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Schrötter, Sandra. "Specificity of developmental- and growth factor-dependent phosphorylation of Akt isoforms in neurons." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17593.
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Ein Signalweg während der neuronalen Entwicklung im adulten Gehirn ist der PI3K-PTEN-Akt Signalweg. Akt ist eine Kinase die drei verschiedene Isoformen besitzt, welche durch die Phosphorylierung von S473 und T308 aktiviert werden. KO Modelle der Isoformen haben gezeigt, dass nicht alle Funktionen von anderen Isoformen kompensiert werden können. Die genaue Rolle der einzelnen Isoformen in einem neuronalen Zusammenhang ist nur wenig untersucht. Ziel dieser Arbeit war, eine detaillierte Analyse der einzelnen Akt Isoformen nach der Aktivierung des PI3K-PTEN Signalweges. Dazu wurde im Labor eine neue Methode zur isoelektrischen Fokussierung etabliert., welche Proteine nach ihrer Ladung trennt und somit eine Analyse der Dynamik von Akt Phosphorylierungen in neuronalen Zellen erlaubt. Im Zuge dieser Arbeit konnten wir bisher unerkannte Merkmale der Akt Aktivierung und Phosphorylierung identifizieren. Wir konnten zeigen, dass die S473 und T308 Phosphorylierung in Neuroblastomazellen unabhängig voneinander auftreten kann und, dass verschiedene Akt1 Moleküle unterschiedlich auf die Inhibition von PI3K reagieren. Außerdem konnten wir Verschiebungen in der Aktivierung und in der Expression der unterschiedlichen Isoformen während der postnatalen Gehirnentwicklung der Ratte feststellen. Des Weiteren konnten wir zeigen, dass die Aktivierung von Akt von dem Signal und dem Alter der Neurone abhängig ist. Noch nicht vollständig differenzierte Neurone reagieren vor allem auf BDNF Stimulation, wohingegen adulte, differenzierte Neurone hauptsächlich auf EGF reagieren und dort explizit Akt2 über EGFR und PI3K-p110α Signale aktiviert wird. Im Gegensatz dazu führt der Verlust von PTEN zu einer Aktivierung von hauptsächlich Akt1. Zusammenfassend zeigt diese Arbeit einen komplexen Zusammenhang der Phosphorylierung von Akt auf, welcher Signal- und Entwicklungsabhängig ist bei dem unterschiedliche Akt Populationen auf Wachstumsfaktoren und auf PTEN Verlust reagieren.A major pathway involved in neuronal development is the PI3K-PTEN-Akt pathway. Akt comprises three isoforms, which are activated by phosphorylation of the residues S473 and T308. KO animals for the isoforms have shown differential as well as redundant functions of the three isoforms. However, their individual role in neuronal signaling pathways has not yet been studied in great detail. The aim of this study was to obtain further insight into differential Akt isoform signaling in response to changes in the activity of PI3K and PTEN pathway. A new isoelectric focusing method was established, which allowed us to separate Akt proteins according to their charge, therefore, providing a refined read-out to study dynamics of Akt phosphorylation in a neuronal background. In the course of this project we were able to identify previously undescribed features of Akt phosphorylation and activation. First, we could provide evidence for an uncoupling of the two activating phosphorylation events at S473 and T308 in neuroblastoma cells and differential sensitivities of Akt1 forms towards PI3K inhibition. Secondly, we found a transient shift in Akt isoform activation and abundance during postnatal rat brain development. Thirdly, we were able to show that the activation of different Akt isoforms is dependent of the upstream signal as well as the age of the neuron. Immature neurons were found to be highly responsive to BDNF treatment, whereas mature neurons were most responsive to EGF stimulation leading exclusively to activation of Akt2 in an EGFR- and PI3K/p110α-dependent manner. Stimulation of Akt phosphorylation by the loss of PTEN led to an activation of mainly Akt1 forms, which suggests inherent differences in the Akt pools that are accessible to growth factors dependent PI3Ks as compared to the pools that are controlled by PTEN. In summary, this thesis demonstrates the presence of complex phosphorylation events of Akt in a developmental- and signal-dependent manner in neurons.
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Lefevre, Carine. "Mécanismes de régulation de la balance prolifération/différenciation érythroïde par les facteurs de transcription GATA-1, FOG-1, E2F et la voie de signalisation Akt." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T010/document.
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Avec plus de 100 milliards de globules rouges produits chaque jour, le lignage érythroïde présente la plus grande capacité de production cellulaire chez le mammifère adulte. Cette production requiert une balance fine entre la prolifération cellulaire, régulée principalement par la voie de signalisation érythropoïétine (Epo)/PI3K/Akt, et la différenciation érythroïde induite par le couple de facteurs de transcription GATA-1/FOG-1. Des interconnexions entre ces deux grands systèmes ont été décrites dans le laboratoire : 1) le facteur de transcription GATA-1 est phosphorylé par Akt en réponse à l’Epo et cette phosphorylation semble avoir un rôle dans la différenciation érythroïde ; 2) GATA-1 est capable d’interagir avec la protéine du rétinoblastome pRb, impliquée dans la régulation du cycle cellulaire, et le complexe formé est nécessaire à l’érythropoïèse terminale.L'objectif de ma thèse était d’étudier les mécanismes moléculaires impliqués dans la balance prolifération/différenciation cellulaire au cours de l’érythropoïèse, et en particulier de déterminer le rôle moléculaire et physiologique de la phosphorylation de GATA-1 par Akt en réponse à l’Epo. Nos travaux ont montré que cette phosphorylation est une des clefs de la dynamique de l’érythropoïèse. Dans sa forme non phosphorylée, GATA-1 ralentit le cycle cellulaire via le complexe GATA-1/pRb/E2F. Cette étape préliminaire est nécessaire à la mise en place de la différenciation érythroïde terminale. La phosphorylation de GATA-1 induit d’une part la dissociation de GATA-1/pRb/E2F favorisant l’expansion cellulaire, et d’autre part la formation du complexe GATA-1/FOG-1 nécessaire à l’activation des gènes érythroïdes. Ce modèle apporte une explication moléculaire au blocage de la différenciation érythroïde terminale induite par le mutant GATA-1V205G qui n’interagit pas avec FOG-1. Ainsi, la phosphorylation constitutive de GATA-1V205G et l’augmentation de la quantité relative de FOG-1 permettent de restaurer la différenciation érythroïde induite par ce mutant in vitro. Enfin, l’étude d’un modèle murin exprimant une protéine GATA-1 non phosphorylable par Akt montre l’apparition d’une anémie létale lorsque la voie IGF-1 est inhibée. Cela démontre l’importance de la dynamique moléculaire induite par la phosphorylation de GATA-1, et met en évidence le rôle majeur de l’IGF-1 dans l’érythropoïèse in vivo.En conclusion, nous proposons un nouveau modèle moléculaire de la régulation de la balance prolifération/différenciation érythroïde dans lequel la phosphorylation de GATA-1 par Akt coordonne la distribution de GATA-1 dans deux complexes protéiques fonctionnels différents : GATA-1/pRb/E2F versus GATA-1/FOG-1. Nous mettons également en évidence l’IGF-1 comme acteur central de la compensation mise en place in vivo pour pallier à l’absence de phosphorylation de GATA-1With more than 100 billion red blood cells generated every day, the erythroid lineage has the largest output of cell production in adult mammals. This production requires a tight balance between cell proliferation, mainly controlled by erythropoietin (Epo)/PI3K/Akt signaling pathway, and erythroid differentiation induced by GATA-1 and FOG-1 transcription factors. Various links between these two processes have been previously demonstrated in the laboratory: 1) Epo-activated Akt directly phosphorylates GATA-1 transcription factors, and this phosphorylation seems to be involved in erythroid differentiation; 2) GATA-1 binds to the cell cycle regulator retinoblastoma protein (pRb), and the resulting complex is essential for terminal erythropoiesis.We investigated the molecular mechanisms involved in the cell proliferation/differentiation balance during terminal erythropoiesis; in particular, we studied the molecular and physiological role of Epo-induced GATA-1 phosphorylation. Our findings suggest that this phosphorylation is one of the key processes in erythropoiesis dynamics. In its unphosphorylated form, GATA-1 can break cell cycle progression via GATA-1/pRb/E2F complex. This preliminary step is necessary for terminal erythroid differentiation. GATA-1 phosphorylation promotes GATA-1/pRb/E2F dissociation, allowing cell cycle progression, and GATA-1/FOG-1 binding, necessary to activate erythroid genes. Our model provides a molecular explanation for the arrest of terminal erythroid differentiation observed in the non-FOG-1-binding mutant GATA-1V205G. We show that the constitutive phosphorylation of GATA-1V205G and the increase of FOG-1 protein amount rescue erythroid differentiation in vitro. Finally, knock-in expression of unphosphorylatable GATA-1 in mice leads to lethal anemia when the IGF-1 signaling pathway is inhibited. This shows the importance of the molecular dynamics of GATA-1 phosphorylation, and highlights the major role of IGF-1 in erythropoiesis, in vivo.In conclusion, we propose a new molecular model for the control of the balance between proliferation and erythroid differentiation. GATA-1 phosphorylation by Akt coordinates the involvement of GATA-1 in two different functional protein complexes: GATA-1/pRb/E2F and GATA-1/FOG-1. We also highlight the major role of IGF-1 in compensating for the lack of GATA-1 phosphorylation in vivo
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Conley, Travis B. "The influence of training status on ERK and AKT phosphorylation in human skeletal muscle." Virtual Press, 2005. http://liblink.bsu.edu/uhtbin/catkey/1319219.
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Exercise induces morphological and metabolic adaptations that are highly specific to the mode of exercise training. These specific phenotypical changes are due to an equally specific molecular response that may depend on the activation and coordination intramuscular signaling pathways. Just as metabolic and morphological changes are influenced by the mode of exercise training, the signaling pathways that mediate exercise adaptation may also be directly related to the training status of skeletal muscle. For example, pre-conditioned skeletal muscle may exhibit a specific intracellular signaling response to an acute bout of exercise that is dependent on past training history. Both Akt (protein kinase B) and extra-cellular signal-related kinase (ERK 1 /2) have been shown to be phosphorylated in response to an acute bout of resistance exercise in human skeletal muscle and have been suggested to mediate the adaptive response to exercise. The purpose of this investigation was to examine the response of Akt and ERKI/2 to an acute bout of resistance exercise in three groups with distinctly different exercise training backgrounds. Twenty one subjects performed 3 sets of 10 repetitions of knee extension exercise at 70% 1-RM. The subjects consisted of a resistance-trained group (RE) (n=7), endurance trained group (END) (n=7) and a sedentary group (SED) (n=7). Muscle biopsies were taken from the vastus lateralis muscle before, immediately after, and 10 min post-exercise and were analyzed for phosphorylation of Akt and ERK1/2. ERK1/2 phosphorylation increased 47%, and 54% from pre-exercise to immediately post-exercise in the SED and RE groups respectively (p < 0.05). ERK1/2 phosphorylation increased 95%, 196%, and 47% from pre-exercise to 10 min post-exercise in the SED, RE, and END groups, respectively. (p < 0.05). The magnitude of ERK1/2 phosphorylation 10 min post-exercise was different between each group and may be linked to the group's training status. (p < 0.05) Akt phosphorylation decreased 42% and 37% from pre-exercise to immediately post-exercise in the SED and END group, respectively (p < 0.05). There was a 40 % increase in Akt phosphorylation from immediate post-exercise to 10 min post-exercise in the END group. In conclusion, training status appears to influence the magnitude and time course of activation of both Akt and ERK1/2 in response to an acute bout of resistance exercise. The immediate response of both ERK1/2 and Akt may play a key role in the adaptive response of skeletal muscle ultimately resulting in metabolic and morphological changes that are dependent on the past training history of the individual.School of Physical Education, Sport, and Exercise Science
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Lyons, Traci Renae. "Analysis of potential substrates for the pro-survival kinase AKT /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2006.
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Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006.Typescript. Includes bibliographical references (leaves 194-209). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;
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Chopra, Ines. "Molecular Mechanisms of AMPK- and Akt-Dependent Survival of Glucose-Starved Cardiac Myocytes." Scholarly Repository, 2012. http://scholarlyrepository.miami.edu/oa_dissertations/710.
