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Michel, Daniel R. "Cytoskeletal Architecture and Cell Motility Remain Unperturbed in Mouse Embryonic Fibroblasts from Plk3 Knockout Mice." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1446546516.
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Primot, Aline. "Etude de la régulation de deux protéines kinases : : GSK-3 et polo." Rennes 1, 2001. http://hal.upmc.fr/tel-01117984.
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Nous avons étudié 2 protéines kinases : GSK-3 et Polo. La "Glycogen Synthase Kinase-3" intervient dans divers processus cellulaires. Nous avons démontré que GSK-3 peut être purifiée par chromatographie d'affinité sur axine fixée sur des billes de séraphose. Nous avons cloné le gène d'une protéine homologue de GSK-3 humaine chez le parasite responsable du paludisme. PfGSK-3 est localisée au niveau de la membrane plasmique de l'érythrocyte et à l'intérieur du parasite. L'utilisation de PfGSK-3 comme cible devrait permettre la découverte d'inhibiteurs sélectifs dont les effets thérapeutiques potentiels seront évalués. Polo intervient à différents niveaux de la régulation cellulaire. Le clonage d'une partie de l'ADNc de Polo d'oursin (Plu1) a permis la synthèse d'anticorps spécifiques. L'immunoprécipitation de la protéine Plu1 au cours des premières divisions d'embryon d'oursin a révélé que Plu1 se lie à différentes protéines au cours du cycle dont l'identité reste à confirmer.
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Randall, Catherine Leah. "Genetic dissection of polo-like kinase 1's functions in human cell division /." Access full-text from WCMC, 2009. http://proquest.umi.com/pqdweb?did=1692357351&sid=1&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Long, Thavy. "Caractérisation structurale et régulation de l'activité de deux Polo-like kinases de Schistosoma mansoni : SmPlk1 et SmSak." Lille 2, 2010. http://www.theses.fr/2010LIL2S007.
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La schistosomiase est l'infection helminthique humaine la plus importante en termes de morbidité et de mortalité dans de nombreux pays. L'émergence récente de cas de résistances à la seule drogue efficace contre le schistosome, le praziquantel (PZQ) fait de cette maladie une priorité pour l'Organisation Mondiale de la Santé (OMS) de rechercher de nouvelles cibles thérapeutiques. La ponte massive des oeufs (jusqu'à 300 oeufs par jour par femelle) est responsable de la transmission de la maladie par des parasites complexes trématodes mais aussi de toute la pathologie de la schistosomiase. Mon travail de Doctorat vise avant tout à déchiffrer les mécanismes complexes de la reproduction des schistosomes et à élucider les voies de signalisation qui régulent cette hyperfécondité, dont les acteurs moléculaires peuvent être des cibles évidentes pour le contrôle de la schistosomiase. Les kinases Polo sont des membres conservés de la famille des Polo-like kinases (Plks) qui sont impliquées dans la progression du cycle cellulaire. Actuellement, les Plks humaines constituent une cible importante dans les traitements anticancéreux de par leur expression aberrante dans les tumeurs et signent d’ailleurs un mauvais pronostic. Sur la base de recherches in silico, nous avons caractérisé SmPlk1 (Schistosoma mansoni Plk1), homologue à la Plk1 humaine. SmPlk1 était principalement transcrit dans des stades de multiplication cellulaire intense et préférentiellement dans les organes reproducteurs des schistosomes adultes suggérant un rôle potentiel de SmPlk1 dans les processus de division. Nous avons montré que SmPlk1 possédait un rôle conservé dans la transition G2/M dans le modèle d’ovocyte de Xénope. L’inhibition dose-dépendante de l'ovogénèse et la spermatogénèse par le BI 2536, un inhibiteur spécifique des Plks, indiquait un rôle de SmPlk1 dans la multiplication et la différenciation des gamètes chez le parasite et suggérait que cette kinase pourrait être une nouvelle cible dans le traitement anti-schistosome. Parallèlement à ces travaux, une seconde Plk, SmSak, a été identifiée dont les résultats préliminaires ont montré une structure et des fonctions différentes de celles de SmPlk1 suggérant un rôle distinct de cette dernière. De plus, nous avons identifié un activateur potentiel de Plk, SmSLK (S. Mansoni Ste20-like kinase) capable d'activer spécifiquement SmPlk1 dans des conditions d'activation particulières dépendant de deux mécanismes originaux, l'un dépendant des caspases et l'autre dépendant d'ARN anti-sensSchistosomiasis is the most important helminthic infection in term of morbidity and mortality in many developing countries and represents the second parasitic disease to malaria. The evidence for praziquantel (PZQ) resistance, the only drug currently used to treat the disease, led the World Health Organization (WHO) to consider as a priority the finding of novel therapeutic targets. Egg production is responsible for disease transmission by complex trematodes parasites but also for the pathology of schistosomiasis. My PhD work contributes to a better understanding of the complex mechanisms that regulate schistosome reproduction. One particularity of schistosomes is that the sexual development of females requires a permanent contact with the male, allowing a level of fecundity exceptionally high. Therefore, the molecular mechanisms implied in this hyperfecundity are obvious targets for the control of schistosomiasis. Polo kinases are serine/threonine kinases, belonging to the Polo-like kinase family (Plks) whose members are conserved from yeast to mammals. During last years, human Plks have been extensively studied and considered as major targets for cancer because of their dramatic overexpression in proliferating cells and many tumors. In silico researches have led us to the characterization in S. Mansoni, SmPlk1, homologous to human Plk1. SmPlk1 was abundantly transcribed in parasite stages containing germinal cells expected to undergo frequent cell divisions, and particularly in the reproductive organs of adult worms suggesting a potential role of SmPlk1 in division processes. We have shown that SmPlk1 induced resumption of meiosis in oocytes of Xenopus. Moreover, the specific Plk1- inhibitor BI 2536 used in clinical trials, induces morphological aberrations in reproductive organs and inhibits oogenesis and spermatogenesis in paired worms, indicating a role of SmPlk1 in gamete multiplication and differentiation in S. Mansoni parasites and so the possibility that this kinase could be a novel potential target for schistosomiasis treatment. In parallel to this work, we recently identified a second Plk in S. Mansoni, SmSak different for its structure and its functions, and notably its role in the centriole duplication. Moreover, we identified a potential activator of Plk, SmSLK (S. Mansoni Ste-20 like kinase) able to activate specifically SmPlk1 in particular conditions. Indeed, two original mechanisms, one dependent on caspases and the other one dependent on antisense RNA, could regulate the kinase activity of SmSLK and therefore, the activity of SmPlk1
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MONTANI, FRANCESCA. "MOLECULAR MECHANISMS UNDERLYING CDC14 ACTIVATION DURING MITOTIC EXIT IN SACCHAROMYCES CEREVISIAE." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/214785.
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In budding yeast, progression through anaphase and exit from mitosis are controlled by the conserved protein phosphatase Cdc14. The activity of Cdc14 is regulated in space and time by changes in its subcellular localization. For most of the cell cycle up to metaphase, the phosphatase is sequestered in the nucleolus, by binding to a competitive inhibitor called Cfi1 (also known as Net1) (Shou et al., 1999; Straight et al., 1999; Visintin et al., 1999). During anaphase, Cdc14 is released from its inhibitor by the sequential activation of two signaling cascades, the Cdc Fourteen Early Anaphase Release (FEAR) network and the Mitotic Exit Network (MEN). Once released Cdc14 spreads throughout the nucleus and the cytoplasm, where it reaches its targets and promotes progression through and exit from mitosis (Pereira et al., 2002; Shou et al., 1999; Stegmeier et al., 2002; Visintin et al., 1999; Yoshida et al., 2002). Several in vivo and in vitro observations suggest that phosphorylation of Cdc14 and/or Cfi1 is responsible for the dissociation of Cdc14 from its inhibitor. Three kinases have been implicated in the process: the polo-like kinase Cdc5, the Clb2-Cdk complex and the MEN kinase Dbf2 (Azzam et al., 2004; Geymonat et al., 2003; Hu and Elledge, 2002; Hu et al., 2001; Mohl et al., 2009; Pereira et al., 2002; Queralt et al., 2006; Stegmeier et al., 2002; Visintin et al., 2003; Yoshida and Toh-e, 2002).
The aim of my project was to assess the contribution of the above-mentioned kinases and to identify the molecular mechanisms by which these kinases mediate the release of Cdc14 from its inhibitor. By modulating the kinases of interest alone or in mutual combination we found that Cdc14 is released from the nucleolus by the combined activity of two kinases, Cdc5 always and either Clb-Cdks or Dbf2. Once active, Cdc14 triggers a negative feedback loop that, in the presence of stable levels of mitotic cyclins, generates periodic cycles of Cdc14 release and sequestration. Similar phenotypes have been described for yeast bud formation and centrosome duplication. A common theme emerges where events that must happen only once per cycle, although intrinsically capable of oscillations, are limited to one occurrence by their coupling with the cyclin-Cdk cell cycle engine.
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Dahmene, Manel. "Caractérisation de la voie de dégradation de l'α-synucléine catalysée par la Polo-Like Kinase 2." Master's thesis, Université Laval, 2018. http://hdl.handle.net/20.500.11794/28320.
