation and phosphatases for dephosphorylation, we hypothesized that the balance between the activation state of the MAPKs and the induction of phosphatases may play a major role in determining the fate of cardiomyocytes exposed to ischaemic stress. The objectives of this study were: (i) to assess the activity of the myocardial protein phosphatases (PSPs and PP1) during sustained ischaemia and during reperfusion of non-preconditioned and ischaemic preconditioned hearts; (ii) to evaluate the significance of these phosphatases in ischaemia/reperfusion as well as in ischaemic preconditioning using available appropriate inhibitors; (iii) to give particular attention to the role of the phosphatase, mitogen-activated protein kinase phosphatase-1 (MKP-1), in ischaemia/reperfusion. MKP-1 is upregulated by stress conditions and selectively inactivates p38 MAPK by dephosphorylation of the regulatory Thr and Tyr residues. The glucocorticoid, dexamethasone which increases MKP-1 expression, was used as agonist to upregulate MKP-1 experimentally. The isolated perfused working rat heart was used as experimental model. After stabilization, hearts were subjected to either a one-cycle or multi-cycle ischaemic preconditioning protocol, followed by sustained global or regional ischaemia and reperfusion. Non-preconditioned hearts were subjected to ischaemia/reperfusion only. For Western blot analysis of MAPKs, PKB/Akt and MKP-1, hearts were freeze-clamped at different times during the perfusion protocol. Endpoints were infarct size, functional recovery and phosphorylation of the MAPKs (ERK and p38 MAPK) and PKB/Akt during reperfusion. Expression of MKP-1 was monitored. The results obtained showed that activation of PSPs and PP1 does not occur during sustained global ischaemia or reperfusion of non-preconditioned and preconditioned hearts. The role of the phosphatases was subsequently further investigated using two inhibitors namely cantharidin (5 μM, a concentration which inhibits both PP1 and PP2A) and okadaic acid (7.5 nM, a concentration which inhibits PP2A selectively). Administration of cantharidin or okadaic acid during the preconditioning phase, completely abolished preconditioning induced cardioprotection as indicated by mechanical failure during reperfusion and increased infarct size, associated with increased phosphorylation of p38 MAPK and PKB/Akt and dephosphorylation of ERK42/44. These results suggest a role for PP2A in the trigger phase of preconditioning. Administration of cantharidin or okadaic acid during early reperfusion of preconditioned hearts improved functional recovery. This was associated with increased phosphorylation of ERK42/44 and PKB, but not p38 MAPK. Dexamethasone, administered intraperitoneally to rats for 10 days (3mg/kg/day) or directly added to the perfusate (1 μM) resulted in significant cardioprotection of hearts subjected to 20 min sustained global ischaemia, followed by 30 min reperfusion. This is associated with a marked upregulation of MKP-1 and dephosphorylation of p38 MAPK during reperfusion. These studies suggest that the phosphatases are definitely involved in the phenomenon of ischaemia/reperfusion and ischaemic preconditioning. However, it also become clear that extensive further research is required to fully elucidate which phosphatases are involved and the mechanisms thereof. Due to the large size of the protein phosphatase family, this may prove to be a formidable task and far beyond the scope of this thesis. The results also suggested that pharmacological targetting of phosphatases involved in phosphorylation of the reperfusion injury salvage kinase (RISK) pathway (e.g. ERK42/44 and PKB/Akt) or dephosphorylation of pro-apoptotic kinases, such as p38 MAPK, may have significant clinical potential.