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Muscle may experience hypoglycemia during ischemia or insulin infusion. During severe hypoglycemia energy production is blocked and an increase in AMP:ATP activates the energy sensor and putative insulin-sensitizer AMP-dependent protein kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique glucose stress defense pathway involving AMPK-dependent, insulin-independent activation of the insulin signaling pathway. Results from my work showed that the central insulin signaling pathway is rapidly activated when cardiac and skeletal myocytes are subjected to conditions of glucose starvation. The effect occurred independently of insulin receptor ligands (insulin and IGF-1). There was a >10-fold increase in the activity of Akt as determined by phosphorylation on both Thr308 and Ser473. Phosphorylation of glycogen synthase 3 beta (GSK3b) increased in parallel, but phosphorylation of ribosomal 70S subunit-S6 protein kinase (S6K) and the mammalian target of rapamycin complex 1 (mTORC1) decreased. We identified AMPK as an intermediate in this signaling network; AMPK was activated by glucose starvation and many of the effects were mimicked by the AMPK-selective activator aminoimidazole carboxamide ribonucleotide (AICAR) and blocked by AMPK inhibitors. Glucose starvation increased the phosphorylation on IRS-1 on Ser789, but phosphomimetics revealed that this conferred negative regulation. Glucose starvation enhanced tyrosine phosphorylation of IRS-1 and the insulin receptor, effects that were blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified proteins confirmed that the insulin receptor is a direct target of AMPK. Insulin receptor kinase activity was essential for cardiac myocytes to survive gluose starvation as inhibition of the IR led to increased cell death in glucose-starved myocytes. Selective activation of mTORC2 by glucose starvation to increase Akt-Ser473 phosphorylation was dependent on the presence of rictor. SIN1 also seemed to be instrumental in the activation of mTORC2 as its levels and binding to rictor increased under glucose starvation. AMPK-mediated activation of the insulin signaling pathway conferred significant protection against the stresses of glucose starvation. Glucose starvation promoted energy conservation, augmented glucose uptake and enhanced insulin sensitivity in an AMPK- and Akt-dependent manner. My results describe a novel ligand-independent and AMPK-dependent activation of the insulin signaling pathway via direct phosphorylation and activation of the IR followed by activation of PI3K and Akt. These results may be relevant in conditions of myocardial ischemia superimposed with type 2 diabetes where AMPK could directly modify the IR to promote cell survival and confer protection.
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Goodwani, Sunil G. "Amoxicillin and Augmentin Reduce Ethanol Intake and Increase GLT1 Expression as well as AKT Phosphorylation in Mesocorticolimbic Regions." University of Toledo Health Science Campus / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=mco1403873055.
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Tamburini-Bonnefoy, Jérôme. "Régulation des voies de signalisation P13K/Akt et mTOR dans les leucémies aiguës myéloïdes : implications physiopathologiques et thérapeutiques." Paris 7, 2009. http://www.theses.fr/2009PA077179.
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Nous avons montré qu'une activation constitutive de la voie PBK/Akt est détectée dans 50% des _ leucémies aiguës myéloïdes (LAM), due le plus souvent à une autocrinie IGF-1/IGF-1R. Néanmoins, l'inhibition spécifique de la PI3K ou de l'IGFlR n'est que cytostatique dans les LAM. L'inhibition spécifique de mTORCl par la rapamycine induit une sur-stimulation de la voie PI3K/Akt dépendante l'IGFl. Ce mécanisme limite l'activité anti-leucémique de la rapamycine dans les LAM. Nous avons ensuite montré que mTORCl ne contrôle pas la traduction dans les LAM. La rapamycine ne réprime donc pas la synthèse de protéines oncogéniques expliquant ses effets limités dans les LAM. Ce mécanisme dépend de la kinase Pim-2, qui contrôle la phosphorylation du régulateur traductionnel 4E-BP1, suggérant l'intérêt potentiel d'inhibiteurs de Pim-2 dans les LAM. De plus, l'inhibition du complexe d'initiation de la traduction par le 4EGI-1 induit l'apoptose des blastes de LAM sans toxicité sur les cellules hématopoïétiques normales, désignant l'inhibition de la traduction comme une nouvelle perspective thérapeutiques dans les LAM. Nous avons ensuite montré que mTOR contrôle 4E-BP1 et ainsi la traduction dans les LAM, indépendamment des complexes mTORCl et mTORC2. Nous avons activé le répresseur physiologique de mTOR, la voie LKB1/AMPK, par la metformine, ce qui se traduit par des effets anti-leucémiques marqués dans les LAM. Ce travail a donc contribué à décrire la dérégulation oncogénique de la traduction comme étant une cible intéressante pour le développement de thérapeutiques — ciblées, par diverses approches basées sur une meilleure compréhension de la physiopathologie des LAMIn acute myeloid leukemia (AML), aberrant activation of signal transduction pathways enhances the survival of leukemic cells. We showed that 50% of primary AML samples had a constitutive activation of -PI3K/Akt generally due to an autocrine IGF-1/IGF-1R loop. However, specific PI3K and ÏGF-1R inhibitors'only showed limited anti-leukemic activity. The specific inhibition of mTORCl by rapamycm induced an IGF 1-dependent overactivation of PI3K that limited thé anti-leukemic potential of rapamycm. This emphasized the potential benefit of dual PI3K and mTORCl inhibitors in AML. Surprismgly, the mRNA translation process was not controlled by mTORCl in AML, and rapamycm failed to reduce the expression of oncogenic proteins. The Pim-2 kinase was involved in this mechamsm suggesting a potential benefit for Pim-2 inhibitors in the future. We showed that directly targeting the translation initiating complex by the 4EGI-1 compound decreased AML cell survival while sparing normal hematopoiesis, suggesting other therapeutic perspectives in AML therapy. We also showed that mTOR controlled by complex phosphorylation events the translation regulator 4E-BP1 m AML, independently of mTORCl and mTORC2 Finally, we activated the LKB1/AMPK pathway using metformin, which represents a physiological repressor of mTOR activity. This molecule markedly impaired the translation of oncogenic mRNA and repressed the growth of AML cells. The present work therefore contributed to emphasize the oncogenic deregulation of mRNA translation as a valuable target m AML that could be inhibited using different physiologic-based approaches
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Lyros, Orestis, Ann-Kristin Lamprecht, Linghui Nie, René Thieme, Katharina Götzel, Mario Gasparri, George Haasler, Parvaneh Rafiee, Reza Shaker, and Ines Gockel. "Dickkopf-1 (DKK1) promotes tumor growth via Akt-phosphorylation and independently of Wnt-axis in Barrett’s associated esophageal adenocarcinoma." e-Century Publishing, 2019. https://ul.qucosa.de/id/qucosa%3A33709.
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Esophageal adenocarcinoma (EAC) is still associated with poor prognosis, despite modern multi-modal therapies. New molecular markers, which control cell cycle and promote lymph node metastases or tumor growth, may introduce novel target therapies. Dickkopf-1 (DKK1) is a secreted glycoprotein that blocks the oncogenic Wnt/β-catenin signaling and its aberrant expression has been observed in many malignancies, including EAC. In this study, we investigated the biological role of DKK1 in EAC. Analysis of DKK1 and active β-catenin expression in human esophageal tissues confirmed a simultaneous DKK1-overexpression together with aberrant activation of β-catenin signaling in EAC in comparison with Barrett’s and healthy mucosa. To elucidate the molecular role of DKK1, the OE33 adenocarcinoma cells, which were found to overexpress DKK1, were subjected to functional and molecular assays following siRNA-mediated DKK1-knockdown. At the functional level, OE33 cell viability, proliferation, migration and invasion were significantly attenuated by the absence of DKK1. At the molecular level, neither DKK1-knockdown nor application of exogenous recombinant DKK1 were found to alter the baseline β-catenin signaling in OE33 cells. However, DKK1-knockdown significantly abrogated downstream Akt-phosphorylation. On the other hand, the Wnt-agonist, Wnt3a, restored the Akt-phorphorylation in the absence of DKK1, without, however, being able to further stimulate β-catenin transcription. These findings suggest that the β-catenin transcriptional activity in EAC is independent of Wnt3a/DKK1 site-of-action and define an oncogenic function for DKK1 in this type of malignancy via distinct activation of Akt-mediated intracellular pathways and independently of Wnt-axis inhibition. Taken together, DKK1 may present a novel therapeutic target in EAC.
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Murakami, Tomoaki. "Angiopoietin-1 attenuates H2O2-induced SEK1/JNK phosphorylation through the phosphatidylinositol 3-kinase/Akt pathway in vascular endothelial cells." Kyoto University, 2006. http://hdl.handle.net/2433/143830.
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Matthews, Jason Aaron. "Investigation of the effects of increased levels of O-GlcNAc protein modification on protein kinase C and Akt." [Tampa, Fla] : University of South Florida, 2006. http://purl.fcla.edu/usf/dc/et/SFE0001723.
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Abbott, Jonathan James. "Aβ modulation of Akt phosphorylation in a transgenic mouse model of AD : the potential role of nicotinic and NMDA receptors." Thesis, King's College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.428583.
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Molina-Calavita, Maria. "Huntingtine et mitose." Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00766423.
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La maladie de Huntington (MH) est une maladie neurodégénérative héréditaire autosomique dominante. Elle résulte d'une expansion anormale de glutamines (polyQ) dans la partie N-terminale de la protéine huntingtine (HTT ; codé par HTT). La MH est caractérisée par la dysfonction et la mort de cellules neuronales dans le cerveau, entraînant l'apparition de symptômes cognitifs, psychiatriques et moteurs, dévastateurs chez les patients. De nombreuses études sur des modèles animaux et cellulaires montrent que l'expansion polyQ dans la protéine mutante conduit à un gain de nouvelles fonctions toxiques, ainsi qu'à la perte de fonctions neuroprotectives de la protéine sauvage. Pendant ma thèse, je me suis intéressée à la description et à la validation fonctionnelle d'un nouvel outil pour étudier la HTT : pARIS-htt. pARIS-htt est un gène synthétique construit pour faciliter le clonage et le marquage de la protéine HTT totale. En utilisant différentes approches cellulaires, nous avons montré que pARIS-htt peut remplacer le rôle de la HTT endogène dans le transport de vésicules du Golgi ainsi que du brain derived neurotrophic factor (BDNF). La version mutante de pARIS-htt ne peut pas restaurer cette fonction. Parallèlement, nous avons généré deux variants de pARIS-htt avec soit une délétion dans la région d'interaction de la HTT avec la dynéine, moteur moléculaire se dirigeant vers l'extrémité négative des microtubules, soit avec la huntingtin associated protein 1 (HAP1), l'un de ses interacteurs. Dans les expériences de remplacement du gène, aucun des deux mutants n'a restauré le transport vésiculaire.Un autre aspect de ma thèse a été d'étudier le rôle de la HTT au cours de la mitose. Nous avons mis en évidence l'importance de la HTT dans le contrôle de l'orientation du fuseau. Cette fonction est perdue lorque la HTT est mutée, mais restaurée lorsque celle-ci est phosphorylée par Akt à la sérine 421. Le contrôle de l'orientation du fuseau est particulièrement important durant la neurogénèse puisque cette orientation ainsi que le mode de division sont impliqués dans la détermination des devenirs cellulaires. Cette fonction de la HTT est conservée chez la D. melanogaster.Cette étude a donc permis de mieux comprendre les fonctions de la HTT, et de proposer de nouvelles cibles thérapeutiques pour traiter la MH.
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Chapuis, Nicolas. "Dérégulation des voies de signalisation P13K/Akt et mTOR dans les Leucémies Aigües Myéloïdes." Paris 7, 2011. http://www.theses.fr/2011PA077010.