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La maladie de Parkinson est une maladie neurologique chronique caractérisée par la dégénérescence progressive des neurones dopaminergiques de la substance noire pars compacta. Une deuxième caractéristique neuropathologique de cette maladie est l’accumulation des agrégats intracellulaires appelés les corps de Lewy (CLs). Ces agrégats sont majoritairement formés par une protéine pré-synaptique, α-synucléine (α-syn). Cette accumulation pathologique interfère avec les voies métaboliques vitales des neurones telles que la transmission synaptique et l’activité mitochondriale, ce qui engendre la mort cellulaire. Par conséquence, éliminer les formes toxiques, diminuer l’expression de la forme native et réduire ainsi la probabilité de la formation d’agrégats pourrait être une stratégie thérapeutique d’intérêt pour le traitement de la maladie de Parkinson et d’autres désordres qui y sont reliés. Dans ce contexte, notre équipe a récemment décrit une nouvelle voie d’élimination de l’α-syn qui est catalysée par l’activité enzymatique de la kinase Polo-like kinase 2 (PLK2). Cependant, les mécanismes cellulaires ainsi que l’identité des molécules impliquées sont encore méconnus. Donc, mes travaux se sont concentrés sur l’étude de cette voie et ses différentes étapes qui mènent à enlever l’effet toxique de l’α-syn. Dans ce mémoire nous montrons que, en plus de la PLK2, la PLK3, un autre membre de la famille des PLKs, est capable de phosphoryler l’α-syn au niveau du résidu S129 et induire son élimination. En plus, nous déclarons que cette action exige une interaction physique entre les 2 protéines (α-syn et PLK2) impliquant le domaine N-terminal et qu’une étape de poly-ubiquitination est essentielle pour que ce complexe protéique se dirige vers la voie de dégradation autophagique. Cette action de la PLK2 est observée également sur des formes mutées de l’α-syn tels que α-syn A30P, α-syn A53T et d’une manière plus accentuée sur la forme mutante α-syn E46K. La caractérisation de cette voie d’élimination offre de nouvelles opportunités pour le développement des traitements qui favorisent, d’une façon spécifique et sélective, la dégradation de l’α-syn et par conséquent la réduction des formes toxiques de cette dernière.Parkinson's disease is a chronic neurological disease characterized by the progressive degeneration of the dopaminergic neurons of the substantia nigra pars compacta. A second neuropathological feature of this disease is the accumulation of intracellular aggregates called Lewy bodies. These aggregates are formed by a pre-synaptic protein, α-synuclein (α-syn). This pathological accumulation interferes with the vital metabolic pathways of neurons such as synaptic transmission and mitochondrial activity, leading to cell death. Consequently, promoting the elimination of the toxic forms, reducing the expression of the native form and decreasing the probability of aggregate formation could be a therapeutic strategy of interest for the treatment of Parkinson's disease and other related disorders. Recently, we have described a novel α-syn degradation pathway that is catalyzed by the enzymatic activity of Polo-like kinase 2 (PLK2). However, the cellular mechanism and the identity of the molecules involved are still unknown. So, my work has focused on studying this pathway and its various steps that lead to remove the toxic effect of α-syn. In this thesis we show that, in addition to PLK2, PLK3, another member of the PLK family, is able to phosphorylate α-syn at S129 and induce its elimination. In addition, we declare that this action requires a physical interaction between the 2 proteins (α-syn and PLK2) involving the N-terminal domain and that a poly-ubiquitination step is essential for the autophagic degradation of the α-syn and PLK2 complex. This effect of PLK2 is also observed on mutated forms of α-syn such as α-syn A30P, α-syn A53T and is more pronounced in the case of the α-syn E46K mutant. The characterization of this elimination pathway offers new opportunities for the development of treatments that allow, in a specific and selective manner, the degradation of α-syn and thus the reduction of its toxic forms.
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Pearson, John Robert. "Metazoan polo-like kinases : their conservation and function during the specialised cell cycles of oogenesis and early embryogenesis." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620065.
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Brassac, Thierry. "Analyse fonctionnelle de la kinase "Polo-like" lors de l'entrée et de la sortie de phase M des ovocytes de Xénope." Montpellier 2, 2000. http://www.theses.fr/2000MON20056.
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L'entree en mitose est declenchee par l'activation du mpf (m-phase promoting factor ou cdc2/cyclineb). Ce complexe est maintenu inactif (pre-mpf) jusqu'en phase g2 du cycle cellulaire par deux phosphorylations inhibitrices, catalysees par les kinases wee1/myt1 chez les eucaryotes superieurs. Lors de l'entree en phase m, la phosphatase cdc25c produit la conversion du pre-mpf en mpf en dephosphorylant les residus threonine 14 et tyrosine 15 de cdc2. Le mpf active lui-meme cdc25c par phosphorylation (boucle de retrocontrole positif). La serine/threonine kinase polo-like de xenope (plx1) fut purifiee par sa capacite a activer cdc25c in vitro (a. Kumagai et al, 1996). L'objet de ce travail etait de preciser les roles de plx1 dans le cycle de division cellulaire des ovocytes de xenope. Nous avons montre que l'activation de plx1 est necessaire a la mise en place de la boucle d'amplification du mpf. Cette activation de plx1 est placee sous le controle positif (probablement indirect) des complexes cdc2/cyclineb. Les processus moleculaires d'activation de cdc2/cyclinea et plx1 sont en revanche independants. Nous avons egalement etudie la fonction de plx1 dans la regulation d'un autre substrat de cdc2/cyclineb : l'anaphase promoting complex (apc). Ce complexe multimerique est responsable de l'ubiquitinylation des cyclines mitotiques qui conduit a leur degradation par le proteasome en fin de mitose. In vitro, il fut prealablement montre que cdc2 et plx1 regulent positivement l'apc (s. Kotani et al, 1999). Nos experiences montrent que plx1 n'est ni necessaire, ni suffisant pour activer l'apc dans les extraits de xenope. En revanche, plx1 prolonge l'activation de l'apc en antagonisant une phosphatase sensible a la microcystine. En presence de microcystine, la fonction de plx1 n'est plus requise pour obtenir la degradation complete des cyclines. Plx1 peut etre considere comme une kinase agissant en synergie avec cdc2 dans la regulation de substrats tels que cdc25c et l'apc.
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Rawal, C. "ROLE OF POLO KINASE CDC5 AND SLX4-RTT107 COMPLEX IN CHECKPOINT SIGNALING DURING DNA DAMAGE IN S. CEREVISIAE." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/335192.
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The integrity of genomic DNA is continuously jeopardized through of environmental stresses such as UV light, ionizing radiations and various chemicals in addition to cellular byproducts such as reactive oxygen species. Furthermore, structural or chemical hindrances also affect the basic cellular processes (replication, transcription and translation) compromising genome stability. All the eukaryotic cells have thus evolved mechanisms to detect such genomic lesions and activate a surveillance mechanisms termed as checkpoint activation to arrest cell cycle, which in term provide time to repair the lesion using a suitable pathway to maintain genome stability. The resumption of cell cycle after the repair is also an important and finely regulated mechanisms. Indeed, resumption of cell cycle in case of faulty/un-repaired damage compromises genome integrity and may lead to cancer.
In this thesis, I studied the role of Polo-kinase Cdc5 and DNA repair scaffold complex-Slx-Rtt107, specifically in response to one of the most deleterious lesion, DNA double strand break (DSB) in budding yeast Saccharomyces cerevisiae. The human counterpart Polo-like kinase 1 is overexpressed in many cancers, while Slx4/FANCP is one of the proteins involved in Fanconi anemia repair pathway.
In first part, we characterized the role of phosphorylation of Threonine 238 in the activation loop of the Cdc5 kinase domain in unperturbed cell cycle and in response to repairable and unrepairable DSB. Using alanine/ aspartic acid mutagenesis and genetic approaches we delineated the requirement of T238 phosphorylation of Cdc5. Interestingly, we discovered that absence of T238 phosphorylation of Cdc5, even though doesn’t affect the normal cell cycle, affects kinase activity and leads to defect in checkpoint adaptation and recovery after one DSB. Importantly, we also found that cdc5-T238A cells also have altered genome stability, assessed by using multiple genetic approaches.
In second part, we characterized the role of Slx4-Rtt107 complex in modulating the level of checkpoint signalling and initial processing of DSB. Indeed in the absence of functional Slx4-Rtt107 complex, we found slower processing of DSB and hyper-activated checkpoint signalling which is due to increased binding of checkpoint adaptor protein Rad9 at the lesion. Importantly, this hyper-activated checkpoint has consequent effect on cell cycle resumption and proliferation in response to various DNA damaging agents.
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Renner, Annelies. "Mise au point de tests "preuve de principe" pour l'étude des inhibiteurs de la Plk1 et caractérisation de la Plk1 en tant que cible dans le traitement des leucémies aigües myéloïdes." Toulouse 3, 2008. http://thesesups.ups-tlse.fr/430/.
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La Polo-like kinase1 (Plk1) est un régulateur mitotique majeur, surexprimé dans différents cancers et souvent associé à un mauvais pronostic. D'un point de vue expérimental, l'expression constitutive de la Plk1 dans des cellules NIH 3T3 entraîne leur transformation et leurs confère la capacité à former des tumeurs chez des souris athymiques. De plus, la déplétion de Plk1 dans des cellules cancéreuses induit souvent leur apoptose. Plk1 est donc une cible potentielle très étudiée dans le cadre des thérapies anti-cancéreuses. L'un des objectifs de ma thèse a été de générer des outils moléculaires et cellulaires afin de mettre au point des tests "preuve de principe" rendant compte de l'activité d'inhibiteurs de Plk1. La construction de ces outils avait également pour objectif de mieux appréhender le rôle de Plk1 dans les différentes étapes de la division cellulaire, et de rechercher de nouveaux substrats et partenaires de cette kinase. Des lignées cellulaires inductibles permettant l'expression de la Plk1 sauvage et de différents mutants (inactifs, suractivés) ont été construites. L'effet de l'induction de ces diverses formes de Plk1 sur la prolifération, la répartition des cellules dans le cycle et les substrats de la kinase a été étudié. Comme attendu, plus la protéine est active, plus l'effet sur ces paramètres est marqué. Divers inhibiteurs de Plk1 ont été testés sur ces modèles cellulaires avec, in fine, la mise au point de tests permettant d'évaluer rapidement et à haut débit l'efficacité de molécules inhibitrices. La technologie Luminex a été notamment mise à profit pour doser Plk1 et certains de ses substrats (phosphorylés ou non), représentatifs de l'activité de la kinase, à partir de lysats cellulaires. Cette approche permet d'évaluer l'effet d'inhibiteurs potentiels de Plk1 in cellulo par une série de dosages réalisés simultanément à partir d'extraits cellulaires. Les Leucémies Aiguës Myéloïdes (LAM) sont des hémopathies clonales liées à la transformation maligne d'une cellule souche hématopoïétique qui présente un blocage de maturation et une prolifération anarchique. Les mécanismes moléculaires impliqués dans la dérégulation du cycle cellulaire dans cette pathologie sont encore mal connus. L'un des objectifs de cette étude a été de caractériser le statut et le rôle de la Plk1 dans la physiopathologie des LAM, afin d'évaluer l'intérêt d'un traitement spécifique ciblant cette kinase dans cette pathologie. .Polo-like kinase 1, a major regulator of mitosis, is found overexpressed in many cancers and its overexpression correlates with a bad prognosis. Experimentally, the constitutive expression of Plk1 induces transformation of NIH3T3 cells and confer them the ability to initiate new tumours in athymic mice. In addition, Plk1 depletion in cancer cells induces apoptosis, suggesting that Plk1 may be a new pharmacological target in anticancer therapies. The first aim of my thesis was to generate molecular and cellular tools in order to characterize the activity of Plk1 inhibitors, evaluate the implication of Plk1 in different steps of cell division and to find new partners Plk1. We generated inducible cell lines allowing the expression of wild type and mutants' forms (inactivated, overactivated) of Plk1, and the analysis of their impact on growth, proliferation, cell cycle and also on known substrates of Plk1. As expected, the higher the activity of Plk1 is, the best its impact on these biological processes. We have tested on these inducible cell lines several inhibitors of Plk1, and developed an assay allowing a rapid and highly efficient evaluation of the effect of these inhibitors on the Plk1 pathway. The Luminex methodology was used on cell extracts in order to assess the activity of Plk1 on its downstream effectors. This methodology appears to be very efficient to analyse the effects of putative Plk1 inhibitors in cellulo. The second aim of my thesis was to analyse the status and role of Plk1 in a malignant hemopathy, Acute Myeloid Leukemia (AML). AML is a clonal hemopathy characterized by a block in differentiation and by an uncontrolled proliferation of immature leukemic cells. Molecular mechanisms involved in the dysregulation of cell cycle in AML are still poorly understood. We have characterized the expression and the role of Plk1 in AML cell lines and primary cells and analysed the consequences of a specific Plk1 inhibition in this pathology. .