AFRIKAANSE OPSOMMING: Proteïenkinases en fosfatases speel 'n belangrike rol in die fosforileringstatus van intrasellulêre proteïene in beide fisiologiese en patofisiologiese toestande. In teenstelling met die groot aantal studies gedoen ten einde die rol van die kinases, veral die mitogeen-geaktiveerde proteïenkinases (MAPKs), in iskemie/herperfusie en iskemiese prekondisionering te ondersoek, is relatief min bekend aangaande die rol van die fosfatases in hierdie scenario. Hoewel verskeie rolspelers in die seintransduksieprosesse van iskemie/herperfusie en iskemiese prekondisionering reeds geïdentifiseer is, is die presiese meganisme van miokardiale beskerming steeds onbekend. Vroeëre studies vanuit ons laboratorium het getoon dat die streskinase, p38 MAPK, 'n tweeledige rol in prekondisionering speel: dit is 'n sneller ("trigger") van die proses, terwyl verlaagde aktivering tydens volgehoue iskemie en herperfusie, noodsaaklik vir beskerming is. Ons hipotese is dus dat die balans tussen die aktiveringstatus van die MAPKs en induksie van fosfatases die oorlewing van kardiomiosiete blootgestel aan iskemiese stres, bepaal. Die doelwitte van hierdie studie was: (1) bepaling van die aktiwiteit van miokardiale proteïen fosfatases (PSPs en PP1) tydens volgehoue iskemie en herperfusie van nie-geprekondisioneerde en iskemies-geprekondisioneerde harte; (ii) evaluering van die belang van fosfatases in iskemie/herperfusie beskadiging sowel as in iskemiese prekondisionering deur van geskikte inhibitore gebruik te maak; (iii) ondersoek na die rol van die fosfatase, mitogeen-geaktiveerde proteïen kinase fosfatase-1 (MPK-1) in iskemie/herperfusie beskadiging. Dit is bekend dat MKP-1 deur strestoestande opgereguleer word en p38 MAPK selektief deur defosforilasie van die regulatoriese Thr en Tyr residue inaktiveer word. Die glukokortikoïed, deksametasoon, wat MKP-1 uitdrukking stimuleer, is as agonis gebruik ten einde MKP-1 eksperimenteel op te reguleer. Die geïsoleerde, geperfuseerde werkende rothart is as eksperimentele model gebruik. Na stabilisasie, is die harte aan 'n enkel- of veelvuldige siklus iskemiese prekondisioneringsprotokol onderwerp, gevolg deur volgehoue globale of streeksiskemie. Nie-geprekondisioneerde harte is slegs aan iskemie/herperfusie onderwerp. Harte is op verskillende tye tydens die perfusieprotokol gevriesklamp vir Western blot analise van die MAPKs, PKB/Akt en MKP-1. Infarktgrootte en funksionele herstel tydens herperfusie is as indikators van iskemiese beskadiging gebruik. Fosforilasie van MAPKs en PKB/Akt sowel as uitdrukking van MKP-1 tydens vroeë herperfusie is gemonitor. Die resultate toon dat aktivering van PSP en PP1 tydens volgehoue iskemie en herperfusie nie plaasvind nie. Die rol van die fosfatases is verder ondersoek deur van twee inhibitore gebruik te maak, naamlik cantharidin (5 μM inhibeer beide PP1 en PP2A) en okadaic suur (7.5 nM inhibeer PP2A selektief). Toediening van of cantharidin of okadaic suur tydens die prekondisioneringsprotokol, hef prekondisionering-geïnduseerde beskerming totaal op, soos aangetoon deur hartversaking tydens herperfusie en 'n toename in infarktgrootte, tesame met 'n toename in die fosforilering van p38 MAPK en PKB/Akt en defosforilering van ERK42/44. Hierdie waarnemings dui op 'n rol vir PP2A as sneller in prekondisionering. Toediening van hierdie inhibitore tydens vroeë herperfusie het ook die miokardium beskerm, soos aangetoon deur 'n verbeterde meganiese herstel van geprekondisioneerde harte, tesame met ‘n verhoogde fosforilering van ERK42/44 en PKB (maar nie p38 MAPK nie). Deksametasoon, intraperitoneaal toegedien, vir 10 dae (3mg/kg/dag) of direk by die perfusaat gevoeg (1μM), het tot 'n hoogs beduidende beskerming teen iskemiese beskadiging gelei van harte blootgestel aan 20 min globale iskemie en 30 min herperfusie. Hierdie toename in funksionele herstel en afname in infarktgrootte het met 'n toename in MKP-1 uitdrukking en defosforilasie van p38 MAPK gepaard gegaan. Bogenoemde resultate dui op 'n definitiewe betrokkenheid van fosfatases in iskemie/herperfusie en iskemiese prekondisionering. Dit is egter ook duidelik dat intensiewe verdere navorsing benodig word om die presiese rol van die fosfatases te bepaal. Vanweë die grootte van die fosfatase familie, val dit egter buite die beskek van hierdie studie. Ten slotte, die resultate toon dat farmakologiese manipulasie van fosfatases betrokke by die fosforileringstatus van anti-apoptotiese kinases soos ERK42/44 en PKB/Akt en defosforilasie van pro-apoptotiese kinases, soos p38 MAPK, besondere kliniese toepassings mag hê.