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Les leucémies aiguës myéloïdes (LAM) constituent un groupe hétérogène de pathologies acquises caractérisées par une prolifération clonale de cellules hématopoïétiques immatures. De nombreuses voies de signalisation sont activées dans les cellules blastiques et la compréhension des mécanismes d'activation et de régulation de ces voies est essentielle en vue du développement de thérapeutiques d'inhibition ciblées. Dans ce contexte, je me suis intéressé aux voies de signalisation PBK/Akt et mTOR qui sont constitutivement activées dans respectivement 50% et 100% des cas. J'ai tout d'abord montré que la dérégulation de la voie PBK/Akt est principalement due à une production autocrine d'IGF-1. J'ai ensuite observé que l'inhibition spécifique de la signalisation mTORCl par la rapamycine induit une sur-stimulation de la voie PBK/Akt dépendante du système IGF-1/IGF-1R. J'ai également démontré que l'inhibition concomitante des voies PBK et mTOR par le NVP-BEZ235 induit un effet anti-leucémique puissant in vitro dans les LAM. Enfin, je me suis intéressé aux mécanismes de régulation des FT FOXO connus pour leurs propriétés de suppresseurs de tumeurs. J'ai observé dans les blastes une inactivation constante du FT FOXO3a due à l'activation constitutive de la kinase IicB. La réactivation des fonctions anti-oncogéniques de FOXO3a via l'inhibition de la kinase LcB pourrait donc constituer une stratégie thérapeutique intéressante dans les LAM. Ce travail montre donc que la compréhension des mécanismes de régulation des voies de signalisation PBK/Akt/mTOR dans les LAM est essentielle pour permettre le développement de nouvelles thérapeutiques dans cette pathologieAcute myeloid leukemia (AML) is a clonal hematopoietic stem cell disorder, characterized by uncontrolled proliferation/survival of immature myeloid progenitors, blocked in their differentiation. Aberrant activation of multiple signalling pathways is frequently found in AML cells. However, a better understanding of the mechanisms leading to constitutive activation of these pathways is required to develop new targeted therapies in this disease. In this work, I investigated the implication in AML cells of both PBK/Akt and mTOR signalling pathways which are constitutively activated in respectively 50% and 100% of cases. I first demonstrated that the PBK/Akt deregulation is mostly due to an IGF1 autocrine production. I then observed that the specific inhibition of mTORCl with rapamycin induces an overactivation of PBK/Akt signalling du to the IGF-1 autocrine loop. I also demonstrated that concomitant inhibition of both PBK/Akt and mTORCl signalling pathways with compounds such as NVP-BEZ235, a dual PBK/mTOR inhibitor induce a promising anti-leukemic effect in vitro and could be use in clinical trials. Finally, I investigated the implication of FOXO proteins, which possess tumours suppressive functions. Interestingly, I observed that FOXO3a is constantly inactivated in AML cells, due to the constitutive activation of the IkB kinase. Réactivation of FOXO3a activity through IkB inactivation represents therefore another therapeutic strategy for AML. Altogether, this work clearly suggests that a better understanding of mechanisms leading to the deregulation o both PBK/Akt and mTOR pathways and their connexions is required to develop new targeted therapies in this disease
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Colin, Emilie. "La voie de signalisation IGF-1/Akt/Calcineurine et les conséquences moléculaires de la phosphorylation de la huntingtine sur le transport axonal." Paris 11, 2007. http://www.theses.fr/2007PA11T028.
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Schrötter, Sandra [Verfasser], Peter-Michael [Gutachter] Kloetzel, Stephan [Gutachter] Sigrist, and Nils [Gutachter] Blüthgen. "Specificity of developmental- and growth factor-dependent phosphorylation of Akt isoforms in neurons / Sandra Schrötter ; Gutachter: Peter-Michael Kloetzel, Stephan Sigrist, Nils Blüthgen." Berlin : Lebenswissenschaftliche Fakultät, 2016. http://d-nb.info/1115767496/34.
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Stechschulte, Lance A. "The Co-chaperones FKBP51 and PP5 Control Nuclear Receptor Phosphorylation and Adipogenesis." University of Toledo Health Science Campus / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=mco1370871316.
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Touati, Sabeur. "Obésité, risque athérogène et effet thérapeutique direct de l’exercice physique : étude sur la contribution des voies signalétiques Akt/eNOS et NADPH oxydase pour expliquer les mécanismes vasculo-protecteurs de l’exercice physique chez le rat rendu obèse par une alimentation enrichie en graisse." Thesis, Avignon, 2010. http://www.theses.fr/2010AVIG0704/document.
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La prévalence de l’obésité est en constante augmentation dans les pays occidentaux, en raison d’une sédentarisation accompagnée d’une alimentation malsaine. L’obésité est souvent associée à une dysfonction endothéliale et à un risque athérogène élevé. Plusieurs observations cliniques ont montré que la modification du mode de vie, incluant la pratique régulière d’une activité physique et l’adoption d’un mode alimentaire sain, représente une stratégie efficace pour combattre l’obésité et ses complications cardiovasculaires. Cependant, de nombreux mécanismes précisant les effets thérapeutiques directs de l’exercice physique sur le risque athérogène lié à l’obésité sont encore largement inconnus. Le but principal de ce travail a donc été d’identifier, en utilisant un modèle de rat rendu obèse par un régime enrichi en graisse, les mécanismes athéro-protecteurs de l’exercice physique seul et/ou associé à une modification du régime alimentaire (du régime riche en graisse au régime standard). Nos résultats montrent que l’exercice physique, indépendamment de la diète utilisée, corrige la dysfonction endothéliale installée au cours de l’obésité. Cet effet bénéfique a été associé à une diminution du stress oxydatif au niveau vasculaire. En effet, nos résultats indiquent que l’exercice diminue l’activité de la NADPH oxydase au niveau aortique. De plus, nous montrons pour la première fois que l’exercice physique seul, indépendamment de la diète utilisée, est capable de moduler la translocation de la sous-unité de la NADPH oxydase p47phox (principal acteur dans l’activation de ce complexe enzymatique) vers la membrane. Nos résultats indiquent également que l’exercice physique, avec ou sans modification du régime, améliore la voie Akt/eNOS phosphorylée, suggérant une augmentation de la production du NO. Ainsi, l’exercice physique, même sans l’associer à un changement du mode alimentaire, peut être considéré comme une stratégie non-pharmacologique efficace pour le traitement du risque athérogène généré par l’obésitéThe prevalence of obesity is increasing at an alarming rate in the western countries. It has been attributed to sedentariness and abundance of unhealthy food. Obesity is often associated with endothelial dysfunction and a high atherogenic risk. Several clinical investigations have reported that life style modification included physical exercise and the adoption of healthydiet was an efficient strategy to combat cardiovascular complications linked to obesity. However, numerous mechanisms by which exercise exerts the direct therapeutic effect on atherogenic risk linked to obesity are still unknown. Using the experimental model of high fat diet-induced obesity rat, the general aim of this study, was to identify the possible molecularmechanisms through which exercise with or without diet modification (high fat to standard diet) exerts an antiatherogenic action. Our results show that exercise independently of diet used, corrected the endothelial dysfunction induced by obesity. This benefit effect was associated with the decreased vascular oxidative stress. In effect, our results show that exercise alone was able to decrease NADPH oxidase activity in aortic tissue. Furthermore, we show for the first time that exercise, independently diet used, was able to modulate the translocation of p47phox subunit to membrane (which plays a pivotal role in NADPH oxidase activation). Ours results show also, that exercise with or without diet modification improves the Akt/eNOS phosphorylation pathway, suggesting that exercise increases NO production. In summary, exercise training even without diet modification, may be a non-pharmacological strategy treatment for atherogenic risk linked to obesity
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Park, Sophie. "Activation des voies PI3K/AKT et mTOR dans les LAM : thérapeutiques ciblées et Implication des facteurs de transcription FOXO dans la leucémogenèse." Paris 7, 2008. http://www.theses.fr/2008PA077107.
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Les leucémies aiguës myéloïdes (LAM) sont des maladies clonales qui atteignent des progéniteurs de la lignée myéloïde. Des activations anormales des voies de signalisation, en particulier des voies PI3K/AKT et mTORd, ont été mises en évidence dans ces hémopathies. La première partie de mon travail montre dans les blastes primaires, les effets biochimiques et fonctionnels anti-leucémiques d'un inhibiteur chimique, le Pl-103, inhibiteur à la fois des PI3K de classe la et de mTORCI. II inhibe la prolifération des blastes leucémiques et leur potentiel clonogénique et induit une apoptose mitochondriale dans le compartiment des cellules leucémiques immatures contenant les cellules souches leucémiques. La deuxième partie de mon travail consiste à déterminer le rôle des facteurs de transcription de la famille FOXO dans la biologie des LAM. J'ai montré dans les blastes primaires et dans une lignée leucémique humaine avec I'IC87114, inhibiteur spécifique de l'isoforme p110δ de PI3K, que l'inhibition de la voie PI3K seule ne permet pas de relocaliser les FOXO dans le noyau et que d'autres voies de signalisation sont impliquées dans ce contrôle. J'ai montré dans une 2e partie qu'une inhibition spécifique de l'activité IKK dans les blastes grâce à un inhibiteur de MEMO pourrait jouer un rôle anti-leucémique en inhibant l'activité NF-KB et en activant la fonctionnalité des facteurs de transcription de la famille FOXO en les relocalisant,dans le noyau. Ces résultats suggèrent que la compréhension des mécanismes d'activation des voies de signalisation dans les blastes primaires et de leur connexion, permettront de définir des stratégies d'inhibition ciblées, dans le traitement des LAMThe PI3K/AKT and mTORCI signaling pathways are frequently activated in AML. MTORd inhibition with RAD001 induces PI3K/AKT activation and both pathways are activated independently, providing a rationale for dual inhibition of both pathways. Pl-103 is a new potent PI3K/AKT and mTOR inhibitor. In blast cells, Pl-103 inhibits leukemic proliferation, the clonogenicity of leukemic progenitors and induces mitochondrial apoptosis, especially in the compartment containing the leukemic stem cells (LSC). Pl-103 has additive pro-apoptotic effects with etoposide in blast cells and in immature leukemic cells. Interestingly, Pl-103 does not induce apoptosis in normal CD34+ cells and has moderate effects on their clonogenic and proliferative properties. FOXO transcription factors have a key role in the control of cell survival. Intracellular localization of FOXO is regulated in part by phosphorylation by différent kinases, especially AKT. I have shown that the specific inhibition of PI3K activity in AML with IC87114, an inhibitor of the p110δ isoform, does not induce apoptosis, does not relocalize FOXOSa in the nucleus and does not induce FasL and Bim target genes expression. Moreover, my data supports the fact that the IKK complex is frequently activated in AML and probably represents the main kinase that controls the localization of FOXOSa in AML, even in AML samples with constitutive PI3K activation. Finally, these results do suggest that the specific targeting of the IKK activity in AML may induce apoptosis by inhibiting NF-KB translocation and by inducing FOXOSa localization in the nucleus
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Affara, Nesrine I. "The role of phosphoinositide 3-kinase/akt signaling pathway in tumor-associated angiogenesis, wound healing, and carcinogenesis." The Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=osu1150469618.
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Baran, Christopher Phillip. "The involvement of Lyn and the SH2-domain-containing inositol 5'-phosphatase 1 (SHIP1) in the negative regulation of M-CSF-induced cellular signaling events." Columbus, OH : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1050605166.
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Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains x, 92 p.: ill. Includes abstract and vita. Advisor: Clay B. Marsh, Dept. of Veterinary Biosciences. Includes bibliographical references (p. 84-92).
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Vitet, Hélène. "Conséquences de la régulation du transport axonal par la huntingtine sur l'homéostasie de réseaux neuronaux et sur le comportement, en conditions saine et pathologique Traffic signaling: new functions of huntingtin and axonal transport in neurological disease Presynaptic APP levels and synaptic homeostasis are regulated by Akt phosphorylation of Huntingtin." Thesis, Université Grenoble Alpes, 2020. http://www.theses.fr/2020GRALV038.