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Martino, Lisa. "Rôle et régulation de la kinase PLK-1 lors de l'entrée en mitose dans l'embryon de Caenorhabditis elegans." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC225.
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Lors de la division cellulaire, une cellule mère doit dupliquer (interphase) puis ségréger son matériel génétique de façon égale entre les deux cellules filles (mitose). Entre ces deux étapes, la cellule subit une réorganisation drastique gouvernée par l’acteur majeur Cdk1-Cycline B, conduisant à l’entrée en mitose. L’activation de cette kinase est régulée par une boucle d’auto-amplification où les premières molécules de Cdk1-Cycline B stimulent l’activation des suivantes. Il a été montré que la kinase Plk1 initie cette boucle d’auto-amplification en stimulant les activateurs et en réprimant les inhibiteurs de Cdk1-Cycline B en amont. Pour que cette kinase soit totalement active, elle doit être elle-même activée par Aurora A, en présence de son co-activateur Bora. Il est crucial de comprendre comment tous ces acteurs se coordonnent dans l’espace et dans le temps pour déclencher l’entrée en mitose car un dérèglement pourrait amener à une ségrégation de l’ADN anarchique, conduisant à la formation de tumeurs et l’apparition de cancers. Au cours de ma thèse, j’ai tout d’abord contribué à la mise en évidence d’un mécanisme conservé d’activation de Plk1 dans les cellules humaines et chez C. elegans (PLK-1), impliquant le co-activateur Bora ou SPAT-1 chez C. elegans. Nous avons montré que la phosphorylation de SPAT-1 par Cdk1-Cycline B induit son interaction avec PLK-1, ce qui promeut la phosphorylation de PLK-1 par Aurora A et donc son activation in vitro. Ce mécanisme phospho-dépendant de SPAT-1 est important in vivo pour contrôler dans le temps l’entrée en mitose. De plus, l’activation de Plk1 in vitro avec les protéines humaines suggèrent fortement une conservation du mécanisme. Nous avons ensuite montré que la phosphorylation de Bora et de SPAT-1 par Cdk1 sur les résidus S41, S112, S137 et S119, S190, T229 respectivement, est nécessaire à leur interaction avec Plk1/PLK-1, déclenchant ensuite l’activation de Plk1/PLK-1 et l’entrée en mitose. Ces résultats démontrent que Bora/SPAT-1 phosphorylée fait partie de la boucle d’auto-amplification de Cdk1-Cycline B via l’activation de Plk1, permettant à terme d’activer de façon irréversible les acteurs de l’entrée en mitose. Par la suite, je me suis focalisée sur le rôle de PLK-1 dans la rupture de l’enveloppe nucléaire en utilisant l’embryon de C. elegans comme système modèle. Après avoir démontré que PLK-1 est cruciale pour la rupture de l’enveloppe nucléaire dans les embryons, j’ai observé une localisation de PLK-1 à l’enveloppe nucléaire avant sa rupture et j’ai identifié un complexe de nucléoporines impliqué dans ce processus. En effet, NPP-1, NPP-4 et NPP-11 dont la fonction est de réguler le transport nucléo-cytoplasmique, ont également un second rôle dans le recrutement de PLK-1 aux pores nucléaires. PLK-1 interagit avec ses substrats phosphorylés par deux types de mécanismes d’amorçage Plk1-dépendant et indépendant, impliquant une autre kinase en amont comme Cdk1-Cycline B par exemple. J’ai montré que le recrutement de PLK-1 aux pores dépend des deux mécanismes, nécessitant donc une coordination entre Cdk1-Cycline B et PLK-1. Une fois que PLK-1 est au centre du pore nucléaire, elle peut alors probablement phosphoryler de nombreuses nucléoporines et participer au désassemblage des pores, conduisant à la rupture de l’enveloppe nucléaireDuring cell division, a mother cell duplicates (interphase) and then segregate its genetic material equally between the two daughter cells (mitosis). Between these two stages, the cell undergoes a drastic reorganization governed by the major actor Cdk1-Cyclin B, leading to mitotic entry. The activation of this kinase is regulated by an auto-amplification loop where the first molecules of Cdk1-Cyclin B stimulate activation of the following. Plk1 kinase has been shown to initiate this self-amplification loop by stimulating activators and repressing upstream Cdk1-Cyclin B inhibitors. For this kinase to be fully active, it must itself be activated by Aurora A, in the presence of its coactivator Bora. It is crucial to understand how all these actors coordinate in space and time to trigger mitotic entry because a disruption could lead to a segregation of anarchic DNA, leading to the formation of tumors and the appearance of cancers. During my thesis, I first contributed to demonstrate a conserved mechanism of Plk1 activation in human cells and in C. elegans (PLK-1), involving the coactivator Bora or SPAT-1 in C. elegans. We have shown that the phosphorylation of SPAT-1 by Cdk1-Cyclin B induces its interaction with PLK-1, which promotes the phosphorylation of PLK-1 by Aurora A and thus its activation in vitro. This phosphory-dependent mechanism of SPAT-1 is important in vivo for controlling the entry into mitosis over time. In addition, activation of Plk1 in vitro with human proteins strongly suggests conservation of the mechanism. We then showed that the phosphorylation of Bora and SPAT-1 by Cdk1 on residues S41, S112, S137 and S119, S190, T229 respectively, is necessary for their interaction with Plk1 / PLK-1, then triggering the activation of Plk1 / PLK-1 and mitotic entry. These results demonstrate that phosphorylated Bora / SPAT-1 is part of the self-amplification loop of Cdk1-Cyclin B via the activation of Plk1, ultimately enabling irreversible activation of the actors of mitotic entry. Subsequently, I focused on the role of PLK-1 in nuclear envelope breakdown using the C. elegans early embryo as a model system. After demonstrating that PLK-1 is crucial for the nuclear envelope breakdown in embryos, I observed a localization of PLK-1 to the nuclear envelope before its rupture and I identified a nucleoporin complex involved in this process. Indeed, NPP-1, NPP-4 and NPP-11 whose function is to regulate nucleo-cytoplasmic transport, also have a second role in the recruitment of PLK-1 to nuclear pores. PLK-1 interacts with its phosphorylated substrates by two types of Plk1-dependent and independent priming mechanisms, involving another upstream kinase such as Cdk1-Cyclin B for example. I have shown that the recruitment of PLK-1 to the pores depends on both mechanisms, thus requiring coordination between Cdk1-Cyclin B and PLK-1. Once PLK-1 is at the center of the nuclear pore, it can probably phosphorylate many nucleoporins and participate in the disassembly of pores, leading to tnuclear envelope breakdown
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Stokes, Jamie Edward. "Small molecule approaches targeting the Polo-box domain of Polo-like kinase 1." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648778.
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Donaldson, M. M. "Multiple roles of polo kinase in Drosophila melanogaster." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598595.
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The polo gene was originally identified as a maternal effect mutation in Drosophila. Flies homozygous for the original polo1 allele display a variety of defects in cell division throughout development that include a failure to correctly assemble centrosomes in syncytial embryos, spindle defects in the larval, CNS, chromosomal non-disjunction and failures in cytokines during male meiosis. These phenotypes are consistent with roles for PLKs (Polo-like kinases) in the separation and maturation of centrosomes to form a bipolar spindle and in cytokinesis, as reported in studies of the homologous enzymes in yeasts, Xenopus and mammalian cells. Here I describe the phenotypes of two new alleles of polo, polo9 and polo10 discovered in a screen of a collection of P-element insertions on the third chromosome of Drosophila. These alleles have defects in spindle architecture and centrosome reflecting the involvement of Polo in organising the centrosomes and the bipolar spindle. They also have a severe mitotic arrest at metaphase, which coincides with high levels of cyclin B and the continued presence of components of the spindle checkpoint mechanism. This is consistent with a role for Polo in regulating some aspects of anaphase-promoting complex (APC) function - a role in which Polo-like kinases have been implicated in other organisms, but not previously in Drosophila. I also present evidence for a synergistic interaction between the polo gene and the makos gene (which encodes Cdc27, a component of the APC). Mutations in makos enhance some phenotypes seen in larval brain cells in polo mutants. Finally I present the initial data from two yeast two-hybrid screens that use the N-terminal and C-terminal halves of Polo as baits to identify interacting proteins. This screen has highlighted new interacting molecules that will help elucidate the roles of Polo kinase.
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Abraham, Anne. "Polo-like kinase interacting proteins in fission yeast." Thesis, University of Edinburgh, 2004. http://hdl.handle.net/1842/10699.
