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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
61

Ellagic Acid-Mediated CK2 Inhibition, A Natural, Multifunctional Strategy to Trigger Cervical Cancer Cell Death in Vitro and in Vivo

Mohammad, Hanan Fathi 23 May 2012 (has links)
No description available.
62

REGULATION DE L'ACTIVITE ET DE LA LOCALISATION DES PHOSPHATASES CDC25B

Theis-Febvre, Nathalie 19 May 2003 (has links) (PDF)
L'activation séquentielle des Kinases Dépendantes des Cyclines (CDK), associées à leur sous-unité régulatrice la cycline, contrôle la progression des cellules eucaryotes dans le cycle cellulaire. L'activité des complexes CDK/cycline est notamment régulée par une balance entre phosphorylation inhibitrice (Wee, Myt) et déphosphorylation activatrice par les phosphatases CDC25. Dans les cellules humaines, trois phosphatases à double spécificité CDC25A, B et C sont impliquées dans la régulation de ces complexes en différents points du cycle cellulaire. CDC25A agit à la transition G1/S alors que CDC25C contrôle l'entrée en mitose. Par contre, CDC25B agirait en phase S ainsi qu'à la transition G2/M. L'existence de trois variants de CDC25B (B1, B2 et B3) issus d'un épissage alternatif pourrait expliquer cette controverse. Afin d'étudier les rôles et les implications de chaque variant de CDC25B, nous avons d'abord étudié leur régulation par la protéine kinase CK2, kinase qui pourrait jouer un rôle dans le contrôle de la transition G2/M. Nos études in vitro ont démontré que CK2 phosphoryle les trois variants de CDC25B mais pas la protéine CDC25C. Une analyse par spectrométrie de masse de CDC25B indique qu'au moins deux résidus, les sérines 186 et 187, sont phosphorylés in vitro par CK2. De plus, CDC25B interagit avec CK2 in vitro et in vivo dans des cellules humaines et d'insectes. Enfin, la phosphorylation de CDC25B par CK2 augmente son activité phosphatase in vitro ainsi qu'in vivo. CK2 est donc un régulateur positif de l'activité catalytique des CDC25B. Au cours du cycle cellulaire ou en réponse aux points de contrôle, CDC25B est également régulée au niveau de sa localisation intracellulaire. En effet, la phosphatase réalise une navette entre le cytoplasme et le noyau, navette qui peut être régulée notamment par phosphorylation. Nous avons montré que la protéine kinase AKT/PKB phosphoryle in vitro CDC25B sur la sérine 353 et qu'elle provoque son accumulation dans le cytoplasme. L'activation d'AKT/PKB par le peroxyde d'hydrogène reproduit la relocalisation de CDC25B. Par contre, si la mutation de la sérine 353 abolit sa phosphorylation par AKT/PKB, elle n'induit qu'un retard dans la relocalisation cytoplasmique de CDC25B ce qui indique que d'autres mécanismes participent à ce phénomène. Ainsi nos différents travaux ont permis d'identifier deux nouveaux régulateurs de CDC25B, CK2 qui régule son activité catalytique et AKT/PKB qui participe au contrôle de sa localisation intracellulaire, et nous permettent de mieux comprendre la régulation de cette phosphatase même si de nombreux partenaires doivent encore être identifiés.
63

Étude fonctionnelle de la sous-unité régulatrice de la protéine kinase CK2 dans les cellules souches embryonnaires murines : survie cellulaire, oligodendrogenèse et cancérogenèse.

Ziercher, Léa 11 October 2007 (has links) (PDF)
La protéine kinase CK2 est un complexe moléculaire dynamique composé de 2 sous-unités catalytiques α et d'un dimère de sous-unités régulatrices β. La structure cristallographique du dimère de CK2β suggère qu'il représente une plate-forme moléculaire, plaçant la protéine au cœur d'un réseau de voies de signalisation.<br />L'invalidation du gène CK2β chez la souris conduit à un défaut de viabilité des cellules souches embryonnaires (ES). Nous avons pu étudier la fonction de différents domaines fonctionnels in vitro dans un modèle cellulaire reposant sur une stratégie d'invalidation conditionnelle du gène CK2β dans les cellules ES murines. L'abolition de l'expression du gène endogène est létale pour les cellules ES et leur viabilité est restaurée par l'expression exogène de la protéine CK2β sauvage. Ce modèle nous permet d'étudier l'implication de divers domaines de CK2β dans la survie, la prolifération et la différenciation des cellules souches embryonnaires, en remplaçant la protéine endogène par diverses formes mutées de la protéine. <br />Parallèlement, l'invalidation conditionnelle du gène chez la souris ciblée dans les précurseurs neuraux a montré que CK2β est un élément clef dans les voies de signalisation qui contrôlent l'oligodendrogenèse. La différenciation des cellules ES nullizygotes exprimant la protéine exogène sauvage nous a permis de montrer que celle-ci restaure la lignée oligodendrocytaire. Nous avons aussi montré que certains domaines de CK2β, impliqués dans la fonction régulatrice du dimère, sont des déterminants structuraux cruciaux pour la prolifération des cellules souches neurales et leur progression dans la lignée oligodendrocytaire.
64