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Les circuits neuronaux régissent les comportements tels que la coordination motrice ou la mémoire et l’apprentissage. Au sein d’un réseau, les neurones communiquent par des processus moléculaires et cellulaires finement réglés à la synapse. Un des mécanismes régulant l’homéostasie synaptique, le transport de vésicules dans les neurones, est dérégulé dans les maladies neurologiques telles que le syndrome de Rett, la maladie d’Alzheimer et la maladie d’Huntington. Ainsi, investiguer la régulation du transport de vésicules dans les neurites dans un contexte physiologique est important pour comprendre, et potentiellement rétablir, les conséquences de ces dérégulations pathologiques.La protéine Huntingtine (HTT), connue pour son implication dans la maladie d’Huntington, est un acteur clé du transport axonal. Elle promeut et influence le transport des vésicules en favorisant le recrutement des adaptateurs et des moteurs moléculaires. Sa phosphorylation sur la sérine 421 (pHTTS421) régule la directionnalité des vésicules de BDNF, d’APP et de VAMP-7 dans des neurones transfectés in vitro. Cependant, les mécanismes et les conséquences de la régulation du transport par HTT comme la spécificité neuritique et les conséquences comportementales restent peu connus. Enfin, nous ignorons si la régulation du transport peut être influencée dans des conditions pathologiques afin de restaurer les phénotypes in vivo.Ce projet de thèse vise à caractériser les mécanismes et les conséquences de la régulation du transport axonal de trois types de vésicules par pHTTS421 et d’investiguer sa propension à restaurer les phénotypes associés à des maladies neurologiques dans des modèles de souris.Dans le but de reproduire in vitro les réseaux associés à des maladies neurologiques, nous avons utilisé des chambres microfluidiques. Nous avons étudié le transport des vésicules d’APP, des précurseurs des vésicules synaptiques (PVSs) ou des vésicules à cœur dense (VCDs) contenant BDNF au sein d’un réseau neuronal dans lequel pHTTS421 a été modifiée. Ces neurones sont issus de souris pour lesquelles la sérine 421 a été remplacée par un acide aspartique ou par une alanine pour mimer respectivement l’état phosphorylé (HTTS421D) ou non phosphorylable (HTTS421A) de la HTT.Dans la maladie d’Alzheimer, l’homéostasie d’APP est dérégulée. Nous avons donc étudié son transport et son accumulation synaptique dans un circuit corticocortical. Nous avons trouvé que la phosphorylation de la sérine S421 par Akt régule la directionnalité des vésicules d’APP uniquement dans les axones : HTTS421A diminue le flux antérograde axonal d’APP ainsi que ses niveaux à la synapse in vitro et in vivo. Réduire le flux antérograde d’APP dans un modèle murin d’Alzheimer restaure l’homéostasie synaptique in vivo et les déficits de mémoire associés (publication 1 ; Bruyère*, Abada*, Vitet* et al., eLife, 2020).Le transport axonal des PVSs régule le nombre de vésicules synaptiques (VSs), ce qui, dans un réseau corticostriatal, est essentiel à l’apprentissage de compétences motrices. Nous avons montré que pHTTS421 augmente le recrutement de la kinésine KIF1A sur les PVSs, augmentant le transport antérograde et la probabilité d’exocytose. En réduisant les niveaux de KIF1A dans le réseau corticostriatal des souris HTTS421D, nous avons trouvé que pHTTS421 augmente le nombre de VSs et altère la mémoire procédurale. Cette étude décrit comment le transport axonal des PVSs impacte les phénotypes comportementaux (publication 2 ; vitet et al., in prep).Enfin, le transport de BDNF est dérégulé dans le réseau corticostriatal des jeunes filles atteintes du syndrome de Rett. Nous avons observé que l’expression endogène de pHTTS421 ou l’injection d’un composé inhibant la calcineurine (FK506) restaure le transport de BDNF dans un réseau corticostriatal, la communication neuronale et les symptômes associés chez les souris modèles du syndrome de Rett (Publication 3 ; Ehinger et al., Embo Mol Med, 2020)Neuronal circuits are at the basis of behaviors such as motor coordination or learning and memory. As being part of a network, neurons communicate at synapses through finely tuned molecular and cellular processes. One key mechanism regulating synapse homeostasis involves transport of vesicles within axons and dendrites which is dysregulated in many neurological disorders such as Rett syndrome, Alzheimer’s (AD) and Huntington’s diseases (HD). Thus, deciphering the regulation of vesicular transport within neurites in physiological context is crucial to understand, and potentially restore, the consequences of these dysregulations in pathological contexts.Huntingtin (HTT) protein, known for its devastating role in HD when mutated, is a key actor of axonal transport. It promotes and regulates vesicular transport in neurites by scaffolding adaptors and molecular motors. Particularly, HTT phosphorylation status on S421 regulates the directionality of BDNF, APP and VAMP-7 vesicles within neurites in cultured and transfected neurons. However, several questions remain to be elucidated regarding the mechanisms and the consequences of this HTT-dependent regulation of axonal transport such as the neuritic specificity (axons or dendrites) and the behavioral consequences of such modifications. Finally, we do not know whether transport regulation can be influenced in pathological conditions to restore disease-associated phenotypes in vivo.This thesis aims at characterizing in vivo the mechanisms and the consequences of axonal transport regulation of three different vesicles through the phosphorylation of Huntingtin at S421 and to investigate its propensity to restore disease-associated phenotypes in mouse models of human neurological disorders.In order to reproduce in vitro the in vivo networks associated to neurological disorders we used microfluidic devices. We investigated the transport of Amyloid Precursor Protein (APP) vesicles, precursors of synaptic vesicles (SVPs) or dense-core vesicles (DCVs) in neurons in which the HTT phosphorylation status was modified. These neurons came from mice in which Serine 421 has been replaced by an aspartic acid to mimic the phosphorylated form of HTT (HTTS421D) or by an alanine to mimic the unphosphorylatable form of HTT (HTTS421A).Transport of APP vesicles is impaired in AD. We investigated APP transport and accumulation at synapses within a corticocortical circuit. We found that Akt-mediated HTT phosphorylation at S421 regulates the directionality of APP containing vesicles in axons but not in dendrites: the phospho-defective form of HTT decreases axonal anterograde flux of APP and reduces its levels at presynaptic zones both in vitro and in vivo. Reducing anterograde flux of APP in familial AD mouse model restored synapse homeostasis in vivo and memory deficits (Publication 21; Bruyere*, Abada*, Vitet* et al., eLife, 2020).SVP axonal transport regulates the number of SVs at the synapse, which, within a corticostriatal synapse, is essential for motor skill learning. We found that HTT phosphorylation increases the recruitment of the molecular motor KIF1A on SVPs, thus promoting anterograde transport and the probability of release. Silencing KIF1A in the corticostriatal network of HTTS421D mice, we found that pHTTS421 increases the number of SVs at the synapse and impairs procedural memory through a specific HTT-KIF1A dependent mechanism. This study defines a pathway by which axonal transport of SVP impact the behavioral phenotype. (Publication 2; Vitet et al, in prep)Finally, it has been shown that BDNF transport within DCVs is dysregulated in the corticostriatal network of Rett syndrome’s patients. We found that endogenous HTT phosphorylation at S421 or a chemical inhibitor of calcineurin (FK506) rescue BDNF transport in the corticostriatal network, neuronal communication, and behaviors of Rett Syndrome mice (Publication 3; Ehinger et al., Embo Mol Med,2020)
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Le, Grand Marion. "La protéine Akt, lien entre mitochondries et microtubules dans le mécanisme d'action des agents anti-microtubules ou quand les MTA s'invitent dans de nouvelles stratégies thérapeutiques." Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM5017/document.
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De nos jours, les agents anti-microtubules (MTA) sont administrés dans de nombreuses pathologiques cancéreuses reflétant ainsi leur grande efficacité anti-tumorale. Cependant, leur utilisation se voit limitée pour deux raisons : (i) l’apparition d’effets indésirables et, (ii) l’émergence de cellules tumorales résistantes. Pour palier ces problèmes, les MTA font l’objet de nombreux travaux de recherche faisant ainsi de ces composés des médicaments toujours dans l’ère du temps. L’objectif principal des travaux présentés dans ce manuscrit repose sur l’étude du mécanisme d’action des MTA afin d’optimiser, par la suite, leur administration. Dans une première partie s’inscrivant dans le domaine de la recherche fondamentale, nous avons caractérisé les mécanismes moléculaires à l’origine de l’efficacité anticancéreuse de ces agents. En effet, nous avons mis en lumière l’existence d’un pont signalétique entre les mitochondries et les microtubules avec un rôle crucial de la voie de signalisation Akt/GSK3β plaçant ainsi, de façon inattendue, la kinase Akt au cœur de l’efficacité des MTA. Ces résultats fournissant un rationnel mécanistique aux stratégies thérapeutiques associant les MTA aux thérapies ciblées anti-Akt, nous avons alors mené une étude oncopharmacologique démontrant que l’association MTA/anti-Akt est fortement synergique in vitro et in vivo.Mieux comprendre le mécanisme d’action des MTA, afin de proposer de nouvelles stratégies thérapeutiques aux cliniciens était l’objectif principal de cette thèse. Les résultats obtenus ici ouvrent ainsi la voie de l’association de ces agents avec les thérapies ciblées anti-Akt nouvelle générationMicrotubule-Targeting Agents (MTA) are a broad group of anticancer drugs that are currently administered in a lot of cancers. Nevertheless, they can cause undesired side effects and can lose their effectiveness as a result of resistance development. The main objective of my PhD work was to characterize the MTA’s mechanism of action in order to optimize their administration in the future. In the first part, we demonstrated the important role of the kinase Akt in MTA effects. In the second part, we evaluated the interest to combine MTA with anti-Akt drugs. We observed that MTA efficacy is highly important with Akt targeting drugs, particularly in lung adenocarcinoma. These promising results will need further explorations in order to develop more convenient cancer therapy strategies
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Boy-Rocher, Géraldine. "Le substrat de ERK IEX-1 est un inhibiteur général des phosphatases PP2A de la famille B56 impliqué dans la signalisation TPO." Paris 7, 2008. http://www.theses.fr/2008PA077003.