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Smith, Anna Louise. "Molecular studies of protein tyrosine phosphatases in the human breast". Thesis, Institute of Cancer Research (University Of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243276.
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Koria, Muntaha. "Identification of PHPT1 in mouse tissues by immunohistochemistry". Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7745.
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Although it has been estimated that protein histidine phosphorylation account for about 6 % of the protein phosphorylation in eukaryotic cells; the knowledge of histidine phosphorylation and dephosphorylation is still limited. Lately, studies have appeared of a mammalian 14-kDa phospho- histidine phosphatase, also named protein histidine phosphatase and molecular cloning have provided some information of its physiological role. The object of the present study was to detect the protein expression of protein histidine phosphatase, PHPT1, in mouse tissue, by using immunohistochemistry. Tissue samples from a 4-week-old mouse (heart, liver, kidney, lung, muscle, and spleen), 5-month-old mouse (testis and intestinal), 8-month-old mouse (uterus) and an embryo from 14.5 days old mouse were obtained and processed for light microscopic examination. An absorption test was also made to confirm the specificity of the antibody. The results reveal that PHPT1 is mainly expressed in epithelium, heart- and skeletal muscle. These results provide new evidences for the understanding of the function of eukaryotic histidine phosphorylation and dephosphorylation.
KEYWORDS
Phosphohistidine, dephosphorylation, protein histidine phosphatase, phosphohistidine phosphatase, protein phosphorylation
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Jin, Lin. "The Bacterial AvrE-Family Type-III Effector Proteins Modulate Plant Immunity via Targeting Plant Protein Phosphatase 2A Complexes". The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1458339056.
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Shouse, Geoffrey P. "Characterization of the functional interaction between two tumor suppressors p53 and B56Gamma-PP2A /". Diss., UC access only, 2009. http://proquest.umi.com/pqdweb?index=31&did=1790085501&SrchMode=1&sid=2&Fmt=7&retrieveGroup=0&VType=PQD&VInst=PROD&RQT=309&VName=PQD&TS=1270138690&clientId=48051.
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Sacher, Michael G. (Michael Gershon). "The effect of protein phosphatase inhibitors on neurofilament assembly". Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=28904.
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Treatment of dissociated cultures of rat dorsal root ganglia with 1 $ mu$M okadaic acid (OA), a serine/threonine protein phosphatase inhibitor, caused a decrease in the electrophoretic mobilities of all three neurofilament (NF) subunits, indicating an increase in their phosphorylation levels. In addition OA treatment led to the asynchronous solubilization of the subunits in Triton X-100 (Triton). This fragmentation corresponded with striking changes in the immunofluorescent staining pattern of NFs. Short-term OA treatment was fully reversible within 10 hrs. upon removal of the inhibitor from the culture medium.Based on the response to varying concentrations of both OA and calyculin A, another serine/threonine protein phosphatase inhibitor, fragmentation of NFs was inferred to be due to the inhibition of protein phosphatase-2A activity while the reduction in electrophoretic mobility of subunits was due to the additional inhibition of protein phosphatase-1. The OA-induced phosphate moieties on the low molecular weight NF subunit (NF-L) were localized, by chemical cleavage analysis, to the amino-terminal head domain and were removed in vitro by the catalytic subunit of protein phosphatase-2A but not by the catalytic subunit of protein phosphatase-1.
Two-dimensional phosphopeptide mapping of the mid-sized NF subunit (NF-M) revealed that the OA-induced phosphate moieties on this subunit were also in the amino-terminal head domain. The Triton-soluble NF fragments were found, by immunoprecipitation under non-denaturing conditions, to be heteromers composed of NF-L/NF-M and NF-L/high molecular weight NF subunit. Similar heteromers were found in the small, naturally occurring Triton-soluble NF fraction from dorsal root ganglion cultures indicating that OA treatment amplifies a natural, dynamic process involving NF assembly/disassembly.
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Dawson, John Francis. "The regulation of protein phosphatase-1 by reversible protein phosphorylation and marine toxins". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ34753.pdf.
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Cox, Sarah Elizabeth. "Protein kinases and protein phosphatases in the central nervous system : identification, characterisation and functional correlates". Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405732.