Molecular mechanisms of nuclear factor-erythroid-2 related factor 2 (Nrf2) regulation phosphorylation by casein kinase 2 (CK2) and interaction with proto-oncogene N-Myc in neuroblastoma cells /

Apopa, Patrick L., January 2007 (has links)
Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains vi, 130 p. : ill. (some col.). Includes abstract. Includes bibliographical references.
65

Developments and applications in computer-aided drug discovery

Ibrahim, Mahmoud Arafat Abd el-hamid January 2012 (has links)
Noncovalent interactions are of great importance in studies on crystal design and drug discovery. One such noncovalent interaction, halogen bonding, is present between a covalently bound halogen atom and a Lewis base. A halogen bond is a directional interaction caused by the anisotropic distribution of charge on a halogen atom X covalently bound to A, which in turn forms a positive region called σ-hole on the A–X axis. Utilization of halogen bonds in lead optimization have been rarely considered in drug discovery until recently and yet more than 50% of the drug candidates are halogenated. To date, the halogen bond has not been subjected to practical molecular mechanical-molecular dynamics (MM-MD) study, where this noncovalent interaction cannot be described by conventional force fields because they do not account for the anisotropic distribution of the charge density on the halogen atoms. This problem was solved by the author and, for the first time, an extra-point of positive charge was used to represent the σ-hole on the halogen atom. This approach is called positive extra-point (PEP) approach. Interestingly, it was found that the performance of the PEP approach in describing halogen bond was better than the semiempirical methods including the recent halogen-bond corrected PM6 (PM6-DH2X) method. The PEP approach also gave promising results in describing other noncovalent halogen interactions, such as C–X···H and C–X···π-systems. The PEP resulted in an improvement in the accuracy of the electrostatic-potential derived charges of halogen-containing molecules, giving in turn better dipole moments and solvation free energies compared to high-level quantum mechanical and experimental data.With the aid of our PEP approach, the first MM-molecular dynamics (MM-MD) study of inhibitors that form a halogen bond with a receptor was performed for tetrahalobenzotriazole inhibitors complexed to cyclin-dependent protein kinase (CDK2). When the PEP approach was used, the calculated MM-generalized Born surface area (MM-GBSA)//MM-MD binding energies for halobenzimidazole and halobenzotriazole inhibitors complexed with protein kinase CK2 were found to correlate well with the corresponding experimental data, with correlation coefficients R2 of greater than 0.90. The nature and strength of halogen bonding in halo molecule···Lewis base complexes were studied in terms of molecular mechanics using our PEP approach. The contributions of the σ-hole (i.e., positively charged extra-point) and the halogen atom to the strength of this noncovalent interaction were clarified using the atomic parameter contribution to the molecular interaction approach. The molecular mechanical results revealed that the halogen bond is electrostatic and van der Waals in nature. The strength of the halogen bond increases with increasing the magnitude of the extra-point charge. The van der Waals interaction’s contribution to the halogen bond strength is most favorable in chloro complexes, whereas the electrostatic interaction is dominant in iodo complexes.The failure of the PM6 semiempirical method in describing noncovalent halogen interactions —not only halogen bonds, but also hydrogen bonds involving halogen atoms— was reported and corrected by the introduction of a second and third generation of noncovalent halogen interactions correction. The developed correction yielded promising results for the four examined noncovalent halogen interactions, namely: C–X···O, C–X···N, C–X···π-system, and C–X···H interactions.
66

Régulation du récepteur nucléaire Nor1 par la SUMOylation et mécanismes de protection neuronale