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La voie ERK joue un rôle important dans les fonctions de la TPO. L'équipe avait isolé le gène précoce IEX-1 en tant que substrat de ERK en réponse à la TPO et démontré une régulation réciproque des activités de ces deux protéines : IEX-1 phosphorylée par ERK acquiert une fonction anti-apoptotique ; IEX-1 en liant ERK, maintien son activation. Ce denier effet est du à la capacité de IEX-1 d'inhiber de façon spécifique la famille des phosphatases PP2A contenant les sous-unités régulatrices B56 (PP2A-B56). IEX-1 inhibe PP2A-B56 en permettant la phosphorylation de la sous-unité B56 par ERK ce qui conduit à la dissociation de la sous-unité catalytique de l'enzyme. Ces résultats suggéraient que IEX-1 pourrait séquestrer les sous-unités B56 de PP2A, les empêchant d'agir sur d'autres substrats. En effet, nous avons observé que IEX-1 bloque la déphosphorylation d'Akt sur Ser473 et Thr308 et augmente son activité kinase. Akt est une cible des PP2A-B56. En effet, la surexpression de sous-unités B56 entraîne une diminution de la phosphorylation d'Akt sur Ser4723 et Thr308. L'effet inverse est retrouvé en utilisant les siRNA dirigés contre B56β et γ. Les PP2A-B56 reversent l'effet de IEX-1 sur Akt. En utilisant des mutants dominant-négatifs de ERK et des mutants de phosphorylation de B56, nous avons montré que l'effet de IEX-1 sur Akt est dépendant de la phosphorylation de B56 par ERK. IEX-1 maintien donc l'activation d'Akt et de ERK par le même mécanisme. Ces résultats montrent que IEX-1 agit comme un inhibiteur cellulaire général des PP2A-B56et identifient un nouveau substrat des PP2A-B56 ainsi qu'une nouvelle coopération entre les voies ERK et Akt. Dans les cellules UT7-Mpl, IEX-1 est induit spécifiquement par la TPO, mais pas par l'EPO ou le GM-CSF, et participe au maintien de l'activation de ERK et Akt en réponse à cette cytokine. IEX-1 est aussi exprimée dans les CSH CD34+CD38- puis au cours de la différenciation des mégacaryocytes. Le nombre et la taille des CFC ainsi que la fréquence des LTC-IC sont augmentés lorsque IEX-1 est surexprimée par infection lentivirale dans les cellules CD34+ de sang de cordon ombilical. Des résultats préliminaires montrent que la surexpression de IEX-1 pourrait compenser de façon spécifique l'absence de TPO pour la prolifération des cellules CD34+. Par contre, la surexpression de IEX-1 n'a pas d'effet sur la différenciation mégacaryocytaire. Ces résultats suggèrent que IEX-1 et la famille des PP2A-B56 pourraient jouer un rôle unique dans les capacités de la TPO à maintenir le compartiment des CSHIEX-1 is an early-response gene involved in survival and proliferation, which is rapidly induced by growth factors, viral infections or chemical carcinogens. IEX-1 was previously isolated as a substrate of the kinase ERK and having a dual role in ERK signaling : IEX-1 acquires anti-apoptotic functions upon phosphorylation by ERK and by binding to active ERK, IEX-1 behaves as a positive regulator of ERK activation, prolonging ERK signal in response to various growth factors. We have elucidated the mechanism by which IEX-1 prolong ERK signal. The major Ser/Thr phosphatase involve in ERK activation is the protein phosphatase 2A (PP2A). It is made of three subunits: the structural (A), the regulatory (B) and the catalytic (C) subunit. IEX-1 binds specifically to the B56 regulatory subunits of PP2A. This association enhances B56 phosphorylation by ERK and trigger dissociation from the catalytic subunit leading to the inactivation of the PP2A. We examine whether IEX-1 could control only the dephosphorylation of associated ERK or have a more general effect on other kinase activities controlled by PP2A. By using IEX-1 surexpression and shRNA targetting IEX-1, we found that IEX-1 had no effect on the phosphorylation of MEK, p38 or JNK, but that it encreased and sustained the activation of the kinase Akt by preventing its dephosphorylation both on in residues the308 and ser473. These similar regulations of Akt and ERK activities by IEX-1 prompted us to examine whether the same mechanism operates on the two pathways. Overexpression of IEX-1 and B regulatory subunit together shows that specifically B56 subunit strongly reduced the capacity of IEX-1 to prolong Akt phosphorylation. This indicates that Akt phosphorylation is controlled by PP2A holoenzyme containing B56 subunits. Next we have explored the mechanism by which IEX-1 prevents the activity of B56 on pAkt. To test that, we use an IEX-1 protein mutated in its ERK binding site, loosing its ability to both bind pERK and to inhibit B56-PP2A activity. This mutant was enable to extained the duration of pAKT signal showing that IEX-1 mediated Akt activation is dependent on its capacity to bind ERK. To confirm this result, we expressed IEX-1 together with ERK1 and ERK2 kinase dead mutant, devoided of catalytic activity. Expression of this mutant reduces the capacity to protect Akt from inactivation. This result indicate that the ability of IEX-1 to encreased ERK mediated phosphorylation of B56 subunit provides a general mechanism leading to inhibition of B56 regulatory subunits containing PP2A enzymes function. IEX-1-mediated ERK-dependent inhibition of B56 containing is responsible for its ability to control both ERK and Akt activation
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Jahani-asl, Arezu. "Influence of phosphorylation on caspase-3-mediated Akt1 cleavage." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/26931.
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Caspase-3, an executioner of apoptosis, is negatively regulated by X-linked inhibitor of apoptosis protein (XIAP), a determinant of cisplatin resistance. XIAP down-regulation in ovarian cancer cells or treatment with cisplatin induces caspase-3-mediated AKT cleavage and apoptosis, while XIAP over-expression suppresses this cleavage and increases phospho-AKT content. The identity of the caspase-3 cleavage site(s) in AM and the possible dependence of caspase-3-mediated cleavage on its phosphorylation status are unknown. The objectives of this thesis were to determine the caspase-3 cleavage site(s) in Akt1 and to examine the influence of phosphorylation on Akt1 cleavage in vitro. Our results suggested presence of three non-consensus (EEEE 117, EEMD119, DAKE398) and one consensus (DQDD456) cleavage sites, and posphorylation of Akt1 influenced the pattern of cleavage in a site-specific manner: Whereas cleavage at site "EEEE117" was facilitated by phosphorylation, that at sites "EEMD119 and DAKE398'' were attenuated. The biological significance of these observations requires future investigation.
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Wallis, Lise J., and n/a. "Regulation of Bub1b phosphorylation by protein phosphatase 2A." University of Otago. Dunedin School of Medicine, 2006. http://adt.otago.ac.nz./public/adt-NZDU20070502.114819.
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The mitotic spindle checkpoint plays a critical role during the cell cycle by protecting the faithful transmission of chromosomes during mitosis. If chromosomes are improperly bound to the spindle microtubules the checkpoint will prevent progress to anaphase by temporarily arresting cells in metaphase until all the chromosomes are correctly aligned. Bub1b is an essential component of the mitotic spindle checkpoint that transiently localises to kinetochores during mitosis and becomes phosphorylated, a response that is sustained during mitotic arrest. Bub1b has been implicated in other processes related to mitotic progression and is thought to regulate mitotic timing and have a role in caspase mediated cell death after prolonged mitotic arrest. The development of aneuploidy and cancer has been associated with mutations in the BUB1B gene and reduction in the level of Bub1b protein. To further our understanding of Bub1b function in the spindle checkpoint and mitosis, new protein interactions involving Bub1b were identified. This thesis describes the search for alternative proteins that interact with Bub1b, and their function in the mitotic spindle checkpoint and regulation of Bub1b activity.
Using a yeast two-hybrid approach, members of the B56 family of regulatory subunits of serine-threonine protein phosphatase (PP2A) were identified as novel interacting partners of Bub1b. Substrate specificity of PP2A is determined by the regulatory subunits. There are five characterised isoforms of the B56 family, each encoded by separate genes. In addition, some isoforms have several recognised splice variants. Confirmation of interactions by alternative methods demonstrated that the isoforms B56γ and B56[epsilon] preferentially interact with phosphorylated Bub1b, whereas the interaction of the remaining B56 isoforms (α, β and [delta]) occurs at a lower affinity with no specificity for the phosphorylated form. It was further demonstrated that B56γ1 associated with phosphorylated Bub1b in vivo.
Induced overexpression of splice variants of B56γ1 and B56γ2 demonstrated a significant reduction in levels of phosphorylated Bub1b during mitotic spindle checkpoint activation. In addition, an associated lower mitotic index was evident in cells with B56γ1 overexpression. Specific inhibition of PP2A activity with okadaic acid was shown to prolong Bub1b phosphorylation during normal mitosis and to restore the levels of phosphorylated Bub1b in arrested cells over expressing B56γ. These findings suggest a role for PP2A activity in regulation of Bub1b function that is mediated through substrate recognition by B56 regulatory subunits.
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Zu, Xin Lin. "Methods for the detection, purification and characterisation of histone H4 histidine kinase and the analysis of protein histidine phosphorylation." University of Western Australia. Biochemistry and Molecular Biology Discipline Group, 2007. http://theses.library.uwa.edu.au/adt-WU2007.0092.
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[Truncated abstract] Protein phosphorylation, one of the most important forms of post-translational modification, has been demonstrated to play crucial roles in regulation of cell function. Phosphorylation of protein serine, threonine and tyrosine residues has been the most thoroughly investigated, taking advantage of the acid-stable character of these phosphohydroxyamino acids. Whereas, the cellular occurrence of acid-labile phosphoamino acids, such as phosphohistidine, phosphoarginine and phospholysine was often underestimated due to the acid treatments employed by most of the traditional phosphoamino acid analysis methods. The biological roles of histidine kinases (HKs) in prokaryotes are well understood in contrast to those of HKs in eukaryotes, especially in mammalian cells. However, the evidence has shown that phosphohistidine comprised 6% of phosphoamino acids of the basic nuclear proteins in eukaryotes (Matthews, 1995) and there was more phosphohistidine than phosphoserine in rat liver mitochondria (Bieber and Boyer, 1966). More significantly, phosphohistidine was revealed to be the major phosphoamino acid in phosphorylated histone H4 in regenerating liver in vivo (Chen et al., 1974) and the Walker-256 carcinosarcoma cells in vitro (Smith et al., 1974). Recently, the histone H4 histidine kinase (HHK) activity of human hepatocellular carcinoma (HCC) tumour tissue was measured to be 400 times higher than the normal liver tissue surrounding the tumour. HepG2 cells (HCC cell line) and PIL-2 cells (a p53 knockout mouse tumorigenic liver progenitor cell line) also displayed high HHK activity (Tan et al., 2004). The above observations suggested that HKs and HHKs are playing important roles in both prokaryotes and eukaryotes, including mammals. One major obstacle in the study of HHK study has been the lack of knowledge of the amino acid sequence of an HHK. Attempts at purifying and identifying the HHK from yeast led to the partial purification of a yeast HHK protein(s) at 32kDa (Huang et al., 1991). However, the amino acid sequence of the HHK has not yet been established. ... The success of the separation was demonstrated by the MALDI-TOF-MS and/or ESI-MS spectra of the RP-HPLC fractions. These achievements suggested that it is possible to detect phosphohistidyl histone H4 in vivo using MS under experimental conditions where phosphohistidine is relatively stable. The study in this thesis represents the progression of HHK research in various aspects, including the yeast HHK purification and identification, mammalian HHK partial purification and the methodological developments in detecting histone H4 histidine phosphorylation using MS. Furthermore, new information regarding the physical characteristics of yeast HHKs and its potential role in cellular biology have been documented. It is anticipated that knowledge generated in these studies will contribute to the insight and the understanding of the biological significance of HHK in yeast and mammalian cells.
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Ajumobi, Taiwo. "Beta-Blockers Act through Clathrin-Dependent Internalization and EGFR Transactivation to Promote ERK Phosphorylation." Digital Commons @ Butler University, 2014. http://digitalcommons.butler.edu/grtheses/267.
Full textAbstract:
For cardiovascular diseases such as high blood pressure, angina pectoris, and left ventricle hypertrophy; long-term activation of beta-adrenergic receptors is strongly linked to the progression of these diseases. A class of antagonistic drugs that target betaadrenergic receptors are collectively called beta-blockers. These drugs are commonly used to reduce the inotropic and chronotropic effects of beta-adrenergic receptor activation. This past decade has revealed that beta-blockers and other ligands are capable of functional selectivity at receptors. Functional selectivity describes the ability of ligands acting at 0 protein-coupled receptors (OPCRs) to preferentially activate or inhibit different signal transduction pathways. The studies on beta-adrenergic 2 receptors that explored functional selectivity showed that beta-blockers can be functionally selective by inhibiting the cAMP pathway while simultaneously activating ERK. The 0 protein coupled to beta-adrenergic receptors are the primary regulators of the cAMP, however there are a variety of pathways that can regulate ERK activity and few studies have tried to determine which pathway(s) the beta-blockers are targeting to cause this ERK activation. This is especially important for beta-adrenergic 2 receptors because they can activate ERK through multiple pathways (0 protein switching from G, to Oi/oprotein, beta-arrestin assisted or EOFR transactivation). ERK activation is linked to reversing cell damage caused by apoptosis signaling that results from G, activation by beta-adrenergic receptors. Understanding the specific pathways these beta-blockers can target for ERK activation would lead to better understanding of their therapeutic benefits. In this study we plan to elucidate the pathways several beta-blockers are targeting to activate ERK. In particular, we will investigate the role of clathrin-mediated receptor internalization and EGFR transactivation in beta-blocker-dependent ERK phosphorylation. In HEK 293 cells transfected with beta-adrenergic 2 receptors, we measured the changes in cAMP and ERK phosphorylation in response to the following beta-blockers labetalol, alprenolol, bucindolol, carvedilol, carazolol, leI 118,551 and propanolol. All of the beta-blockers studied inhibited isoproterenol-stimulated cAMP accumulation but stimulated the phosphorylation of ERK to varying degrees. Beta-blocker-mediated ERK phosphorylation was shown to be dependent on clathrin-dependent internalization and EGFR transactivation.