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Humphries, A. C. "The total synthesis of the protein phosphatase inhibitor okadaic acid". Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604785.
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This thesis is divided into seven chapters. The first is a brief discussion of the structure, regulation and rôle of the protein phosphatases in biological systems, with particular emphasis on the important contribution made by the protein phosphatase inhibitor okadaic acid. The second chapter is a review of published synthetic work towards okadaic acid by other research groups. Chapter three begins with a brief review of some synthetic strategies towards the spiroketal unit. The general approach adopted within these laboratories for the total synthesis of okadaic acid is then described, with disconnections giving three distinct spiroketal units; followed by the synthesis of the central fragment. The fourth chapter describes the novel, intermolecular C-glycosylation of a 2-oxy substituted glycosyl sulfone. The application of this methodology to the introduction of a formyl unit into the anomeric position of the central spiroketal fragment is then described. Chapter five details a novel strategy towards the synthesis of the C15-C38 portion of okadaic acid, using the C-glycosylation methodology for the direct coupling of the C14-C26 and the C27-C38 fragments. It also contains a brief summary of the synthesis of the C27-C38 fragment from these laboratories. Chapter six begins with a review of the synthesis of the C1-C14 fragment from these laboratories. The coupling of the two advanced fragments is then detailed, together with the completion of the total synthesis.
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Trautmann, Susanne. "Functions of the Cdc14-Family Phosphatase Clp1p in the Cell Cycle Regulation of Schizosaccharomyces pombe: A Dissertation". eScholarship@UMMS, 2005. https://escholarship.umassmed.edu/gsbs_diss/10.
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In order to generate healthy daughter cells, nuclear division and cytokinesis need to be coordinated. Premature division of the cytoplasm in the absence of chromosome segregation or nuclear proliferation without cytokinesis might lead to aneuploidy and cancer.
The cyclin dependent kinases, CDKs, are a main regulator of the cell cycle. Timely increase and decrease in their activity is required for cell cycle progression. To enter mitosis, mitotic CDK activity needs to rise. CDK activity stays elevated until chromosome segregation is completed and exit from mitosis requires decrease in CDK activity.
Observations in several experimental systems suggest that coordination of cytokinesis with the nuclear cycle is regulated through CDK activity. Prolonged high CDK activity, as it occurs when chromosome segregation is delayed, was found to oppose cytokinesis. Prevention of cytokinesis through high CDK activity may therefore provide a mechanism to prevent precocious cell division in the absence of chromosome segregation. To prevent polyploidy when cell division is delayed, progression through the next nuclear cycle should be inhibited until cytokinesis is completed, presumably by the inhibition of CDK activity.
In the fission yeast Schizosaccharomyces pombe, a signaling cascade called Septation Initiation Network (SIN) is required for the coordination of cytokinesis with the nuclear cycle. The SIN is essential for cytokinesis, triggering the execution of cell division through constriction of the actomyosin ring. The activation of the SIN signaling cascade, and thus cytokinesis, is opposed by high CDK activity, preventing precocious cytokinesis.
S. pombe delay entry into the next nuclear division in response to delayed cytokinesis due to defects in the contractile ring until cytokinesis is completed thereby preventing the accumulation of multinucleate, non viable cells. This safeguard against multinucleate cells is termed the cytokinesis checkpoint. The cytokinesis checkpoint keeps CDK activity low, preventing nuclear cycle progression. The SIN is required for the cytokinesis checkpoint and therefore is a key coordinator between nuclear cycle and cytokinesis. How the SIN functions in the cytokinesis checkpoint was not known.
Cdc14-family phosphatases are highly conserved from yeast to humans, but were only characterized in Saccharomyces cerevisiae at the time this thesis was initiated. Cdc14 had been identified as the effector of a signaling cascade homologous to the SIN, called the mitotic exit network (MEN), which is required for exit from mitosis. This thesis describes the identification of the S. pombe Cdc14-like phosphatase Clp1p as a component of the cytokinesis checkpoint. Clp1p opposes CDK activity, and Clp1p and the SIN activate each other in a positive feedback loop. This maintains an active cytokinesis checkpoint and delays mitotic entry. We further found that Clp1p regulates chromosome segregation.