Gagnon, Jonathan 04 1900 (has links)
Afin de répondre correctement aux nombreux changements se produisant à chaque instant dans leur environnement, les cellules utilisent une panoplie de messagers moléculaires dont la synchronisation est essentielle à la signalisation cellulaire appropriée. La superfamille des récepteurs nucléaires compte quarante-huit membres impliqués dans ces processus de signalisation et influence ainsi plusieurs fonctions physiologiques. Les récepteurs nucléaires de la sous-famille NR4A composée de Nur77/NR4A1, Nurr1/NR4A2 et Nor1/NR4A3 sont des facteurs critiques du développement et de la maintenance du système nerveux. Nor1/NR4A3 en particulier est essentiel aux processus de guidage axonal et de survie neuronale au niveau de l’hippocampe. Les NR4A se démarquent des autres récepteurs nucléaires puisqu’ils sont considérés comme des récepteurs orphelins constitutivement actifs, ce qui veut dire qu’ils ne nécessitent pas d’interaction avec un ligand afin d’être activés. Il devient ainsi important d’identifier de nouveaux mécanismes de régulation pour cette sous-famille de récepteur afin d’améliorer notre compréhension et potentiellement contrôler leurs activités dans un contexte neuronal. L’activité des récepteurs nucléaires peut être régulée de plusieurs façons, indépendamment de leur association avec un ligand endogène. Les modifications post-traductionnelles représentent un aspect crucial de la signalisation cellulaire permettant de réguler la fonction des protéines cibles de manière spécifique au contexte. La SUMOylation et la phosphorylation sont des exemples de modifications post-traductionnelles avec le potentiel de réguler l’activité transcriptionnelle, la stabilité et l’expression des gènes cibles des récepteurs nucléaires. Dans cette thèse, l’impact de la SUMOylation retrouvée sur un motif consensus ainsi que sur un motif non-consensus de SUMOylation phosphorylé du récepteur Nor1 est étudié. Dans la première étude, un motif de SUMOylation non-consensus nouvellement découvert sur Nor1 est décrit. Ce nouveau motif nommé pSuM a été identifié pour la première fois sur le récepteur nucléaire des estrogènes ERβ et sur le récepteur farnésoÏde FXR. Nous avons identifié un motif pSuM situé à la lysine 137 de Nor1 qui sert de cible fonctionnelle de SUMO2. Le pSuM se démarque du motif de SUMOylation consensus puisqu’il nécessite une phosphorylation afin d’être SUMOylé. Dans le cas de Nor1, nos résultats démontrent que la sérine 139 est phosphorylée par la voie des MAPK. La SUMOylation sur ce site mène à une réduction de l’activité transcriptionnelle et du recrutement à la chromatine de Nor1 ainsi que de l’expression des gènes sensibles à Nor1. Une particularité intéressante du pSuM de Nor1 est qu’il possède également une extension phosphorylable par la kinase CK2 qui est essentielle au processus de SUMOylation. Cette extension a également un effet sur la stabilité et la compétence transcriptionnelle de Nor1. En utilisant des lignées SH-SY5Y exprimant de manière stable différents mutants SUMO de Nor1, il est démontré que la SUMOylation du pSuM diminue la prolifération et la survie cellulaire en réponse au stress oxydant. Dans la seconde étude, la SUMOylation de Nor1 sur un motif de SUMOylation canonique situé sur la Lysine 89 est caractérisée. Il est démontré que ce site de SUMOylation est ciblé principalement par SUMO1 et qu’il est important afin de maintenir une compétence transcriptionnelle et une stabilité optimale du récepteur. Cette SUMOylation régule également la prolifération et la survie en réponse à un traitement au nocodazole des lignées stables ainsi que la stabilité des microtubules. En conclusion, ces études identifient de nouveaux mécanismes de SUMOylation et phosphorylation utilisés dans la régulation de l'activité du récepteur nucléaire Nor1. Elles permettent également d’approfondir nos connaissances des rôles joués par Nor1 dans la neuroprotection en réponse au stress oxydant ainsi que dans la régulation de la stabilité du réseau de microtubules, ce qui apporte une nouvelle fonction de Nor1. Puisque Nor1 et les autres