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Court, Naomi Wynne. "The subcellular localisation, tissue expression, substrate specificity and binding partners of stress-activated protein kinase-3." University of Western Australia. School of Biomedical and Chemical Sciences, 2004. http://theses.library.uwa.edu.au/adt-WU2004.0084.
Full textAbstract:
[Truncated abstract] Cells need to be able to detect changes in their surrounding environment and transduce these signals into the appropriate cellular compartments. One of the major ways that the cell achieves this signal transduction is through the process of phosphorylation. Protein kinases are the enzymes responsible for catalysing this transfer of phosphate groups from ATP to amino acid residues of their specific substrates. A subfamily of serine/threonine kinases known as the Mitogen-Activated Protein Kinases (MAPKs) is essential in a diverse range of cell processes including growth, metabolism, differentiation and death. The first identified MAPKs, the Extracellular Signal-Regulated Kinases (ERKs), were found to be activated in response to mitogenic stimuli such as growth factors. However, since the discovery of the ERKs, other pathways leading to the activation of related kinases have been recognised. These kinases are preferentially activated in response to stress, and are thus termed “Stress-Activated Protein Kinases” or SAPKs. They consist of the c-Jun N-terminal kinase isoforms 1, 2 and 3 (also called SAPK1γ, SAPK1α and SAPKβ respectively) and the p38 MAPKs, p38α, p38β, p38γ and p38δ (also called SAPK2a, SAPK2b, SAPK3 and SAPK4 respectively). A major challenge in this field has been to identify the substrates and functions of the SAPKs. This has been partly achieved by the development of inhibitors for the JNK MAPKs and SAPK2a/b. However, no inhibitors currently exist that specifically inhibit SAPK3 and SAPK4. Therefore, elucidating the function of these SAPKs has proved more difficult. Recent studies suggest that SAPK3 may play a unique role in the cell compared to other members of the p38 MAPK family. For example, several signalling proteins appear to specifically activate SAPK3 in certain circumstances while not activating other members of the p38 MAPK family. In addition, SAPK3 contains a unique sequence motif that allows it to bind to specialised domains known as PDZ domains. The interaction of SAPK3 with proteins containing these domains may regulate its subcellular localisation and interactions with other proteins in the cell. This project was undertaken to expand the knowledge on the expression, localisation, substrate specificity and binding partners of SAPK3. In Chapter 3 of this thesis, a SAPK3 monoclonal antibody was evaluated for its ability to specifically recognise endogenous SAPK3 protein. SAPK3 was found to be expressed in immortalised cell lines and primary cultures of neonatal rat myocytes, and to be colocalised with the mitochondria of these cells. This co-localisation remained unaltered in response to treatment with the nuclear export inhibitor Leptomycin B, and with exposure to osmotic shock, suggesting that SAPK3 substrates may be localised at the mitochondria
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Bostner, Josefine. "The Akt/mTOR Pathway and Estrogen Receptor Phosphorylations : a crosstalk with potential to predict tamoxifen resistance in breast cancer." Doctoral thesis, Linköpings universitet, Avdelningen för kliniska vetenskaper, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-100903.
Full textAbstract:
Estrogen receptor α content is the primary breast cancer biomarker distinguishing the patients responsive from the non-responsive to endocrine treatments. Tamoxifen is an estrogen competitor with large potential to treat breast cancer patients and prolongs time to recurrence. Despite the estrogen receptor positivity and tamoxifen treatment, many women face recurrence of the disease. An important mechanism of resistance to endocrine treatments is upregulated growth factor signaling, and the subsequent effect on the estrogen receptor, rendering an active receptor that stimulates cell proliferation or reduced estrogen-receptor dependence. This thesis concerns the investigation of biomarkers, as a complement to the existing markers, for determining optimal treatment for patients with primary invasive breast cancer. Randomized patient tumor materials were used in order to measure variations in gene copies, proteins, and protein phosphorylations and to further relate these variations to time-to-recurrence. Endocrine untreated groups within the patient tumor sets gave us the opportunity to study the prognostic potential of selected markers and to compare tamoxifen-treated patients with endocrine untreated, thus obtaining a treatment-predictive value of each marker or marker combination. In endocrine-dependent cancer the 11q13 chromosomal region is frequently amplified, harboring the genes encoding the cell cycle stimulator cyclin D1 and the estrogen receptor phosphorylating kinase Pak1, respectively. Amplification of the genes was associated with reduced time-torecurrence, indicating a prognostic value, whereas PAK1 gene amplification predicted reduced response to tamoxifen treatment. Moreover, the protein expression of Pak1 tended to predict treatment response, which led to the investigation of this protein in a larger cohort. Together with one of its targets, the estrogen receptor phosphorylation at serine 305, Pak1 predicted reduced response to tamoxifen treatment when detected in the nucleus of tumor cells, suggesting activation of this pathway as a mechanism for tamoxifen-treatment resistance. The estrogen receptor is phosphorylated by several growth factor stimulated kinases. The role of serine-167 phosphorylation has been debated, with inconsistent results. To study the biomarker value of this site the upstream activity of Akt, mTOR, and the S6 kinases were analyzed individually and in combinations. As a prognostic factor, serine 167 indicated an improved breast cancer survival, and as a treatment predictive factor we could not detect a significant value of serine 167 as a single marker. However, in combination with serine 305, and Akt/mTOR-pathway activation, the response to tamoxifen treatment was reduced. The mTOR effector protein S6K1 was found to be associated with HER2 positivity and a worse prognosis. In the group of patients with S6K1 accumulation in the tumor cell nuclei, treatment did not prolong time-to-recurrence, similarly as observed with expression of active S6 kinases. In vitro, a simultaneous knockdown of the S6 kinases in estrogen receptor-positive breast cancer cells resulted in G1 arrest, and tamoxifen-induced G1 arrest was in part S6 kinase dependent. The results presented herein suggest biomarkers that would improve treatment decisions in the clinic, specifically for estrogen receptor-positive breast cancer and tamoxifen treatment but in a broader perspective, also for other endocrine treatments and targeted treatments.
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Wouterlood, Madeleine. "Carboxylates in the rhizosphere of chickpea (Cicer arietinum) in relation to P acquisition." University of Western Australia. School of Plant Biology, 2005. http://theses.library.uwa.edu.au/adt-WU2005.0029.
Full textAbstract:
[Truncated abstract] The highly weathered, phosphorus-fixing soils of Western Australia require large amounts of P fertiliser to produce acceptable crop yields. Chickpea (Cicer arietinum L.) is an important leguminous crop that is increasingly used in rotations with wheat (Triticum aestivum L.), Western Australia’s major crop. Chickpea and a range of other species exude P-mobilising carboxylates into the rhizosphere. Plants that exude carboxylates may need less P fertiliser or may use P in the soil that is unavailable to other plants. There is a wealth of information about P mobilisation and carboxylate exudation by white lupin; in contrast, research on carboxylate exudation by chickpea is fairly limited. The major aim of this PhD research project was to investigate the relationships between exudation of carboxylates and soil and plant P status for chickpea ... In conclusion, whereas carboxylate exudation of plants such as white lupin is clearly targeted at P acquisition, chickpea showed constitutive carboxylate exudation mainly of malonate into the rhizosphere in a series of experiments, each with a different design. Unlike white lupin, chickpea forms associations with mycorrhizal fungi that may improve plant P status. Some of the functions of constitutive carboxylate exudation by chickpea may include P acquisition and deterring microorganisms, but the exact reasons and mechanisms remain unresolved.
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Pearse, Stuart James. "Carboxylates in the rhizosphere of canola, wheat, lupins and pulses : their role in P acquisition from sparingly soluble forms." University of Western Australia. Faculty of Natural and Agricultural Sciences, 2006. http://theses.library.uwa.edu.au/adt-WU2006.0069.
Full textAbstract:
[Truncated abstract] Native Australian soils contain very low amounts of phosphorus. The soils of southwestern Australia are ancient and highly weathered. Consequently, the availability of phosphorus in these soils is too low for cropping purposes, so the application of P is necessary to maintain productivity. When P is applied to soil, typically as soluble superphosphate, it tends to be transformed to increasingly less soluble forms over time. Sparingly soluble forms of soil P are relatively inaccessible to Triticum aestivum; however, many grain legumes have a higher P-acquisition efficiency, allowing them to access pools of soil P that T. aestivum cannot. The P-acquisition efficiency of some grain legumes has been attributed in part to their ability to release large quantities of carboxylates, coupled with the development of cluster roots for species such as Lupinus albus. There are a number of unexplained observations in terms of the P-acquisition efficiency of grain legume species and the way that those species respond to P fertilisation. This PhD project aimed to study carboxylate release from a range of crop species, and investigate its role in variation among species for acquisition of phosphorus from sparingly soluble forms (chapter 1). ... L. albus (chapter 5). There was considerable variation in P acquisition among accessions. The variation cannot be attributed to differences in carboxylate release, cluster-root development or whole root system rhizosphere extract pH as measured in this study. We hypothesise that the variation might be attributed to differences in the ratio of release of protons and other cations localised around cluster roots. In conclusion studies of carboxylate exudation and sparingly soluble forms should use more than a single form if the aim is to draw generalised conclusions on P-uptake efficiency from sparingly soluble forms. Comparative studies of a range of species are a useful tool for enhancing our understanding of root physiology. While the benefit of carboxylates for providing access to poorly soluble P has been demonstrated, questions remain as to potential other roles for carboxylates, particularly in species that do not form cluster roots. Variation in P uptake among accessions of L. albus is present, and more work on proton release and ion balance of root clusters is necessary to understand intraspecific variation.
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Huang, Kuan-Yu, and 黃冠宇. "Telomere Shortening by Akt-mediated TPP1 phosphorylation." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/m64344.
Full textAbstract:
碩士國立陽明大學
生化暨分子生物研究所
97
Telomeres are dynamic protein-DNA complexes, which protect eukaryotic chromosome ends from illegitimate rearrangements and degradation. Telomeres are composed of double-stranded telomeric DNA (TTAGGG repeats in human) and single-stranded telomeric overhang that are maintained by telomerase. Telomere integrity and length maintenance is essential for cellular growth, and its regulation involved in aging and tumor progression. Once telomeres become sufficiently short, they lose the ability to protect the ends of chromosomes from end-to-end fusion or nucleic degradation. The shelterin protein complex models the dynamic structure “T-loop”, which including six direct or indirect telomeric DNA-binding proteins TRF1, TRF2, POT1, TIN2, TPP1 and Rap1. Among them, TPP1 and protector of telomeres 1(POT1) protect human telomere single-strands (ss) DNA overhangs from degradation. Recent work has highlighted the regulation of telomere length and genome integrity, TPP1 and POT1 formed heterodimer and specifically bind to single-strands telomeric DNA. However, the regulation of TPP1/POT1 activity in physiological and pathological conditions remains unknown. Akt/Protein kinase B(PKB) are Serine/Threonine protein kinase, which include AKT1, AKT2, and AKT3, are key intermediates of signaling pathways that regulate many cellular function. They regulate a wide range of target proteins controlling cell proliferation (e.g., FOXOs), cell survival (e.g., BAD), cell growth (e.g., mTOR), and angiogenesis (e.g., eNOS). The PI3K-PKB pathway regulates diverse cellular processes involved in tumorigenesis and cancer progression. Genome instability has been linked to cancer progression. Akt activation cause genome instability may associate with its downstream target mTOR. Telomere dysfunction was shown to resulting genomic instability. However, The linkage between Akt and telomere length control is little known. In this article, we show the direct interaction between Akt and TPP1. In vitro kinase assays showed that Akt phosphorylate of a putative Akt phophorylation site (Serine500) in wild type TPP1, but not TPP1 mutant (S500A). We also show phosphorylation of TPP1 does not result in change of TPP1-POT1 binding to single-strands telomeric DNA(TTAGGG repeats) and nor TIN2-TPP1 interaction. Interestingly, phosphorylation of TPP1 may change the TPP1-POT1 localization, promotes telosome assembly. These results indicate that Akt plays an crucial role in telomere length regulation and genome integrity, eventually contributing to tumorigenesis and cancer progression.