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Kms1 and Kms2 are important for integrity of the SPB. To find the biological significance of the interactions between Plo1 and these SPB proteins, attempts were made to disrupt the interaction by mutations. For this purpose, firstly regions responsible for the interaction were identified, and then mutations were made in the SPB proteins by random mutagenesis of this region. For Sid4, I isolated two point mutations, which had greatly weakened interaction with Plo1. To study the effect of disrupting the interaction in vivo, the two sid4 mutants were expressed from a fission yeast promoter in the sid4 temperature-sensitive mutant. Both point mutants of Sid4 that had weakened interaction with Plo1 were able to rescue the temperature-sensitive sid mutant with a similar strength as that of wild-type sid4 under the same promoter. The identification of potential Plo1 interacting proteins and mutants defective in these interactions will be an important step to understand cell cycle control by Plo1.
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Reindl, Wolfgang. "Inhibition of polo-like kinase 1 by small molecules targeting the polo-box domain." kostenfrei, 2008. http://mediatum2.ub.tum.de/doc/650458/650458.pdf.
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Nguyen, Annie, Vijay Gokhale, and Gregory Rogers. "Therapeutic Molecular Targeting of Polo-Like Kinase 4 for Cancer Treatment." The University of Arizona, 2015. http://hdl.handle.net/10150/614130.
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Class of 2015 AbstractObjectives: Two characterized peptide substrates were assayed with human Polo-like kinase 4 to determine phosphorylation activity. A pilot library of Type-II kinase inhibitors designed to fit into the ATP-binding pocket will be screened to determine HsPlk4 inhibition activity, which will help characterize a novel drug compound. Methods: Two peptide substrates of varying concentrations (2 uM, 1 uM, and 0.5 uM) were each combined with serial dilutions of HsPlk4 (1.25 uM, 0.625 uM, 0.313 uM, 0.156 uM, 0.078 uM, and 0.039 uM). EZ Reader detected phosphorylation activity by measuring fluorescence of both substrate and product, which separated at respective time points based on electrophoresis. The subsequent part of the experiment will be to inhibit the kinase activity with molecular inhibitors. Results: The results showed HsPlk4 activity with the modified PLKtide, (5FAM)KKKTPSDSLYDDGLSKK(CONH2). All reactions with the various concentrations of substrate 1 and HsPlk4 showed phosphorylation activity. The reaction started within the first 10 minutes, quickly reaching maximal phosphorylation of substrate. No p-values were calculated due to lack of data. Conclusions: No overall conclusions can be drawn based on the current results. Results showed the reaction reached its saturation point, so methods need to be refined to obtain data within the first 10 minutes. HsPlk4 phosphorylation of PLKtide confirmed the presumption that PLK family is a conserved family of Ser/Thr kinases. There are practical limitations for obtaining good kinetics data depicting enzyme activity, such as having EZ Reader quickly sample the reaction.
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Eckerdt, Frank. "Funktionelle Charakterisierung von Interaktionspartnern der Polo-like-Kinase 1 (Plk1)." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=96938193X.
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Zhang, Yun [Verfasser], and Andreas [Gutachter] Gießl. "Novel insights into the aberrant activation of fibroblasts in systemic sclerosis - Casein kinase 2, Poly(ADP-ribose)-Polymerase 1, and Janus kinase1 mediated transactivation of Janus kinase2 as novel players / Yun Zhang ; Gutachter: Andreas Gießl." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2016. http://d-nb.info/1114499722/34.
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Schmidt, Andreas. "Functional characterization of a novel Xenopus polo-like kinase interacting protein." Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-59773.
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Robert, Kin Yip Cheng. "Structural and functional studies of the mitotic Polo-like kinase 1." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432557.
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Neutzner, Melanie. "Regulatoren des Zellteilungszyklus der Hefe Saccharomyces cerevisiae : die Polo-Kinase Cdc5 und der Ubiquitinierungsfaktor Hct1 /." [S.l. : s.n.], 2003. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10605153.
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Warnke, Silke. "Funktionelle Charakterisierung der humanen Polo-Kinase Plk2-Snk im Zell- und Centrosomenzyklus." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971267154.
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Habedanck, Robert. "The Human Polo-like Kinase 4 is a Regulator of Centrosome Duplication." Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-57757.
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Seeburg, Daniel P. (Daniel Philip). "The role of Polo-like kinase 2 in synaptic function and plasticity." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/38993.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.Includes bibliographical references.
Homeostatic forms of plasticity keep the spiking output of neurons within an optimal range in the face of changing activity levels of the surrounding network, but little is known about the underlying molecular mechanisms, particularly during heightened activity. We report in Chapter 2 that in hippocampal neurons experiencing elevated activity, the activity-inducible protein kinase, Polo-like kinase 2 (Plk2), was required for synaptic scaling in dissociated culture and for reducing membrane excitability in slice culture-two principal compensatory mechanisms underlying homeostatic plasticity. Inhibition of Plk2 activity in slice culture during elevated activity resulted in increased dendritic spine size and density as well as a "run-up" in synaptic strength that prevented subsequent LTP. Thus, the homeostatic functions of Plk2 allow synapses to remain within a modifiable range during prolonged heightened network activity. In Chapter 3, we show that the homeostatic prevention of run-up during elevated activity also depended on CDK5, which acted as a "priming" kinase for the phospho-dependent binding of PIk2 to its substrate SPAR, a postsynaptic RapGAP.
(cont.) Overexpression of SPAR strengthened synapses, whereas RNAi knockdown of SPAR weakened synapses and disrupted homeostasis. Thus CDK5-dependent recruitment of Plk2 to SPAR, followed by Plk2-mediated degradation of SPAR, constitutes a likely molecular mechanism for Plk2-dependent homeostatic plasticity in neurons.
by Daniel P. Seeburg.
Ph.D.
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Argunhan, Bilge. "Interplay between Dbf4-dependent Cdc7 kinase and polo-like kinase unshackles mitotic recombination mechanisms by promoting synaptonemal complex disassembly." Thesis, University of Sussex, 2016. http://sro.sussex.ac.uk/id/eprint/59019/.
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Meiotic recombination is initiated by self-inflicted DNA breaks and primarily involves homologous chromosomes, whereas mitotic recombination involves sister chromatids. Whilst the mitotic recombinase Rad51 exists during meiosis, its activity is suppressed in favour of the meiosis-specific recombinase, Dmc1, thus establishing a meiosis-specific mode of homologous recombination (HR). A key contributor to the suppression of Rad51 activity is the synaptonemal complex (SC), a meiosis-specific chromosomal structure that adheres homologous chromosomes along their entire lengths. Here, in budding yeast, we show that two major cell cycle kinases, Dbf4-dependent Cdc7 kinase (DDK) and Polo-kinase (Cdc5), collaborate to link the mode change of HR to the meiotic cell cycle by. This regulation of HR is through the SC. During prophase I, DDK is shown to maintain SC integrity and thus inhibition of Rad51. Cdc5, which is produced during the prophase I/metaphase I transition, interacts with DDK to cooperatively destroy the SC and remove Rad51 inhibition. By enhancing the interaction between DDK and Cdc5 or depleting DDK at late prophase I, meiotic DNA breaks are repaired even in the absence of Dmc1 by utilising Rad51. We propose that the interplay between DDK and Polo-kinase reactivates mitotic HR mechanisms to ensure complete repair of DNA breaks before meiotic chromosomem segregation.
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Westendorf, Jens. "Regulation of Polo-like kinase 4 via phosphorylation and ubiquitin-dependent proteolytic degradation." Diss., lmu, 2009. http://d-nb.info/1000276333/34.
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Gheghiani, Lilia. "Mécanisme de contrôle de l'entrée en mitose par Polo-like kinase 1 (PlK1)." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066385/document.
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L'exigence principale du cycle cellulaire est une coordination étroite entre l'achèvement du processus de réplication et l'entrée des cellules en mitose, afin de maintenir l'intégrité génétique, l'identité et à la survie cellulaire. Toutes les cellules somatiques exécutent de manière reproductible une phase G2 intermédiaire, d'une durée constante pendant les divisions cellulaires successives au sein d'un type cellulaire donné. Cependant, les mécanismes moléculaires qui contrôlent précisément sa durée au cours d'un cycle cellulaire non perturbé, restent mal caractérisés. La kinase Cycline B1-Cdk1, facteur universel permettant l'entrée en mitose, est sous le contrôle, chez les mammifères, d'un ensemble de régulateurs directs, les kinases inhibitrices Wee1 et Myt1 et les phosphatases activatrices Cdc25A, B et C. Son activation soudaine, en fin de phase G2, révèle qu'une modification rapide de l'équilibre entre ses régulateurs opposés prend place par des mécanismes moléculaires qui restent à élucider. Dans ce cadre, j'ai étudié le rôle potentiel de la kinase Polo-like kinase 1 (Plk1) pour l'initiation de l'activation de Cycline B1-Cdk1. Bien que les rôles de Plk1 au cours de la mitose soient bien caractérisés, sa contribution dans la régulation de l'entrée des cellules en mitose reste controversée. Au niveau moléculaire, Plk1 phosphoryle au moins in vitro, plusieurs régulateurs de Cycline B1-Cdk1, tels que Cdc25B & C, et Wee1 et Myt1. Cependant, il reste largement inconnu si ces événements de phosphorylation se produisent in vivo et s'ils contribuent de manière significative au processus de l'activation de Cycline B1-Cdk1 permettant l'entrée en mitoseA main requirement of the cell cycle is a tight coordination between the completion of the replication process and entry into mitosis in order to maintain genetic integrity and the identity and survival of cell progeny. All somatic cells reproducibly execute an intermediate G2 phase of constant duration during successive cell divisions in a given cell type. However the molecular mechanisms controlling precisely its duration during unperturbed cell cycle remains poorly characterized. In this context, the main objective of my PhD project was to decipher signaling pathways controlling entry into mitosis during normal cell cycles as well as their spatiotemporal regulation. CyclinB1-Cdk1, the universal master mitotic driver, is under the control of direct inhibitors (Wee1 and Myt1) and activators (Cdc25A, B and C). Previously, it was determined that CyclinB1-Cdk1 is suddenly activated in very late G2 phase, suggesting that a rapid modification in the equilibrium between its opposite regulators is reproducibly taking place in late G2 by poorly elucidated mechanisms. During my PhD, I investigated the potential role of Polo-like kinase 1 (Plk1) in the initial activation of CyclinB1-Cdk1. Even though its roles during mitosis are well characterized, its contribution for the regulation of entry into mitosis remains controversial. At the molecular level, Plk1 was shown to phosphorylate at least in vitro, several regulators of CyclinB1-Cdk1 including Cdc25B&C, and Wee1 and Myt1. However, it remains largely unknown if these phosphorylation events are taking place in vivo and whether they significantly contribute to the activation process of CyclinB1-Cdk1 leading to mitotic entry
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Pahlavan, Golbahar. "Polo-like kinase 1(Plk1) et la maturation méiotique de l'ovocyte de souris." Paris 7, 2001. http://www.theses.fr/2001PA077229.