NR4A sont fortement impliqués dans la formation et maintenance du système nerveux et que les modifications post-traductionnelles peuvent réguler ces fonctions, la découverte et la caractérisation de nouveaux mécanismes de régulation de ces récepteurs ont le potentiel de nous fournir des nouvelles connaissances utiles dans le cadre des maladies neurodégénératives et autres conditions pathologiques. / To answer the many changes happening every instant in its surroundings, cells require a fine-tuned array of molecular messengers to carry on proper signal transduction and homeostasis. The superfamily of nuclear receptors contains forty-eight members implicated in a wide variety of cellular and physiological functions. The nuclear receptors of the NR4A subfamily containing Nur77/NR4A1, Nurr1/NR4A2 and Nor1/NR4A3 are heavily implicated in the development and maintenance of the nervous system. In particular, Nor1/NR4A3 has been shown to be essential for axonal guidance and neuronal survival in the hippocampus. This subfamily also operates differently from other nuclear receptors as they are considered constitutively active orphan nuclear receptors without known endogenous ligand. Therefore, there is an increasing need to identify critical mechanisms that regulate NR4A nuclear receptors and to better understand the control of their activities in a neuronal context. Nuclear receptor activity can be regulated in various ways independently of their interaction with an endogenous ligand. One is through post-translational modifications which allow the regulation of protein function depending on the cellular context. SUMOylation and phosphorylation are post-translational modifications with the potential to regulate nuclear receptor activity, stability and target gene expression. In this thesis, the impact of a canonical SUMOylation site and a phosphorylation dependant SUMOylation motif on the orphan nuclear receptor Nor1 are studied. In the first study, a newly identified non-canonical SUMOylation motif on Nor1 was described. This new motif named pSuM was first identified on the nuclear estrogen receptor ERβ and farnesoid X receptor FXR. We report that this pSuM is located at Lys-137 on Nor1 and is a target of SUMO2. The pSuM differs from traditional SUMOylation motif since it requires to be phosphorylated for SUMOylation to occur. For Nor1, our evidence showed that the obligate phosphorylation of the pSuM on Ser-139 occurred through the MAPK pathway. SUMOylation of Nor1 pSuM reduced Nor1 transcriptional competence, responsive gene expression and chromatin binding. Interestingly, the pSuM of Nor1 also possesses an extension phosphorylated by the CK2 kinase, which is essential to achieve the SUMOylation process. This extension also affected Nor1 protein stability and transcriptional activity. Using stable SH-SY5Y cell lines expressing different SUMO mutants of Nor1, we also showed that Nor1 pSuM SUMOylation reduced cell proliferation and survival to oxidative stress. In the second study, the SUMOylation of Nor1 on a canonical SUMOylation site found at Lys-89 was characterized. This SUMOylation site was found to be targeted mainly by SUMO1 and to be important in maintaining optimal transcriptional competency and stability of the receptor. This SUMOylation also regulated proliferation and survival to a nocodazole treatment of stable cell lines, as well as microtubule network stability. In conclusion, these studies provide novel mechanisms in the regulation of Nor1 activity by SUMOylation and phosphorylation. They also helped to expand our knowledge on the role played by Nor1 in neuroprotection in response to oxidative stress, as well as in the regulation of microtubule stability, which identified a new function of Nor1. Since Nor1 and other NR4A receptors are implicated in the formation and maintenance of the nervous system, the identification of post-translational modifications as a regulatory mechanism uncovers novel opportunities in our understanding of these receptors and provide new insights for neurodegenerative diseases and other neuropathological conditions.
67