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叶力夏提, 阿德力别克, and Adelibieke Yelixiati. "Indoxyl Sulfate Counteracts Endothelial Effects of Erythropoietin Through Suppression of Akt Phosphorylation." Thesis, 2013. http://hdl.handle.net/2237/18992.
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Mohanty, Sarina. "Akt Phosphorylation of Drosophila Heat-Shock Factor: A Signature for Stress Resistance." Thesis, 2008. https://thesis.library.caltech.edu/5218/8/Sarina_Thesis_Full_Final.pdf.
Full textAbstract:
The heat-shock response is vital to cellular homeostasis. Drosophila melanogaster heat-shock factor (dHSF) is the primary transcriptional activator in the stress response pathway for induction of heat-shock-mediated gene transcription. This work investigates the potential for dHSF to undergo post-translational modification by phosphorylation and lysine tagging, specifically, direct phosphorylation by kinases and covalent-lysine tagging by ubiquitin, acetyl, and SUMO groups. Direct phosphorylation of, and binding to, dHSF was demonstrated by Akt/PKB kinase. Knock-down of this kinase by RNAi resulted in a heat-shock phenotype for dHSF and the acquired DNA-binding ability characteristic of activated transcription factor. Site-directed mutagenesis of lysines within a putative nuclear localization sequence (NLS) revealed two potential sites for regulation of dHSF activation by post-translational modification. The functional consequences of synergistic Akt phosphorylation and lysine modifications are discussed – this work implicates a role for direct kinase phosphorylation in regulating the stability of dHSF.
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Su, Chih-Hao, and 蘇致豪. "Akt Phosphorylation at Thr308 and Ser473 Links Proteasome-mediated Degradation of the Kinase." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/82422692013911590302.
Full textAbstract:
碩士國立陽明大學
生化暨分子生物研究所
98
The master cellular kinase Akt tightly regulates several signal transduction pathways which involve in protein synthesis, cell survival and proliferation, glucose metabolism, etc through its kinase activity. Phosphorylation at Thr308 and Ser473 is known to activate Akt, and its activity is closely related to carcinogenesis. Recent studies have shown that the Akt level increases with age in the brain, so that inactivated Akt is able to promote phosphorylation of the tau protein in the neuron cells, leading to accumulation of hyperphosphorylated tau, a major cause of Alzheimer’s disease. Of late, Hsp90 inhibitors have been tested to treat Alzheimer’s disease, raising an issue of Akt degradation. Akt activation depends on PDK1 and mTOR-rictor complex phosphorylate its Thr308 and Ser473. Once activated to turn on downstream signaling pathways, Akt returns to an inactive pool via PP2A-mediated dephosphorylation. We show here that Thr308 and Ser473 phosphorylations promote Akt to enter the ubiquitin-proteasome pathway. Insulin stimulates Thr308 and Ser473 phosphorylation but promotes Akt to bind the U-box E3 ubiquitin ligase CHIP (C-terminal Hsp70-interacting protein). Mutation at Thr308 and Ser473 dampens CHIP-mediated ubiquitination. Our study unveils that the well-known phosphorylations responsible for Akt activation turn out to transduce recognition signals for Akt-CHIP binding and ensuring degradation.
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Hsiao, Jui-kang, and 蕭瑞康. "Overexpression of Akt and JNK associated with low phosphorylation of Erk1/2 in papillary thyroid carcinoma." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/37906382762621835970.
Full textAbstract:
碩士國立成功大學
生理學研究所
90
Papillary thyroid carcinomas (PTC) is the most common follicular cell-derived thyroid cancer which is often caused by somatical rearrangement of ret gene that results in a constitutive activation of rearranged RET. Rearrangement induced transformation (RET) is a receptor tyrosine kinase involved in the development of kidney and enteric nervous system and associated with several neoplasia. Whether RET expression in PTC is elevated is not certain yet. We collected 27 cases of PTCs and 8 cases of normal thyroid. To delineate signal pathways in formation of PTC, we analyzed expression and phosphorylation of several signal proteins in downstream of RET, such as Akt and JNK, Erk1/2, and p85. We observed that levels of Akt and JNK were significantly higher in PTC than in normal thyroid tissue. However, the phosphorylation/expression ratio of Akt and JNK were not different between normal thyroid tissue and PTC. In contrast, the phosphorylation/expression ratio of Erk1/2 was markedly lower in PTC than normal thyroid tissue. Therefore, abnormally high expression of Akt and JNK in PTCs might be associated with the pathogenesis of PTC. Furthermore, there was an inverse correlation between Akt levels and phospho-Erk1/2, indicating that overexpression and activation of Akt may inhibit phosphorylation of Erk1/2 in PTC. To clarify the regulating mechanisms involved in higher expression of Akt and JNK in PTCs, mRNA of Akt and JNK was assessed by RT-PCR. The results showed that Akt and JNK isoforms between normal thyroid tissue and PTC were not different. The up-regulation of Akt and JNK in PTC might be regulated at post-transcriptional level. We also analyzed the expression of wild-type RET in PTC and normal thyroid by RT-PCR. Wild-type RET mRNA seemed to be augmented in PTC, suggesting that the expression of wild-type RET mRNA might be important in pathogenesis of PTC.
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Azakir, Bilal Ahmad. "Régulation et rôle de la ligase de l'ubiquitine Itch dans la signalisation cellulaire." Thèse, 2008. http://hdl.handle.net/1866/6603.
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Chen, Chia-Hung, and 陳家弘. "MEK inhibitors induce Akt activation and drug resistance via suppressing negative feedback ERK-mediated HER2 phosphorylation at Thr701." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/has8rp.
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王智琦. "The Role of MAP Kinasesand Akt, eNOS Phosphorylation Pathways in Oxysterols Induced Cytotoxicity in Human Umbilical Vein Endothelial Cells." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/83874923782337270489.
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碩士臺北醫學大學
藥學系
92
Oxidation products of cholesterol have been shown to be present in oxidized low density lipoprotein and in atherosclerotic lesions. Here we showed three oxysterols, Cholesterol-5a-6a-epoxide (Epoxide), 7-keto-cholesterol (7-Keto) and Cholesterol-3b-5a-6b-triol (Triol) induce cytotoxicity in human umbilical endothelial cells (HUVECs). And the cytotoxicity effect were regulated by MAP kinases, Akt, and eNOS phosphorylation through diversal pathways. Three oxysterols showed different mechanisms and dual aspects of regulation pathways, apoptosis or necrosis, and cell protection, in HUVECs. Epoxide did not cause cytotoxicity within 36 hours and showed the induction of cell protection proteins, ERK, Akt in HUVECs. Besides, Epoxide showed a feedback effect between ERK and eNOS, and thus it might show no cytotoxicity in HUVECs. This unique effect caused by Epoxide made it different from other two oxysterols. Triol showed obvious dual effects in HUVECs. One is cytotoxicity effect including apoptosis and necrosis. JNK, c-Jun, p-38 and caspase-3 were activated by Triol and those were believed to contribute the cytotoxicity. In addition, the generation of ROS also involved in Triol-induced cytotoxicity. Antioxidants such as N-acetylcystein (NAC), vitamin C and vitamin E could diminish the cytotoxicity of Triol. The other is the induction of cell protection proteins, like ERK and Akt. 7-Keto significantly induced apoptosis and JNK, c-Jun, p-38, caspase-3 activation in HUVECs. 7-Keto also activated ERK phosphorylation, and the p-JNK activation seems to contribute the beneficial effect in HUVECs. In summary, the role of oxysterols in HUVECs is controversial. We found that Triol and 7-Keto could induce cytotoxicity, but there is still a cell protection pathway going in the same time. Different with Triol and 7-keto, Epoxide only showed the beneficial effect in this study. The effect of oxysterols on HUVECs seems to be complicated and need further investigation.
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ROHAN. "NEW INSIGHT: IN-SILICO ANALYSIS OF PHYTOCHEMICALS AS A THERAPEUTIC INTERVENTION AGAINST MENINGIOMA." Thesis, 2023. http://dspace.dtu.ac.in:8080/jspui/handle/repository/20118.
Full textAbstract:
Drug development for conventional delivery targets is the major challenge for
therapeutic progress. PI3K signaling has shown a prominent role in progression of
meningioma which subsequently makes these receptors a target for efficient treatment
approach. Loss or alteration of NF2 (neurofibromin 2) with respect to its gene product
merlin is crucially implicated in tumor development eventually leads to the
meningioma. However, the interactions of meningioma pathophysiology (merlin) to
many pathways makes it complicated. Different signalling pathways interacting with
merlin are the hippo pathway, PI3K /Akt route and mTORC pathway. We thesis on the
inhibitory approach for P13K pathway and phosphorylation of Akt to terminate merlin
polyubiquitination and promote its expression and association with PIKE-L thereby
activating tumor suppressive action. Our work emphasizes on exploring biological
compounds that target the PI3K pathway using the in-silico method.
Here, we implemented 50 phytochemicals from various plant sources against PI3K for
molecular docking and assessed as a potential anticancer drug. The docking was
performed showing binding energy, drug likeness and affinity of different
phytochemicals. The following study demonstrated various phytochemicals (such as
Leachianone A, withaferin A, kurarinol and gardenoilic acid B) along with comparing
their ADME score, bioavailability radar and score that provides insights on possible
therapeutic approach of these biological compounds.
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Mayemba, Christian Ndofusu. "Rôle de la phosphorylation des kératines 8/18 dans la modulation de la voie de survie PI3K/AKT/NF-KB en réponse au stress cytotoxique." Thèse, 2019. http://depot-e.uqtr.ca/9089/1/032314919.pdf.
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Lévesque, Marie-Pierre. "Étude génétique et fonctionnelle de variantes de la région chromosomique 3p21 associée aux maladies inflammatoires de l'intestin." Thèse, 2011. http://hdl.handle.net/1866/5881.
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Des études de liaison et d’association génétiques ont permis d’identifier certains des facteurs de risque génétiques aux maladies inflammatoires de l’intestin (MII) dans la région chromosomique 3p21. Dans cette région, le polymorphisme nucléotidique simple (SNP) codant non-synonyme du gène MST1, rs3197999, encodant pour la mutation R689C, a été associé et répliqué à la fois à la colite ulcéreuse (CU) et à la maladie de Crohn (MC). Un autre SNP, corrélé à des SNP codants non-synonymes du gène MST1R, a également été associé à la MC. Afin de déterminer si d’autres variantes des gènes MST1 et MST1R sont associés à la CU, nous avons testé pour association des SNP de ces gènes. Seul un proxy de R689C a montré un signal d’association significatif aux MII, ce qui suggère que R689C est la variante causale aux MII dans le gène MST1. En cherchant à déterminer si la région 3p21 contenait plusieurs signaux d’association mutuellement indépendants, trois SNP ont été identifiés comme possible facteurs de risque indépendants, et ont été génotypés dans des cas de CU et de MC et des témoins, puis nos résultats d’association ont été combinés à ceux provenant de trois autres cohortes indépendantes. Les trois SNP, R689C (MST1), rs6802890 et rs7629936 (CDHR4), sont associés aux MII, mais une étude d’association conditionnelle suggère qu’il existe en fait deux signaux d’association mutuellement indépendants dans la région 3p21. Le signal principal provient de R689C, une mutation de la protéine MSP. Cette protéine a un rôle dans l’inflammation chez les macrophages murins, et la migration, la cicatrisation et la survie chez les cellules épithéliales. Dans cette étude, le rôle de la MSP a été investigué dans des modèles de macrophages humains et de cellules épithéliales de côlon, et seule la phosphorylation d’AKT, un acteur dans la voie de signalisation de la survie cellulaire, a été modulée par la MSP dans nos modèles. Ce projet a donc permis d’apporter des connaissances sur les facteurs de risques génétiques aux MII dans la région 3p21, en identifiant 2 signaux d’association indépendants, et en nous informant sur le rôle de MST1, duquel provient le signal d’association principal, chez les cellules humaines.Linkage studies and association studies allowed the discovery of some of the genetic risk factors of inflammatory bowel disease (IBD) in the chromosomal region 3p21. In this region, the non-synonymous coding single nucleotide polymorphism (SNP) rs3197999, situated in the gène MST1 and encoding for the mutation R689C, has been associated to UC and CD multiple times, and an other SNP, correlated to non-synonymous coding SNPs in the gene MST1R, has also been associated to CD. In order to verify if other variants of MST1 and MST1R are associatied to UC, we tested the association of some of their SNPs. Apart from R689C, only its proxy showed a significative association signal to IBD. It suggests that R689C might be the causal variant of IBD in the region 3p21. In the aim to determine if the region 3p21 has multiple independant association signals, 3 SNPs have been identified, from the results of a published meta-analysis of UC genome-wide association studies, as being possibly independant risk factors for UC based on their correlation. Their association to IBD and their independance have been tested by genotyping them in a cohort composed of controls, and UC and CD cases. The results of the association tests have been combined, in a meta-analysis, to the results of 3 other independent association studies. The 3 SNPs, R689C (MST1), rs6802890 and rs7629936 (CDHR4) are associated to IBD, but the results of the subsequent conditional association tests suggest that there is only 2 independant association signals in the region 3p21. The main signal is raising from R689C, a mutation of the protein MSP. According to published studies, this protein has a function in the inflammation in murine macrophages, and also in the scattering, wound healing and survival of epithelial cells. In this thesis, we investigated the role of MSP in human macrophage models and in human côlon epithelial cells, and it has been show that MSP modulates the phosphorylation of AKT, an actor in the pathway of cellular survival. This project brought some knowledges about the IBD genetic risk factors in the region 3p21. We identified 2 independent association signals to IBD in this region, and the main signal is coming from a SNP in MST1, a gene which has a role, based on our results, in the survival in human colon epithelial cells.