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Ullrich, Andrea [Verfasser]. "Expressionsmuster der Polo-like-Kinase 1 und 3 im humanen Magenadenokarzinom / Andrea Ullrich." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2010. http://d-nb.info/1024007022/34.
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Zhu, Kaiyuan, and 祝开元. "Polo-like kinase 1 (Plk1) phosphorylates VCP T76 during mitosis for the fragmentation of Golgi in mammalian cell." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208019.
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Myer, David. "Role of the Mammalian Polo-Like Kinase 3(Plk3) in Cell Cycle Regulation and DNA Damage Checkpoints." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1141395911.
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Lee, Cathy. "Molecular targeting of polo-like kinase 1 (PLK1) for the treatment of brain tumours." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44820.
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Brain cancer is a disease that is difficult to treat and has a high mortality rate. Two of the major challenges facing current treatments are disease relapse and drug resistance. Disease relapse may be attributable to brain tumour initiating cells (BTICs), which are multi-potent stem-like cells endowed with the ability to self-renew and generate differentiated cells that form the bulk of a tumour. BTICs are notably resistant to conventional chemotherapy and radiation and may therefore survive treatments, repopulate new tumours and cause disease relapse. Failure to respond to Temozolomide (TMZ), a DNA alkylating agent used as a front-line therapy for glioma, is frequently observed in glioma patients with O⁶-methylguanine-DNA methyltransferase over-expression. Furthermore, despite being largely ineffective in the pediatric population, TMZ is routinely used in clinic due to the lack of therapeutic agents that cross the blood-brain-barrier. There is an imperative need to seek alternative therapeutic strategies to address these issues. In the studies described herein, we identified polo-like kinase 1 (PLK1) as a novel molecular target for the treatment of the most common malignant brain tumours in adults and children, glioblastoma multiforme (GBM) and medulloblastoma (MB), respectively. In our studies, PLK1 is highly expressed in GBM and MB cultured cell lines, tumour tissues and patient-derived primary isolates but not in the normal brain cells including astrocytes, neurons and human neural stem cells. Targeting PLK1 by siRNA or the small molecule inhibitor BI2536 suppressed cell growth and induced cell cycle arrest accompanied by apoptosis in both GBM and MB. Of note, PLK1 inhibition had the unique capacity of obliterating GBM and MB BTICs, as well as TMZ-resistant GBM cells. To further the study, we demonstrated that BI2536 delayed disease progression and prolonged the survival of mice bearing orthotopic GBM or MB brain tumours. In addition, we established the prognostic importance of PLK1 as an independent marker for patient survival in both GBM and MB, lending further support to targeting this kinase in brain cancer treatment.
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Zalles, Nicole. "Effects of MicroRNA modulation of PLK1 in Breast Cancer in combination with PLK1 inhibitor NMS-P937." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1554130079416561.
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Spänkuch-Schmitt, Birgit. "Funktionelle Studien zur Hemmung von PLK1 (Polo-like-Kinase 1) in vitro und in vivo." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=968909922.
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Triscott, Joanna Catherine Caprio. "Polo-like kinase 1 as a prognostic and therapeutic target in high-grade brain tumors." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/54512.
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High-grade brain tumors have some of the highest rates of cancer-related death. Occurring predominately in adults, patients with glioblastoma (GBM) are not expected to survive longer than two years. Similarly, medulloblastoma (MB) is the most commonly occurring malignant brain tumor in children and, although these cases have a much better probability of survival, the severe impact of high-intensity treatment often causes long-term negative side effects. Unfortunately, high-grade brain tumors are frequently resistant to standard treatments like temozolomide (TMZ). Further, the development of new drugs is hindered by the blood-brain barrier and the astronomical cost of drug discovery and clinical evaluation. The immediate need for new treatment options encouraged our approach of querying compounds that have previously been tested in clinical trials. Herein, we found that TMZ resistant GBM cells express high levels of the mitotic kinase, Polo-Like Kinase 1 (PLK1) and that TMZ resistance can be overcome using PLK1 kinase inhibitors, such as BI-2536. An assessment of off-patent drugs revealed that the anti-alcoholism treatment, disulfiram (DSF), also had efficacy in eliminating PLK1-high cells and this work proposes DSF can be repurposed for cancer treatment. The importance of PLK1 was further investigated in a retrospective study of MB patient samples that were assessed with the NanoString nCounter system. Cases with high PLK1 expression were more likely to relapse and had worse overall survival. This work suggests that stratification of these high-risk cases can identify patients that may benefit from PLK1 inhibitors, which cause G2/M arrest and apoptosis. Notably, both DSF and BI-2536 treatment had no negative growth effects on normal brain cells. Finally, translationally controlled tumor protein (TCTP) is a substrate of PLK1 that is used as a marker of kinase activity. An exploration of the clinical and mechanistic impact of TCTP demonstrated a striking association with the sonic hedgehog (SHH) MB subtype, as well, as establishing its role in cancer cell proliferation. In conclusion, the studies outlined in this thesis encourage the investigation of PLK1 as a therapeutic target in high-grade brain tumors and emphasizes the potential benefit of fighting cancer with repurposed drugs.Medicine, Faculty of
Medicine, Department of
Experimental Medicine, Division of
Graduate
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Patel, Avinash. "Exploiting classical and chemical genetics to interrogate the polo kinase phospho-proteome of fission yeast." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/exploiting-classical-and-chemical-genetics-to-interrogate-the-polo-kinase-phosphoproteome-of-fission-yeast(c6666a88-fb38-47f2-bfc8-62c864823073).html.
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Commitment to mitosis is universally regulated by the activation of a protein complex Mitotic Promoting Factor (MPF). Activation of MPF activates the downstream mitotic kinases (e.g. Polo, Aurora and NIMA related kinases) that lead to a number of different mitotic events. As in other systems, the fission yeast Polo kinase, Plo1 regulates diverse mitotic events including mitotic commitment, spindle formation, the cytokinetic ring formation/contraction, and the exit from mitosis. The aim for my project was to use a genetic and biochemical approach to assess the impact of Plo1 upon the composition of the mitotic phosphoproteome. Novel temperature sensitive plo1 alleles were generated. All known Plo1 dependent events were abolished in these mutants upon incubation at the restrictive temperature. One mutant was subsequently used in analysis where cells were arrested in mitosis with and without Polo Kinase activity and phosphopeptides whose levels were differentially regulated in the two conditions were identified through exploitation of SILAC technology. A parallel SILAC experiment identified phosphopeptides whose levels were altered when Plo1 activity was increased by mutation of the spindle pole body component Cut12. The range of phosphorylation events identified, not only fits existing models for the mitotic controls exerted by Plo1 in fission yeast, but highlight candidates for novel functions for Plo1.An analogue sensitive plo1 allele was also generated. This allele was used to determine the consequences of selective inhibition of Plo1 kinase activity following the addition of an non-hydrolysable ATP analogue to the culture. This modified kinase domain was also used to specifically target and activate Plo1 to specific subcellular locations to demonstrate that localised Plo1 activity on the Spindle Pole Body (SPB) in late G2 phase triggers commitment to mitosis. In conclusion, these studies of Plo1 function have considerably extended our current insight into established Plo1 dependent events and pathways and identified important avenues that could lead to the identification of novel functions for Plo1 in future.
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Klebba, Joseph Earl. "A Comprehensive Analysis of Polo-like Kinase 4's Regulation and Role in Centriole Biogenesis." Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/318808.
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Plk4 has been termed a `suicide kinase' because it promotes its own destruction to regulate protein levels. We identified numerous autophosphorylated residues within a region of Plk4 called the Downstream regulatory element (DRE). We find that phosphorylation of a single residue is sufficient for Slimb recruitment and phosphorylation of the surrounding residues builds a high affinity Slimb-binding site. These autophosphorylation events are dependent on Plk4 homodimerization, although the domains that mediate this dimerization are unknown. We show that Plk4 homodimerization is mediated by interactions between the PB1-PB2 cassette. We find that like all Polo kinases, Plk4 encodes a mechanism of kinase autoinhibition. Unlike other Polo kinases, which rely on external inputs for relief of inhibition, Plk4 is self-sufficient in relieving kinase inhibition. This relief of autoinhibition is regulated by PB3 of Plk4 and is dependent on homodimerization, thereby making homodimerization a necessary step in formation of the Slimb phosphodegron on Plk4. Polo Boxes are known as multifunctional domains, and the Polo Boxes of Plk4 are no different. We identified numerous Slimb-mediated ubiquitination sites on PB1 as well as PB2. Furthermore, the PB1-PB2 cassette mediates the interaction between Plk4 and the N-terminus of Asterless. In Drosophila cells, Plk4 requires Asterless for centriolar localization and Asterless overexpression drives centriole amplification in a Plk4 dependent manner. This is a fascinating result as endogenous Plk4 protein levels are undetectable in S2 cells, making it hard to envision a scenario where overexpression of Asterless could shuttle a non-existent Plk4 population to the centriole to initiate duplication. We found that in addition to shuttling Plk4 to the centriole, Asterless stabilizes Plk4, likely protecting Plk4 at the centriole to allow it to `license' the centriole for duplication. Moreover, we show that Asterless encodes two distinct Plk4 binding sites: the previously described N-terminal binding site as well as a novel C-terminal binding site. We found that the interaction between the C-terminal of Asterless and Plk4 is necessary for centriole duplication while the interaction between the N-terminal of Asterless and Plk4 is expendable. Together these findings provide significant insight into Plk4 biology and the mechanisms which limit its activity.