神經滋養因子BDNF在PC12細胞中與蛋白激酶CK2對SRE所調控之基因轉錄作用的機制探討 / Neurotrophic factor BDNF up-regulates SRE-mediated gene transcription through protein kinase CK2 in PC12 cells

楊淑萍, Yang, Shu Ping Unknown Date (has links)
神經系統裡,神經滋養因子在調控細胞分化與生存作用中扮演非常重要的角色,近年來的研究顯示 BDNF 的神經保護效果是透過細胞外訊息調控激酶 (extracellular signal-regulated kinase, ERK) 及磷脂酰肌醇3-激酶 (phosphatidylinositol-3 kinase, PI3K) 訊息傳遞路徑調控,然而,還有許多其他的細胞信號傳遞路徑可能參與 BDNF 的保護作用機制中。而蛋白激酶 CK2 (casein kinase 2) 是一種普遍存在於細胞且具有高度保留序列的絲胺酸/蘇胺酸蛋白質激酶,在細胞中具有非常重要的地位。近來研究有非常多證據支持 CK2 是細胞凋亡的抑制者。此外,血清反應因子 (serum response factor, SRF) 是一種轉錄因子,會與保留序列 SRE (即 CArG box) 相結合,而此段序列過去曾在早期即時表現基因 (如 c-fos、Egr ) ,或是抗細胞凋亡基因-Mcl-1-上的啟動子被發現, SRF 調控著基因的活化,進而與細胞增生、存活、突觸活性相關聯,然而調節 SRE 調控之基因的作用機制尚未十分明瞭。因此,本論文研究主要探討在 PC12 細胞中, BDNF調節 SRE 調控轉錄作用機轉 CK2 是否參與其中? 由冷光酶活性試驗結果顯示 BDNF 會顯著地促進 SRE 的轉錄活性,並且當 CK2α 過度表現亦會促進 SRE 調控的轉錄活性,而利用小干擾 RNA 抑制內生性 CK2α 生成,則會降低 SRE 的轉錄活性,更進一步證明 CK2α siRNA 會降低 BDNF 促進 SRE 調控的轉錄活性。此外,將 CK2α 與 SRF S99A 質體一同轉染至細胞中,會減緩 CK2 促進的 SRE 啟動子轉錄活性。為了探討 CK2 調控 SRE 的轉錄活性在神經保護作用裡扮演的角色,因此,將 CK2 蛋白表現量增加是否會保護 PC12 細胞對抗Rotenone所誘發的細胞凋亡傷害?結果顯示 CK2 表現量增加會保護細胞對抗Rotenone誘導的細胞凋亡,並減緩 Rotenone 對 SRE 調控的轉錄活性降低,但是,突變型 SRF S99A 蛋白會降低 CK2α 的影響作用。這些結果顯示 BDNF 促進 SRE 調控的基因表現是會透過 CK2 訊息傳遞路徑。 / The neurotrophins play an important role in cell differentiation and survival of the nervous system. Among them, the neuroprotective effects of brain-derived neurotrophic factor (BDNF) is showed to be mediated by extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) signaling pathway in the recent studies. However, other cellular signaling pathways might be involved in these effects of BDNF. Protein kinase CK2 (casein kinase 2) is a ubiquitous and highly conserved serine/threonine protein kinase and is indicated as a vital cellular role. In recent years, evidences have been mounted in support of the importance of CK2 in the suppression of apoptosis. Serum response factor (SRF) is a transcription factor binding to a consensus DNA sequence SRE (known as a CArG box) which was found in the promoters of some immediately early genes (such as c-fos, Egr) and anti-apoptotic Mcl-1 gene. The activations of SRF-regulated genes were associated with cell proliferation, cell survival and perception of synaptic activity. However, the regulatory mechanism of SRE-mediated genes is not well studied. The SRE-mediated transcription activity through CK2 signaling by BDNF treatment was studied in the PC12 cells in the present study. Results revealed that BDNF significantly increased the SRE promoter activity by luciferase report assay. The SRE-mediated transcription activity was increased by overexpression of CK2α, and the inhibition of endogenous CK2α by small interfering RNA was also shown to reduce this transcription activity. Furthermore, CK2α siRNA treatment antagonized the up-regulation effects of BDNF on SRE-mediated transcription activity. The co-transfection of CK2 and mutant SRF S99A plasmids significantly diminished up-regulatory effects of CK2 on SRE promoter activity. To test this CK2 induction in SRE-mediated transcription plays a role in neuroprotecion, we determined whether over-expression CK2 protects PC12 cells against rotenone-induced apoptosis. The results revealed that the over-expression of CK2α protected cells against rotenone-induced apoptosis and rescued the SRE-mediated transcription activity. Further, these effects of CK2α were blocked by co-transfection of mutant SRF S99A. These above results demonstrate that the up-regulation of BDNF on SRE-mediated genes is through CK2 signaling pathway.
68

蛋白激酶 CK2 與轉錄因子 SRF 所調控之抗細胞凋亡蛋白 Mcl-1 對 PC12 神經細胞之保護機制的探討 / Anti-apoptotic effects of Mcl-1 through CK2-mediated SRF pathway in PC12 cells