Concluding, this thesis describes discoveries adding to the characterization of the cytokinesis checkpoint and the function of Clp1p. While others found that Cdc14-family phosphatases, including Clp1p, have similar catalytic functions, we show that their biological function may be quite different between organisms, possibly due to different biological challenges.
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Lu, Kuojung Gordon. "Investigations into the role of protein phosphatases in the EGFR signaling network". Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45309.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.Includes bibliographical references (leaves 39-43).
Protein phosphatases regulate the phosphorylation state of intracellular signaling molecules in conjunction with protein kinases. In order to better understand the role of phosphatases in the ErbB signaling network, experimental studies were carried out to validate assays and phosphatase inhibitors for gathering dynamic, system-wide data of phosphatase activity. We verified the use of In-Cell Westerns and phospho-ErbB ELISAs for these studies, as well as the use of different cell lines and both general and specific phosphatase inhibitors in system-wide experiments. The phosphatase inhibitors we used perturbed cellular systems in complex ways that can help elucidate the regulation and activity of specific phosphatases in the ErbB signaling pathway in the future.
by Kuojung Gordon Lu.
S.M.
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Besco, Julie Ann. "Genomic structure and alternative splicing of type R2B receptor protein tyrosine phosphatases, and the role of RPTPrho". Columbus, Ohio : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1041353035.
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Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xvii, 227 p. Includes abstract and vita. Advisor: Andrej Rotter, Dept. of Pharmacology. Includes bibliographical references (p. 201-227).
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Zhao, Xiaotao. "The role of protein phosphatase signaling in the formation of the neuromuscular junction /". View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202004%20ZHAO.
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Sloss, Callum. "Control of subcellular distribution of the MAP kinase phosphatase, MKP-2". Thesis, University of Strathclyde, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288715.
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Riordan, Fiona Anne. "The role of protein kinases and protein phosphatase in the regulation of leukaemia cell apoptosis". Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286143.
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Joyce, Bradley Ryan. "The Regulation of Alkaline Phosphatase during the Development of Dictyostelium". Diss., Virginia Tech, 2006. http://hdl.handle.net/10919/27646.
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Regulation of gene expression is known to be a critical factor involved in proper development, responses to environmental cues, metabolism, energy conservation, and disease. Gene expression is regulated at several levels including transcription, mRNA splicing, post translational modification, and the rate of protein degradation. The developmental control of alkaline phosphatase (alp) in Dicytostelium has provided a focal point for the study of gene regulation at the level of de novo synthesis.
The localization of alkaline phosphatase (alp) expression during development was characterized by fusing the 5' flanking sequence to the lacZ reporter and using an in situ β-galactosidase staining method. The localization of lacZ expression corresponds with that of the endogenous ALP enzyme suggesting that alp is regulated at the level of transcription. In order to identify temporal regulatory elements within the alp promoter a series of 5' and internal promoter deletions were generated and fused to the lacZ reporter. The data from these promoter deletion constructs indicated a regulatory element within the -683 to -468 bp sequence that is required for normal expression of alp during development. A series of small internal and 5' promoter deletions were designed within the -683 to -468 bp regulatory sequence. The results from these promoter deletion-reporter gene fusions suggested a DNA regulatory element is located within a 26-bp sequence beginning at the -620 bp site.
The function of cis-acting regulatory elements were evaluated using the electromobility shift assay (EMSA) to identify sequence specific DNA-protein interactions on the alp promoter. We report the characterization of three DNA-binding activities with the 20% ammonium sulfate (AS) slug nuclear fraction. These DNA-binding activities appear to be related as they all require magnesium or calcium for effective binding to the alp promoter. Interestingly, the DNA-binding proteins appeared to interact with a GT-rich sequence that contained a G-box binding factor (GBF) consensus element.
Additionally, a DNA-binding activity observed in the 80% AS slug nuclear extract was characterized and sequentially purified using conventional and affinity chromatography techniques. The DNA-binding protein was identified as TFII, a protein that was previously identified during the investigation of glycogen phosphorylase-2 (gp2) regulation. A comparison of the alp and gp2 probes used to identify TFII suggests a DNA-binding site, ACAATGN₈₋₁₂CACTA. The ability of TFII to bind specifically with the promoter of two functionally different genes suggests that it may regulate the temporal and/or spatial expression of several Dictyostelium genes.Ph. D.