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Labella, Sara. "Characterization of the functions of polo-like kinase 2 during meiotic chromosome pairing in C. elegans." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114400.
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The two rounds of cell division that constitute meiosis are a conserved process that generates the gametes required for sexual reproduction. Given the importance of this specialized cell division in forming new life it is imperative for meiotic cells to ensure that the homologous chromosomes (at meiosis I) and subsequently the sister chromatids (at meiosis II) are properly segregated to avoid aneuploidy and infertility. As a first step in this process the maternal and paternal chromosomes (the homologs) have to be able to recognize each other and align along their length (pairing). In many organisms this culminates in the formation of the synaptonemal complex (SC) to further stabilize their interactions. Since SC formation is independent of chromosome homology, pairing and synapsis processes must be coordinated to ensure SC formation only after homology assessment. During the chromosome pairing process in the nematode C. elegans, one end of each chromosome (the pairing centers, PCs) interacts with the nuclear envelope (NE) and is thought to generate their movement within the nucleus through a connection to cytoskeletal forces across an intact NE, a process well conserved from yeasts to mammals. The function of chromosome movement and how it is established and regulated is still poorly understood in any system and here I present my contribution to understanding the mechanism and regulation of meiotic chromosome pairing and synapsis and the role of chromosome movement during these events in C. elegans.My initial work focused on the characterization of polo-like kinase 2 (PLK-2); PLK-2 localizes to the PCs associated with the NE upon meiotic entry and loss of plk-2 function severely disrupts homologue pairing and results in nonhomologous synapsis. Previous work has shown that at meiosis I onset, the NE is reorganized such that integral NE SUN-1/ZYG-12 modules that bridge the NE and interact with cytoskeletal forces aggregate in the vicinity of chromosome PCs to form mobile foci that can further coalesce into patches. I showed that PLK-2 activity at the PCs is required for the meiotic reorganization of SUN-1/ZYG-12 complexes within the NE and I directly show that these bridges connect nuclear chromosomes with the cytoskeletal forces that are required to generate chromosome movement during pairing stages. Using a kinase dead PLK-2, I found that PLK-2 kinase activity is required for chromosome motion and loss of this motion results in nonhomologous synapsis between the unpaired chromosomes. Using a separation-of-function allele of PLK-2, I demonstrate for the first time that chromosome movement per se is not sufficient for homologous pairing. In these mutants, the chromosomes retain wild-type like movements, despite the failure to reorganize the NE and form SUN-1/ZYG-12 foci and patches. Analysis of the chromosome movement indicates that chromosome ends undergo fewer encounters and separate more rapidly in comparison to wild-type. Consequently, I propose that SUN-1/ZYG-12 patch formation is not required for chromosome movement but to restrain this movement in order to provide a window of opportunity for the chromosomes to undergo homology assessment. The balance of forces between NE protein aggregates that constrain chromosome ends together and cytoskeletal microtubules that try to separate them might be at the basis of chromosome homology establishment. Since many of the proteins participating in these events are conserved, including PLK-2, this mechanism may be a conserved feature of meiotic chromosome pairing in different species.Les deux cycles de division cellulaire qui constituent la méiose représentent un processus conservé au cours de l'évolution des espèces qui permet de créer les gamètes nécessaires à la reproduction sexuelle. Compte tenu de l'importance de ce processus dans la formation de toute nouvelle vie, il est essentiel pour les cellules méiotiques d'assurer la séparation correcte des chromosomes homologues lors de la première division de méiose (MI), puis des chromatides sœurs lors de la deuxième division de méiose (MII) afin d'éviter aneuploïdie des gamètes et infertilité. Au cours de la méiose I, les chromosomes maternels et paternels homologues se reconnaissent et s'alignent sur toute leur longueur (appariement). Dans de nombreux organismes cette première étape aboutit à la formation du complexe synaptonémal (SC) qui stabilise davantage leurs interactions. Puisque la formation du SC est indépendante de l'homologie des chromosomes, les processus d'appariement et de synapse doivent être dûment coordonnés pour que la formation du SC n'ait lieu qu'après vérification de l'homologie. Pendant le processus d'appariement des chromosomes chez le nématode C. elegans, une extrémité de chaque chromosome (les centres d'appariement, PC) interagit avec l'enveloppe nucléaire (NE) et génère vraisemblablement leur mouvement à l'intérieur du noyau par le biais d'une connexion au cytosquelette via l'enveloppe nucléaire, un processus bien conservé des levures aux mammifères. La fonction du mouvement des chromosomes et la façon dont il est établi et réglementé est encore mal comprise dans tout système. Je présente dans ce manuscrit ma contribution à la compréhension du mécanisme et de la fonction précise de ce processus dans le mouvement des chromosomes lors de la méiose chez C.elegans.Mon travail initial a porté sur la caractérisation d'une protéine polo-like kinase 2 (PLK-2). PLK-2 se localise au niveau des centres d'appariement associés à l'enveloppe nucléaire au début de la méiose; son absence perturbe gravement l'appariement des homologues et conduit à la formation de synapse non-homologue. Des travaux antérieurs avaient montré que à l'entrée en méiose l'enveloppe nucléaire est réorganisée de telle façon que les complexes protéiques SUN-1/ZYG-12 qui permettent de créer la liaison physique entre le cytosquelette à l'extérieur du noyau et les chromosomes à l'intérieur s'agrègent entre eux au voisinage des centres d'appariement des chromosomes. J'ai montré que c'est l'activité de PLK-2 qui préside à la réorganisation de ces complexes SUN-1/ZYG-12 dans l'enveloppe nucléaire, et que ces complexes sont directement nécessaires au mouvement des chromosomes durant le processus d'appariement. Par mutagénèse dirigée j'ai créé une protéine PLK-2 sans activité kinase, et j'ai ainsi pu démontrer qu'une réaction de phosphorylation par PLK-2 est essentielle au mouvement des chromosomes; en son absence les chromosomes forment des synapses non homologues. La découverte et l'étude d'un allèle non nul de plk-2 (vv44) a aussi permis de comprendre que le mouvement des chromosomes n'est pas suffisant à la reconnaissance des homologues, puisque dans ces mutants vv44 les chromosomes, bien que mobiles, ne créent pas de réorganisation des complexes SUN-1/ZYG-12 dans l'enveloppe nucléaire et forment aussi des synapses non homologues. Une analyse détaillée du mouvement des chromosomes dans ces mutants montre que les chromosomes se séparent plus rapidement que dans le type sauvage. Je propose donc un modèle dans lequel la formation des agrégats SUN-1/ZYG-12 n'est pas nécessaire au mouvement des chromosomes mais bien à leur rétention entre eux. Ainsi il existe un équilibre des forces entre les agrégats qui contraignent les extrémités des chromosomes dans un micro-environnement et les forces du cytosquelette qui permettent de séparer les chromosomes non homologues; cet équilibre offre une fenêtre d'opportunité pour tester l'homologie des chromosomes.
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McKenzie, Lynsey. "G2/M checkpoint associated repression of polo-like kinase-1 mediated by the tumour suppressor, p53." Thesis, University of Dundee, 2010. https://discovery.dundee.ac.uk/en/studentTheses/fea09569-1f92-4084-8dbe-d85de3c86c6d.
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Polo-like Kinase-1 (PLK1) is an important mediator of the G2/M phase of the cell cycle that is down-regulated in a DNA damage dependent manner. In cancer cells PLK1 is overexpressed and allows for continued proliferation of the cell by overriding this checkpoint. Here I show that PLK1 is down-regulated in a p53-dependent manner and that can occur both in response to DNA damage and to a non-genotoxic stimulus of the p53 pathway. My data show that p53 is able to repress PLK1 through a responsive element in the promoter and that p53 is necessary and sufficient to cause PLK1 repression. When examined in the context of a PLK1 promoter/reporter fusion, wild type but not mutated forms of p53 can repress expression. EMSA shows that p53 binds to the p53-responsive element and that mutation of this element reduces p53 binding. Furthermore, PLK1 repression occurs independently of p21-mediated arrest at G1/S, a stage of the cell cycle where PLK1 levels are physiologically low. PLK1 repression mediated by p21 through the CDE/CHR element in the promoter does not appear to cause significant repression of PLK1 but may play a minor role. Down-regulation of PLK1 is relieved by the HDAC inhibitor TSA and supports the transcriptional repression mechanism described in this thesis. Silencing of PLK1 expression by siRNA interferes with cell cycle progression consistent with a role in the p53-mediated checkpoint. This thesis provides two distinct and perhaps overlapping mechanisms by which p53 may repress PLK1: 1)through competitive displacement of an unidentified transcription factor that is essential for normal PLK1 expression and 2) through HDAC recruitment leading to local repression-associated changes in the chromatin structure. These data establish PLK1 as a transcriptional target of p53 that is required for efficient G2/M arrest.
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Dedieu, Fabien. "Régulation de l'expression du facteur eIF4GII par le protéasome : rôle de la Polo-like Kinase Snk." Toulouse 3, 2007. http://thesesups.ups-tlse.fr/131/.