曾惠敏, Tseng, Hui Min Unknown Date (has links)
蛋白質激酶 CK2 是一種多功能的絲胺酸/蘇胺酸蛋白激酶,且普遍存在於哺乳類動物細胞中,CK2 受質眾多,對於細胞週期的發展、轉錄作用以及抗細胞凋亡等過程中扮演很重要的角色。SRF 是一種哺乳類動物的轉錄因子,它會結合到血清反應元素 SRE 上進而調控一些促進細胞存活的基因轉錄作用。Mcl-1歸類於抗細胞凋亡 Bcl-2 家族,具有促進細胞存活的能力。過去研究顯示 SRF 的 DNA 結合活性會受到蛋白激酶 CK2 的磷酸化而增加,且 SRF 對 Mcl-1 的活性調控作用也被描述在其他的研究中,然而,對於細胞的訊息目前還沒有更詳細的研究。在本實驗中,我們探討是否可以藉由 CK2 調控 SRF 的路徑來影響 Mcl-1 的表現以作為抗細胞凋亡的機制。利用 CK2 抑制劑 TBB 處理的結果顯示,在 4 hr 後,phospho-SRF 蛋白質表現的降低具有劑量相關性。而相似的降低也可以從 Mcl-1 的 mRNA 和蛋白質表現量觀察到。處理 24 hr 後,phospho-SRF 的蛋白質表現量有顯著降低,而 Mcl-1 的 mRNA 表現量相較 Mcl-1 的蛋白質影響層面微弱。另一方面,轉染野生型 CK2α 會增加 phospho-SRF,相反的,轉染抑制催化活性的突變型 CK2αA156 則會顯著降低 phospho-SRF 的表現。更進一步,野生型 CK2α 同時增加 Mcl-1 的 mRNA 及蛋白質層級,而 CK2αA156 則會降低 Mcl-1 的表現。突變型的 SRF99A 轉染作用降低 Mcl-1 的 mRNA 及蛋白質,並經由共同轉染的實驗顯示具有抵抗上游野生型CK2α 對 Mcl-1 蛋白質的影響。綜合這些結果我們認為 CK2α對 SRF 的訊息調控影響包括對 Mcl-1 的表現。且這條訊息路徑所促進的 Mcl-1 蛋白質表現可能對魚藤酮處理所引發的細胞凋亡作用具有保護的效果。 / Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrates and is ubiquitously expressed in mammalian cells to play an important role in cell cycle progression, transcription, and anti-apoptosis. The serum response factor (SRF) is a mammalian transcription factor which binds to serum response element (SRE) and mediates some gene transcriptions relevent to promote the cell survival. The Myeloid cell leukemia 1 (Mcl-1) belongs to the anti-apoptotic Bcl-2 family and its effect are involved in promoting cell viability. Previous studies have revealed that the DNA-binding activity of SRF is enhanced when it is phosphorylated by protein kinase CK2. The activation regulation of Mcl-1 by SRF has also been reported in other studies. However, the detailed cellular signaling has not been studied well. In the present study, we investigate whether the regulation of Mcl-1 expression through CK2-mediated SRF pathway is involved in its anti-apoptotic effects. The results from CK2 inhibitor TBB revealed that the phosphorylated SRF were reduced in a dose-dependent manner after 4 hr of TBB treatments in PC12 cells. The similar decreases were also observed in the mRNA and protein levels of Mcl-1. After a 24 hr exposure of PC12 cells to TBB, a decreased in phosphorylated SRF and Mcl-1 mRNA were observed; a decreased in Mcl-1 protein level was also detected, albeit to a lesser extent. On the other hand, transfection of the wildtype CK2α increased, whereas transfection of the catalytically inactive CK2αA156 mutant decreased phosphorylated SRF. Further, wildtype CK2α increased, whereas CK2αA156 mutant decreased the mRNA and protein levels of Mcl-1. Furthermore, the mutant SRF99A transfection decreased, the mRNA and protein levels of Mcl-1 and antagonized the up-regulatory effects of wildtype CK2α on Mcl-1 protein level in the co-transfection experiments. These results together suggest that CK2α-mediated SRF signaling is involved in the regulation of Mcl-1 expression, and this signaling pathway may involves the anti-apoptotic effects of Mcl-1 against rotenone treatment.
69

蛋白激酶 CK2 調控受質蛋白 DARPP-32 磷酸化對 PC12 細胞株之抗凋亡機制的探討 / DARPP-32 phosphorylation by protein kinase CK2 mediates the anti-apoptotic effects in PC12 cells