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Chez les Eucaryotes, l'initiation de la traduction se déroule en trois étapes essentielles : la formation d'un complexe de pré-initiation (43S), sa liaison à l'ARNm et son positionnement sur le codon initiateur. Ce complexe est recruté à l'extrémité coiffée de l'ARNm grâce au facteur protéique eIF4F. EIF4F est composé de trois sous-unités : eIF4E qui interagit directement avec la coiffe; eIF4A, une ARN hélicase; eIF4G, une protéine d'assemblage qui s'associe à la fois à eIF4E, eIF4A et eIF3, un autre facteur d'initiation lui même lié à la sous-unité 40S du ribosome. Ainsi, eIF4G joue le rôle d'un adaptateur permettant l'interaction entre l'ARNm (via la coiffe et eIF4E) et l'ARNr (via eIF3). Deux homologues fonctionnels d'eIF4G ont été clonés: eIF4GI et eIF4GII. La régulation de l'initiation par le complexe eIF4F s'effectue par le biais de phosphorylations. Le facteur eIF4G est une phosphoprotéine dont les fonctions sont régulées suivant son état de phosphorylation et peut ainsi soit activer ou réprimer la traduction Cap-dépendante. Les voies de signalisation régulant la phosphorylation des facteurs eIF4GI et eIF4GIIsont mal connues. La recherche de partenaires d'interaction (double hybride) avec les facteurs eIF4G nous a permis d'identifier la polo-like kinase 2 (Plk2 aussi appelée Snk), pour laquelle seule la protéine SPAR est connue comme substrat spécifique. Par homologie de séquence, Snk est classée dans la famille des polo kinases, laquelle compte 3 représentants majeurs : Plk1, Plk2 (ou Snk) et Plk3 (FGF-inducible kinase ou Fnk). Ces protéines kinases lient puis phosphorylent des substrats mitotiques, contrôlant ainsi la progression à travers la mitose. L'étude de l'interaction entre Plk2 et eIF4G a permis d'identifier les domaines impliqués dans cette interaction soulignant l'originalité de Plk2 car à la différence des autres polo-like kinases cette interaction ne requiert pas son domaine Polo. La localisation cytoplasmique de Plk2 et non pas nucléaire renforce encore cette originalité. .The eukaryotic translation initiation factors (eIF) 4GI and eIF4GII are scaffolding proteins critical for the assembly of translation initiation complexes. EIF4GI and eIF4GII are the target of different signaling pathways, and a recent report indicates that they may possess distinct functions. Here we show that the polo like kinase (Plk) 2 interacts directly with eIF4GII. In the cell, Plk2 targets eIF4GII to destruction. Plk2-dependent degradation of eIF4GII is sensitive to proteasome inhibition and necessitates both a physical interaction with and ubiquitination of Plk2 (but not that of eIF4GII), suggesting that Plk2 acts as a specific adaptor for the proteasome. We also show that both eIF4GII and Plk2 are destructed by the proteasome upon oxidative stress. Furthermore, although eIF4GI also interacts with Plk2, Plk2 does not target eIF4GI to destruction suggesting that eIF4GI binding to Plk2 may serve another function. Thus, we propose that Plk2 can control translation initiation rates at least through the regulation of eIF4GII cellular content
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Tavernier, Nicolas. "Régulation de SPAT-1/Bora, l'activateur de la polo-kinase, dans l'embryon précoce de Caenorhabditis elegans." Paris 7, 2013. http://www.theses.fr/2013PA077133.
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Le mécanisme de division cellulaire, appelé mitose, est un processus fondamental par lequel une cellule mère donne deux cellules filles. De nombreux cas de cancer peuvent être imputés au dérèglement du processus de division des cellules. Pour assurer une division, un nombre important de réactions biochimiques est mis en jeu. Ces réactions sont le fruit de l'activité d'une myriade de protéines intervenant très temporairement et dans un ordre précis. La coordination de ces réactions est donc finement régulée. Afin d'élucider le fonctionnement de ces mécanismes, nous utilisons l'embryon précoce de Caenorhabditis elegans comme organisme modèle. Les avantages de ce système sont multiples. Premièrement, la communauté scientifique dispose d'une pléthore d'outils éprouvés (extinction de gènes par ARN interférence, protéomique, banques de mutants, génome entièrement séquencé et annoté) pour travailler efficacement sur ce modèle. Deuxièmement, nous étudions les processus de divisions des cellules dans un contexte de développement c'est-à-dire au sein d'un tissu organisé, où les divisions des cellules sont coordonnées. Ceci nous rapproche fortement de ce qui se passe au sein d'un tissu structuré contrairement à des analyses effectuées sur des cellules individualisées en culture. Enfin, le coût d'utilisation est plutôt faible. Le fait que le cycle cellulaire soit un mécanisme extrêmement bien conservé au sein du monde vivant permet de transposer le plus souvent les découvertes au modèle humain, mais également de clarifier et compléter des concepts développés à partir d'études réalisées chez d'autres organismes modèles et ainsi accélérer considérablement la rechercheThe mechanism of cell division, called mitosis, is a fundamental process by which a mother cell gives two daughter cells. Many cancers can be attributed to the disruption of the process of cell division. To ensure a division, a large number of biochemical reactions is put in. These reactions are the result of the activity of a myriad of proteins involved very temporarily and in a specific order. The coordination of these reactions is tightly regulated. In order to elucidate the functioning of these mechanisms, we use Caenorhabditis elegans early embryos as a model organism. The advantages of this system are many. First, the scientific community has a plethora of tools (gene silencing by RNA interference, proteomics, mutant libraries, fully sequenced and annotated genome) to work effectively. Second, we study the process of cell divisions in the context of development where cell divisions are coordinated with polarity. This brings us strongly of what is happening within a structured contrast to analyzes of individual cells in tissue culture. Finally, the cost of use is rather low. The fact that the cell cycle is an extremely well-preserved in the world living mechanism can transpose most often discovered human model, but also to clarify and supplement the concepts developed from studies in other model organisms and thus greatly accelerate research
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Thrum, Stephan. "In vitro-Versuche mit dem Polo like-Kinase 1 Hemmstoff BI 2536 an Zelllinien von Gallenwegskarzinomen." Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-134869.
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Karzinome der Gallenwege sind mit einer schlechten Prognose assoziiert. Eine potentiell kurative chirurgische Resektion ist bei der Mehrzahl der Patienten aufgrund des späten Zeitpunkts der Diagnosestellung nicht möglich, so dass derzeit vorrangig palliative Therapieansätze Anwendung finden. Das nur geringe Ansprechen auf konventionelle Radio- oder Zytostatikatherapie begründet die Notwendigkeit neuer Therapieansätze. Einen möglichen Angriffspunkt stellt hierbei die Polo-like-Kinase 1 (Plk1) dar, da ihre zentrale Rolle in der Regulation des Zellzyklus zunehmend erkannt und eine vermehrte Expression in malignem Tumorgewebe verglichen mit gesundem Gewebe nachgewiesen wurde. Das Dihydropteridinon BI 2536 ist ein poten-ter, niedermolekularer und selektiver Hemmstoff der Plk1 und sollte daher auf seine Wirksamkeit an Gallenwegskarzinomen untersucht werden.
In der vorliegenden Arbeit konnte gezeigt werden, dass BI 2536 die untersuchten 14 Zelllinien von Gallenblasen- und Gallengangskarzinomen wirkungsvoll hemmt. Das Ansprechen unterschied sich zwischen den Zelllinien und ordnet sich vergleichbar zu Veröffentlichungen an anderen malignen Tumoren ein. Die Expression von Plk1 und dessen assoziierten Transkriptionsfaktor FoxM1 konnte bei Westernblot-Versuchen bei allen Zelllinien nachgewiesen werden, was eine Bedeutung in der Onkogenese vermuten lässt. Die Behandlung mit BI 2536 beeinflusste jedoch die Proteinmenge beider nicht. An für die folgenden Versuche ausgewählten drei Zelllinien zeigten sich in der reversen Transkription mit anschließender Echtzeit-Polymerase-Kettenreaktion (qRT PCR) ähnliche Ergebnisse in Bezug auf die exprimierte mRNA von Plk1. Westernblot-Analysen ermittelten keine signifikanten Veränderungen der an wichtigen intrazellulären Kaskaden beteiligten Proteine p42/44 und Akt sowie deren phosphorylierten Formen. Obwohl die Proteinmenge des Mitosemarkers Phospho-Histon H3 ebenso unverändert blieb, führte die Behandlung mit BI 2536 – dies zeigen Ergebnisse der Durchflusszytometrie – zu einer signifikanten, dosisabhängigen Zunahme der G2/M Fraktion des Zellzyklus und Zunahme der Apoptose-rate. Der maximale Hemmeffekt in der Behandlung von BI 2536 lag bei einer Inkubations-dauer von vier Tagen.
Die Empfehlungen aus den klinischen Studien der Phase II von BI 2536 sowie dem Ziel der Vermeidung von Resistenzen ergibt sich die Notwendigkeit von Kombinationsversuchen mit Zytostatika, die in einer anderen Phase des Zellzyklus angreifen. Die in der Behandlung von Gallenwegskarzinomen etablierten Antimetaboliten 5-Fluorouracil und Gemcitabin wurden hierzu ausgewählt und es zeigten sich für 5 Fluorouracil synergistische, für Gemcitabin hingegen additive Kombinationseffekte. Zusätzlich wurde die Wechselwirkung mit dem IGF 1-Rezeptor-Inhibitor NVP-AEW541 untersucht, der ebenfalls einen neuen Behand-lungsansatz in der Krebstherapie darstellt und bei Gallenwegskarzinomen in vitro wirksam ist. Auch hier zeigen sich synergistische Effekte, die jedoch erst in höheren Behandlungs-dosen auftraten.
Die Ergebnisse dieser Arbeit zeigen, dass die Hemmung der Plk1 bei Gallenblasen- und Gallengangskarzinomen einen wirksamen Behandlungsansatz darstellt. Auf der Grundlage der in dieser Arbeit beschriebenen Ergebnisse wird eine weitere präklinische und klinische Testung von selektiven Plk1-Hemmstoffen wie BI 2536 an Gallenwegskarzinomen empfohlen.
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CLAUDI, CECILIA. "POLO-LIKE KINASE CDC5 CONTRIBUTES TO MITOTIC SPINDLE ELONGATION VIA THE KINESIN-5 MOTOR PROTEIN CIN8." Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/607694.
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Proper chromosome segregation requires an orderly sequence of events, whereby spindle elongation follows the dissolution of sister chromatid linkages. Chromosome segregation starts at the onset of anaphase when the separase triggers the cleavage of cohesin, a protein complex that holds sister chromatids together. Next, chromatids are segregated into the daughter cells by the pulling force of the mitotic spindle. The mitotic spindle is a sophisticated and complex machinery built of microtubules, microtubule associated proteins and motor proteins. Despite the fundamental role of the mitotic spindle, the molecular mechanisms underlying its regulation remain elusive. Proper spindle function requires that microtubule dynamics are stabilized at anaphase. This change in microtubule dynamics is perceived as dictated by a shift in the balance of kinase and phosphatase activities in favor of the phosphatases. The finding that cells simultaneously lacking the polo-like kinase Cdc5 and the phosphatase Cdc14 cannot progress through anaphase albeit having cleaved cohesin due to defects in spindle elongation, challenges the view of mitotic exit as a time for protein dephosphorylation.