李曉怡, Lee, Hsiao Yi Unknown Date (has links)
蛋白激酶 CK2 是一種具有多種功能的絲胺酸/蘇胺酸蛋白質激酶,其作用的受質眾多且普遍存在於哺乳類動物細胞中。從許多的研究結果顯示,蛋白激酶 CK2 參與調節許多的神經系統功能其中包括有神經保護作用,但是其分子層面的機制目前尚未釐清。DARPP-32(Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa)主要表現在紋狀體中型多刺狀 GABA 神經元中的蛋白質,參與調控與藥物成癮相關的多巴胺訊息傳遞路徑,不過,近年來的一些研究報告指出DARPP-32亦參與了細胞的抗凋亡作用。雖然先前已有研究發現DARPP-32 Ser102胺基酸是CK2的磷酸化作用受質,但是並沒有進一步的研究證實,該胺基酸的磷酸化作用是否參與CK2所調控的細胞機制。屬於抗細胞凋亡蛋白Bcl-2 家族成員之ㄧ的bcl-x基因會經由pre-mRNA選擇性剪裁機制(alternative splicing)而產生兩種異構蛋白Bcl-xL和Bcl-xS,其中Bcl-xL蛋白被證實會促進細胞存活;而Bcl-xS蛋白則會造成細胞死亡。實驗室先前的研究結果發現,在神經滋養因子BDNF的刺激下,CK2可以促進Bcl-xL基因的表現,因此本論文欲進一步探討CK2對DARPP-32 Ser102的磷酸化作用是否參與CK2的抗細胞凋亡訊息傳遞,進而影響Bcl-xL和Bcl-xS的表現。實驗結果顯示,轉染野生型CK2α DNA質體會增加DARPP-32 Ser102的磷酸化現象、Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例;而處理 CK2 抑制劑 TBB 或轉染 CK2α siRNA則會降低 DARPP-32 Ser102的磷酸化現象、Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例。此外,轉染 DARPP-32 siRNA會降低 Bcl-xL的蛋白質表現。轉染模擬之磷酸化構型的DARPP-32 S102D DNA質體會增加Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例;但是,轉染突變型DARPP-32 S102A DNA質體則會降低Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例。進一步利用野生型CK2α和DARPP-32 S102A DNA質體進行細胞共同轉染的實驗結果則發現,DARPP-32 S102A會拮抗野生型 CK2α對促進Bcl-xL蛋白質表現的作用;另外,利用過氧過氫產生細胞氧化逆境下,CK2α或DARPP-32 siRNA處理可以顯著降低 DARPP-32 Ser102的磷酸化現象、Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例,同時會顯著造成細胞凋亡。綜合本論文的實驗結果,顯示CK2會透過DARPP-32 Ser102的磷酸化作用而調控Bcl-xL以及Bcl-xS的表現,而且在氧化逆境下,此條細胞訊息傳遞路徑應參與了細胞的抗凋亡機制。 / Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrares and is ubiquitously expressed in mammalian cells. Many studies have shown that CK2 is involved in many neuronal functions including neuroprotection, but its cellular mechanisms are not well-studied. DARPP-32 (Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa) is highly enriched in striatal medium-size spiny GABA neurons and is a prominent mediator of dopamine signalling which relates with drug abuse. Beside its well-known function in drug abuse, recent studies also reveal that DARPP-32 may be involved in the anti-apoptotic effects. Although the Ser102 residue of DARPP-32 is a phosphorylation site for CK2, this phosphorylation-mediated CK2 signaling has not been studied yet. The bcl-x gene, one member of the Bcl-2 family, encodes two isoform proteins Bcl-xL and Bcl-xS by the pre-mRNA alternative splicing. The former increases cell survival and the later enhances cell apoptosis. Our previous study found that CK2 can increase Bcl-xL expression by BDNF treatment. In the present study, we investigate whether DARPP-32 ser102 phosphorylation also mediates the CK2 signaling for cell survival. Our results revealed that DARPP-32 Ser102 phosphorylation, Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio were all increased by wild-type CK2α plasmid DNA transfection. Meanwhile, CK2 inhibitor TBB treatment or CK2α siRNA transfection decreased DARPP-32 Ser102 phosphorylation, Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio. On the other hand, DARPP-32 siRNA transfection decreased Bcl-xL protein level. Furthermore, transfection of DARPP-32 S102D, which mimics the constitutive phosphorylation form, increased whereas transfection of mutant S102A decreased the Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio. Further, the mutant DARPP-32 S102A antagonized the up-regulatory effects of wild-type CK2α on Bcl-xL protein level in the co-transfection experiments. From the results of H2O2-induced oxidative stress experiments, we also found that prior knock-down of CK2 or DARPP-32 can aggravate the decrease in DARPP-32 Ser102 phosphorylation, Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio by H2O2 treatment. These results together suggest that DARPP-32 mediates CK2α signaling in regulating Bcl-xL/Bcl-xS expression and this signaling pathway might be involved in cell survival under oxidative stress.
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蛋白磷酸水解酶PP1在蛋白激酶CK2a調控 抗凋亡蛋白Bcl-xL基因表現過程中的角色 / The role of protein phosphatase 1 in the protein kinase CK2a-mediated anti-apoptotic Bcl-xL gene expression