The aim of my work is to understand the molecular mechanism by which the two proteins contribute to anaphase spindle elongation, with a particular focus on the role of Cdc5. We identified the kinesin 5 motor protein Cin8 as a key target of the “Cdc14-Cdc5” spindle elongation pathway. We show that besides being dephosphorylated by Cdc14, Cin8 is also phosphorylated by Cdc5 on residues S409 and S441, and that this phosphorylation is crucial for the function of the kinesin in anaphase spindle elongation, likely because of the impact it has on the interaction between Cin8 and microtubules. Since these residues, S409 and S441, are located within a highly conserved stretch of amino acids, it will be interesting to test whether this regulation is conserved in other vertebrates as well. The finding that Cin8 is simultaneously a substrate of a kinase and a phosphatase sheds light on the complexity of mitotic exit regulation and is in complete agreement with recent data showing that approximately equal numbers of phosphosites are phosphorylated and dephosphorylated during mitotic progression and exit. Since it appears that phosphorylation and dephosphorylation events are equally important to the point that kinases and phosphatases cooperate to regulate the same substrates, the view of mitotic exit as the realm of phosphatases is dismantled and the continuous need for single molecule studies in addition to global analyses investigation is put forward.
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Reindl, Wolfgang [Verfasser], Horst [Akademischer Betreuer] Kessler, Thorsten [Akademischer Betreuer] Berg, and Bernhard [Akademischer Betreuer] Küster. "Inhibition of Polo-like Kinase 1 by Small Molecules Targeting the Polo-Box Domain / Wolfgang Reindl. Gutachter: Bernhard Küster ; Horst Kessler. Betreuer: Horst Kessler ; Thorsten Berg." München : Universitätsbibliothek der TU München, 2008. http://d-nb.info/1054351910/34.
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Chumduri, Cindrilla. "Mechanism of cell death in Burkitt lymphomas." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2010. http://dx.doi.org/10.18452/16086.
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Apoptoseresistenz ist einer der Gründe für ein Versagen von Chemotherapie bei vielen Krebserkrankungen, darunter das Burkitt Lymphom. Um die molekularen Mechanismen der Apoptoseresistenz aufzuklären, wurde die Apoptoseinduktion in 15 Burkitt-Lymphom-Zelllinien nach Behandlung mit den Spindelgiften Taxol (Paclitaxel), Nocodazol und Vincristin untersucht. Interessanterweise entwickelten Zellen, die sich als resistent gegenüber Taxol- und Nocodazol-induzierter Apoptose erwiesen, nach Behandlung eine Polyploidie (>4N DNA), was eine inverse Relation von Apoptose und Polyploidie aufzeigt. In den sensitiven Zelllinien war die Taxol- und Nocodazol-induzierte Apoptose von Caspase-Aktivierung, Bid-Spaltung und Herunterregulation von Mcl-1 begleitet. Im Gegensatz zu den sensitiven Zelllinien wiesen die meisten apoptoseresistenten Zellen einen Verlust von Bax und Bak auf und waren durch einen anhaltenden mitotischen Arrest mit Auftreten eines >4N DNA-Gehalts nach Behandlung charakterisiert. Um weitere Einblicke in den Mechanismus der Spindelgift-induzierten Apoptose zu erhalten, wurde die Rolle der mitotische Kinase PLK1 (polo-like kinase) näher untersucht. Eine dominant-negative PLK1-Mutante induziert Apoptose. Allerdings zeigte eine zusätzliche Behandlung mit Spindelgiften keinen synergistischen Effekt, was darauf schließen lässt, dass sowohl Inhibierung von PLK1 als auch Mikrotubuli-destabilisierende Agenzien den gleichen Stress-Signalweg aktivieren. Andererseits unterstützte Überexpression von Wildtyp-PLK1 in Taxol behandelten Zellen die Zellzyklus-Progression. Dies deutet auf eine Verbindung zwischen Zelltodresistenz und genetischer Instabilität (Aneuplodie) hin. Inhibition von Apoptose in sensitiven Zelllinien durch Caspase-Inhibierung förderte Polypoidie, welche die inverse Relation bestätigte. Medikamente, welche die Caspase-Aktivierung unabhängig von Bax und Bak induzieren, könnten eine weitere Möglichkeit zur Behandlung von resistenten Burkitt-Lymphomen darstellen.Apoptosis resistance is the major cause of chemotherapy failure in most kinds of cancers, including Burkitt lymphomas (BL). To elucidate molecular mechanisms regulating the development of apoptosis resistance, a panel of 15 BL cell lines was investigated for apoptosis induction upon treatment with microtubule inhibitors taxol, nocodazole and vincristine. Significant differences were observed in the extent of apoptosis induction among BL cell lines examined. Interestingly, cell lines exhibiting resistance to taxol- or nocodazole-induced apoptosis, showed development of polyploidy (>4N) and vice versa, displaying an inverse relationship between apoptosis and polyploidy induction. Further, in sensitive cell lines taxol-induced apoptosis was accompanied by caspase activation, Bid cleavage and Mcl-1 down-regulation. In contrast, most apoptosis resistant cell lines exhibited a loss of Bax and Bak expression and showed prolonged mitotic arrest with >4N DNA content upon treatment. To gain mechanistic insights into microtubule inhibitor-induced cell death, the role of the mitotic kinase PLK1 was addressed. Dominant negative PLK1 mutant induced apoptosis, however, failed to show synergism in induction of apoptosis in combination with microtubule inhibitors. This indicates that PLK1 inhibition and spindle toxins might trigger a similar mitotic stress pathway. Conversely, overexpression of wildtype PLK1 promoted cell cycle progression in cells treated with taxol. Remarkably, inhibition of apoptosis in sensitive cell lines by caspase inhibition promoted polyploidy confirming the inverse relationship between apoptosis and polyploidization. Considering targets to induce Bax/Bak independent caspase activation would be of great importance to avoid undesirable events leading to chromosomal imbalances in treating resistant cancers.
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Reynolds, Nicola. "Functional dissection of the conserved domains of polo-like kinase and its interacting proteins in fission yeast." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/11305.
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The polo-like kinases are a family of highly conserved serine/threonine protein kinases with multiple mitotic functions. Polo-like kinases are recognisable by the presence of two conserved domains: a catalytic, kinase domain at the amino terminus of the protein and at the carboxy terminus, a non-catalytic domain. In the fission yeast, Schizosaccharomyces pombe, there is one member of this family, Plol. The aim of this study was to define the function of the non-catalytic domain of the protein and to identify unknown mitotic roles for Plol. Site directed mutagenesis of Plol illustrated the roles of the two conserved domains in different aspects of the Plol function. Localisation, regulation of kinase activity and in vivo function of mutant proteins were analysed. Two hybrid analysis was carried out to identify interacting proteins. This was successful in revealing previously uncharacterised functions for Plol such as an interaction with the anaphase promoting complex subunit, Cut23. Reverse two hybrid screening combined with random mutagenesis was carried out to determine the in vivo consequences of eliminating specific interactions.
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Vidanes, Genevieve M. "Suppression of the DNA damage checkpoint by the Saccharomyces cerevisiae polo-like kinase, CDC5, to promote adaptation." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3352477.
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Pina-Mimbela, Ruby Melisa. "Association of Polyphosphate (poly P) Kinases with Campylobacter jejuni Invasion and Survival in Human Epithelial Cells." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1385978458.
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Hyland, Paula Lisa. "Sequence analysis of the adenine phosphoribosyltransferase gene locus in wild-type and thymidine kinase-deficient friend erythroleukaemia cells." Thesis, University of Ulster, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390158.
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Ahmadi, Pour Malek. "Role of the mitotic Polo-like kinase PLK1 in the control of the interferon induction pathway mediated by the mitichondria-bound MAVS protein and the IKKε protein kinase." Paris 6, 2009. http://www.theses.fr/2009PA066318.
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A la suite d’infections virales ou de conditions de stress, les cellules montent une réponse immune innée qui conduit à la production d’interféron et de cytokines pro-inflammatoires. MAVS, une protéine adaptatrice liée aux mitochondries et les IRF3/7 kinases TBK1 et IKKε, situées en aval, sont d’importants effecteurs de cette réponse. Une particularité de IKKε, par rapport à TBK1, est de s’associer avec MAVS à la mitochondrie au cours de leur activation. Une recherche de partenaires pour MAVS et IKKε par crible double hybride en levure a révélé la polo-like kinase PLK1 mitotique comme leur unique partenaire commun. L’objectif de ce travail a été de caractériser ces deux associations et de définir leur importance dans la réponse immune innée. Dans une première partie, nous montrons que PLK1 s’associe avec MAVS via son domaine Polo-box (PBD) sur un motif STP où le résidu T central est phosphorylé, une caractéristique commune à de nombreuses cibles de PLK1. PBD s’associe aussi à la partie C terminale de MAVS mais de façon indépendente de phosphorylation. Ceci conduit à l’inhibition de l’association de MAVS avec son partenaire aval TRAF3 et à l’inhbition de la capacité de MAVS à activer les voies NF-B et IRF3 ainsi que l'induction de l’IFN-. Dans une deuxième partie, nous montrons que PLK1-PBD interagit avec le domaine catalytique de IKKε, indépendamment de son site de liaison aux phosphopeptides. Cette association dépend de l’activité catalytique de IKKε mais non de celle de PLK1. PLK1 inhibe la capacité de IKKε à induire l’IFN- et à activer les voies IRF3 et NF-B. Au contraire de la situation avec MAVS, cette inhibition n’est pas médiée par PLK1-PBD mais implique l’activité catalytique de PLK1. L’activité de IKKε peut être restaurée par la molécule BI2536, un compétiteur de la liaison de l’ATP au domaine catalytique de PLK1. Dans l’ensemble, nous démontrons un nouveau mode de régulation de la réponse immune innée par la protéine kinase mitotique PLK1. Nos données peuvent conduire au développement d’inhibiteurs capables d’empêcher l’interaction PLK1/MAVS et/ou PLK1/IKKε afin de soutenir et/ou de moduler la réponse immune innée.