許焙琹 Unknown Date (has links)
蛋白激酶 CK2 是一種具有多功能的絲胺酸/蘇胺酸蛋白激酶,大量表現於哺乳類動物的腦中,對於調控細胞週期的發展、基因表現、訊息傳遞以及抗細胞凋亡機制扮演相當重要的角色。許多研究顯示 CK2 也參與調節許多神經系統功能,包括神經保護及神經存活,但是其中調控機制目前尚未釐清。DARPP-32 (dopamine- and cAMP- regulated phosphoprotein with a molecular mass of 32 kDa) 主要表現在紋狀體中型多刺狀神經元中,過去研究已證實 DARPP-32 Ser102 胺基酸是CK2 的磷酸化作用受質。雖然DARPP-32 被發現主要透過抑制蛋白磷酸水解酶 PP1 參與藥物成癮的細胞調控機制,但近年研究指出DARPP-32 也參與抗細胞凋亡作用。PP1 是真核細胞的絲胺酸/蘇胺酸磷酸水解酶,能調節多種細胞功能,如轉錄、細胞訊息傳遞及細胞凋亡。過去文獻已指出 PP1 可以調節 Bcl-x 基因的 pre-mRNA 選擇性剪切,再經由轉譯過程合成抗細胞凋亡 Bcl-xL 異構蛋白,研究也發現抑制 PP1 可以防止細胞週期的停滯及細胞凋亡,強調細胞在壓力的情況下,PP1 扮演了相當關鍵性的角色。因此論文以人類神經母細胞瘤 SH-SY5Y 為實驗模式,探討透過 CK2 調控 DARPP-32 Ser102 的磷酸化是否具有抑制 PP1 的活性並促進細胞存活的作用。實驗結果顯示,抑制 CK2或DARPP-32 蛋白含量會導致細胞存活率下降,轉染 CK2 siRNA 會降低 DARPP-32 Ser102 的磷酸化現象、Bcl-xL 的蛋白質表現;轉染DARPP-32 siRNA 及突變型DARPP-32 S102A DNA 質體也會降低 Bcl-xL 的蛋白質表現,PP1 活性則會因轉染突變型DARPP-32 S102A DNA 質體而增加;此外,給予 PP1 抑制劑的實驗結果發現會促進 Bcl-xL/Bcl-xS mRNA 的比例以及 Bcl-xL 的蛋白質表現量。利用過氧化氫誘導細胞造成氧化壓力狀況下,同時給予 PP1 抑制劑,發現 Bcl-xL 的蛋白質表現量會回復以及促進細胞存活。轉染 CK2-EGFP 或 DARPP-32 S102D DNA 質體可以顯著回復Bcl-xL 的蛋白質表現量及Bcl-xL/Bcl-xS mRNA 的比例,轉染 DARPP-32 S102D DNA 質體亦可降低 PP1 的活性。論文的實驗結果提供 CK2 調節抗細胞凋亡基因表現的新機制,是經由促進 DARPP-32 Ser102 磷酸化作用進而抑制 PP1 活性,此條細胞訊息傳遞路徑將可提供應用於在氧化壓力下提升神經存活的臨床治療。 / Protein kinase casein kinase II (CK2) is a multifunctional serine/threonine protein kinase and is highly abundant expression in the mammalian brain. CK2 plays an important role in the regulation of the cell cycle, gene expression, signal transduction and anti-apoptotic mechanisms. A number of studies have indicated that CK2 is involved in several neuronal functions including the neuroprotection and neuron survival, but its cellular mechanisms are not well-studied. The DARPP-32 (dopamine- and cAMP-regulated phosphoprotein with a molecular mass of 32 kDa) is highly enriched in the striatal medium spiny neurons and the Ser102 residue is identified as the phosphorylation site for CK2. Although DARPP-32 is known as a prominent cellular mediator of drug abuse through the inhibition of protein phosphatase 1 (PP1), the recent studies indicate that DARPP-32 may also be involved in the anti-apoptotic effects. Protein phosphatase PP1 is a major eukaryotic serine/threonine phosphatase that regulates diverse cellular functions such as transcription, cell signaling and apoptosis. PP1 is indicated to regulate the pre-mRNA alternative splicing of Bcl-x gene to encode the anti-apoptotic Bcl-xL isoform. Inhibition of PP1 prevents the induction of cell cycle arrest and apoptosis, underlines the crucial role of PP1 in the cellular response to the stress. In my thesis study, the neuroblastoma SH-SY5Y cell line system was used to investigate whether the promotion of cell survival by PP1 inhibition is through the signaling pathway of DARPP-32 Ser102 phosphorylation by CK2. The current results reveals that the cell viability is decreased under down-regulations of CK2 and DARPP-32. The Ser102 phosphorylation status of DARPP-32, Bcl-xL mRNA and protein level are decreased by CK2 siRNA transfection. Transfection of either DARPP-32 siRNA or mutant DARPP-32 S102A plasmid DNA decreased the Bcl-xL protein level. The PP1 activity was increased by mutant DARPP-32 S102A plasmid DNA transfection. Furthermore, the PP1 inhibitor treatment increased the Bcl-xL/Bcl-xS mRNA ratio and Bcl-xL protein level. Under oxidative stress, inhibition of PP1 activity can reverse the H2O2-induced decrease in Bcl-xL protein level and promote the cell viability. The transfection of CK2-EGFP or DARPP-32 S102D plasmid DNA both can antagonize the effects of H2O2 on Bcl-xL protein level and the Bcl-xL/Bcl-xS mRNA ratio. The DARPP-32 S102D plasmid DNA transfection also attenuated the induction of PP1 activity under oxidative stress. These findings provide another insight for the regulation of anti-apoptotic gene expression by inhibition of PP1 activity through DARPP-32 phosphorylation on Ser102 by CK2. This signaling pathway might be applied in the clinical therapy for neuronal survival under oxidative stress.

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