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Truong, Vanessa. "Rôle de la protéine kinase B (Akt) dans la phosphorylation des histones désacétylases 5 (HDAC5) et l’expression de l’early growth response protein-1 (Egr-1) induites par l'angiotensine II dans les cellules musculaires lisses vasculaires." Thesis, 2020. http://hdl.handle.net/1866/24569.
Full textAbstract:
Une augmentation de la concentration de l’angiotensine II (Ang II) contribue à la prolifération, la migration et l’hypertrophie des cellules musculaires lisses vasculaires (CMLVs) par l’activation des voies des mitogen-activated protein kinases (MAPK) et de la phosphoinositide 3-kinase (PI3K)/protéine kinase B (PKB/Akt). L’Ang II induit l’activation du facteur de transcription early growth response protein-1 (Egr-1) et sa suractivation est remarquée dans les lésions athérosclérotiques et les modèles animaux de lésions vasculaires. La régulation des facteurs de transcription est effectuée par des histones désacétylases (HDACs) qui désacétylent les lysines des histones et protéines non-histones. L’Ang II induit la phosphorylation et l’export nucléaire de la classe IIa des HDACs, particulièrement les HDAC5, et une augmentation de celles-ci est observée dans les maladies vasculaires. L’Ang II est un puissant activateur des voies des MAPK et de la PI3K/Akt, toutefois l’implication de ces voies dans la phosphorylation des HDAC5 et l’expression de l’Egr-1 dans les CMLVs reste inexplorée. Dans cette étude, l’Ang II a induit la phosphorylation des HDAC5 sur la sérine 498 dans les A10 CMLVs. Un blocage pharmacologique de l’extracellular signal-regulated kinase 1/2 (ERK1/2) par U0126 n’a montré aucun effet significatif sur la phosphorylation et l’exclusion nucléaire des HDAC5 induite par l’Ang II. Par contre, l’inhibition de la voie PI3K par wortmannin, de l’Akt par SC66 ou le knockdown de l’Akt par des petits ARN interférents (siRNA) a atténué la phosphorylation et l’export nucléaire des HDAC5 induits par l’Ang II. Par ailleurs, l’inhibition de l’Akt ou le knockdown de cette kinase a diminué l’expression de l’Egr-1 induite dans les CMLVs stimulées par l’Ang II. L’inhibition des HDACs de la classe IIa par MC1568 ou TMP-195 ou bien le knockdown des HDAC5 a diminué l’expression de l’Egr-1 induite par l’Ang II. De plus, le blocage de l’export nucléaire des HDAC5 par la leptomycine B ou la KPT-330 a empêché la localisation cytoplasmique des HDAC5 et a atténué l’expression de l’Egr-1 en réponse à une stimulation de l’Ang II. L’hypertrophie vasculaire induite par l’Ang II a pu être inhibée par la suppression de l’HDAC5 et l’Egr-1. En conclusion, l’Ang II induit la phosphorylation et l’exclusion nucléaire des HDAC5 par la voie PI3K/Akt et non celle de ERK1/2; de plus, l’Ang II induit l’expression de l’Egr-1 à l’aide des HDAC5 via la voie Akt contribuant ainsi à l’hypertrophie des CMLVs.Elevated concentration of angiotensin II (Ang II) contributes to vascular smooth muscle cells (VSMCs) proliferation, migration and hypertrophy by the activation of the mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathways. Ang II induced the expression of early growth response protein-1 (Egr-1), which is a transcription factor that is upregulated in atherosclerosis lesions and in animal models of vascular injuries. The activation or derepression of gene transcription is mediated by histone deacetylases (HDACs), which deacetylate lysine residues from histone and non-histones proteins. Ang II-induced the phosphorylation and nuclear export of class IIa HDACs, notably HDAC5, and its elevated activation is observed in vascular pathologies. Ang II is a potent activator of the MAPK and PI3K/Akt pathways, however their implication in the phosphorylation of HDAC5 and Egr-1 expression in VSMCs remain unexplored. In this study, Ang II-induced HDAC5 phosphorylation at serine 498 in A10 VSMCs and pharmacological blockade of the extracellular signal-regulated kinase 1/2 (ERK1/2) by U0126 did not affect the phosphorylation and nuclear exclusion of HDAC5 in response to Ang II. Whereas, pharmacological inhibition of the PI3K by wortmannin, Akt by SC66 or small interfering RNA (siRNA)-induced silencing of Akt attenuated Ang II-induced HDAC5 phosphorylation and its nuclear export. Furthermore, inhibition or knockdown of Akt suppressed Ang II-induced Egr-1 expression. In addition, the inhibition of class IIa HDAC5 by MC1568, TMP-195 or HDAC5 knockdown by siRNA reduced Ang II-induced Egr-1 expression. The blockade of the nuclear export of HDAC5 by leptomycin B or KPT-330 prevented the cytoplasmic localization of HDAC5 and attenuated the expression of Egr-1 by Ang II in VSMCs. Moreover, HDAC5 or Egr-1 depletion prevented Ang II-induced cell hypertrophy. In summary, Ang II-induced HDAC5 phosphorylation and its nuclear export is mediated by the PI3K/Akt and not the ERK1/2 pathway, in addition, Ang II-induced Egr-1 expression involves the implication of HDAC5 via the Akt pathway which subsequently leads to VSMC hypertrophy.
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Sanchez, Melanie. "Growth factor activation of ErbB2/ErbB3 signaling pathways regulate the activity of Estrogen Receptors (ER)." Thèse, 2010. http://hdl.handle.net/1866/4472.
Full textAbstract:
La signalisation par l’estrogène a longtemps été considérée comme jouant un rôle critique dans le développement et la progression des cancers hormono-dépendants tel que le cancer du sein. Deux tiers des cancers du sein expriment le récepteur des estrogènes (ER) qui constitue un élément indiscutable dans cette pathologie. L’acquisition d’une résistance endocrinienne est cependant un obstacle majeur au traitement de cette forme de cancer. L’émergence de cancers hormono-indépendants peut est produite par l’activation de ER en absence d’estrogène, l’hypersensibilité du récepteur aux faibles concentrations plasmique d’estrogène ainsi que l’activation de ER par des modulateurs sélectifs. L’activité du ER est fortement influencée par l’environnement cellulaire tel que l’activation de voie de signalisation des facteurs de croissances, la disponibilité de protéines co-régulatrices et des séquences promotrices ciblées. Présentement, les études ont principalement considérées le rôle de ERα, cependant avec la découverte de ERβ, notre compréhension de la diversité des mécanismes potentiels impliquant des réponses ER-dépendantes s’est améliorée. L’activation des voies des kinases par les facteurs de croissance entraîne le développement d’un phénotype tumoral résistant aux traitements actuels. Nos connaissances des voies impliquées dans l’activation de ER sont restreintes. ERα est considéré comme le sous-type dominant et corrèle avec la plupart des facteurs de pronostic dans le cancer du sein. Le rôle de ERβ reste imprécis. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre l’implication de ERβ dans la prolifération cellulaire par l’étude du comportement de ERβ et ERα suite à l’activation des voies de signalisation par les facteurs de croissance.
Nous démontrons que l’activation des récepteurs de surfaces de la famille ErbB, spécifiquement ErbB2/ErbB3, inhibe l’activité transcriptionnelle de ERβ, malgré la présence du coactivateur CBP, tout en activant ERα. De plus, l’inhibition de ERβ est attribuée à un résidu sérine (Ser-255) situé dans la région charnière, absente dans ERα. Des études supplémentaires de ErbB2/ErbB3 ont révélé qu’ils activent la voie PI3K/Akt ciblant à son tour la Ser-255. En effet, cette phosphorylation de ERβ par PI3K/Akt induit une augmentation de l’ubiquitination du récepteur qui promeut sa dégradation par le système ubiquitine-protéasome. Cette dégradation est spécifique pour ERβ. De façon intéressante, la dégradation par le protéasome requiert la présence du coactivateur CBP normalement requis pour l’activité transcriptionnelle des récepteurs nucléaires. Malgré le fait que l’activation de la voie PI3K/Akt corrèle avec une diminution de l’expression des gènes sous le contrôle de ERβ, on observe une augmentation de la prolifération des cellules cancéreuses. L’inhibition de la dégradation de ERβ réduit cette prolifération excessive causée par le traitement avec Hrgβ1, un ligand de ErbB3. Un nombre croissant d’évidences indique que les voies de signalisations des facteurs de croissance peuvent sélectivement réguler l’activité transcriptionnelle de sous-types de ER. De plus, le ratio ERα/ERβ dans les cancers du sein devient un outil de diagnostique populaire afin de déterminer la sévérité d’une tumeur. En conclusion, la caractérisation moléculaire du couplage entre la signalisation des facteurs de croissance et la fonction des ERs permettra le développement de nouveaux traitements afin de limiter l’apparition de cellules tumorales résistantes aux thérapies endocriniennes actuelles.It has long been appreciated that estrogenic signaling plays a critical role in the development of hormone-dependent cancers such as breast cancer. Two-thirds of breast cancers express estrogen receptor (ER) which has been demonstrated to play an irrefutable role in tumour development and progression. However the acquisition of endocrine resistance has become a major obstacle in the treatment of hormone-dependent cancers that have acquired a hormone-independent state. Hormone-independent cancers emerge from an array of pathways involving ER activation in the absence of estrogen, hypersensitivity of ER to low serum levels of estrogen and activation by estrogen antagonists. The activity of ER is critically influenced by the cellular environment such as growth factor signaling pathways, availability of coregulatory proteins and the promoter sequence of target genes. The mechanisms studied have mostly considered the role of ERα, however with the discovery of the second subtype, ERβ, the understanding on the diversity of potential mechanisms involving ER-dependent responses have improved. Hormonal-independent activation of ER can occur in estrogen-dependent breast tumours, with concomitant rise in kinase signaling pathways, resulting in the acquisition of a therapeutic resistant phenotype in treated women. Our knowledge is relatively limited on which pathways trigger ER signaling and how these phosphorylation-coupled events affect ER activity. ERα is considered the dominant subtype and correlates with most of the prognostic factors in breast cancers. Conversely the role of ERβ remains unclear. The results presented in this thesis were carried out with the objective of gaining a better understanding of ERβ’s role in cellular proliferation by examining the behavior of ERβ and ERα during the activation of growth factor signaling pathways by cell-surface receptor-tyrosine kinases. We demonstrate here that the activation of cell surface receptors of the ErbB family, specifically ErbB2/ErbB3, inhibits the transcriptional activity of ERβ despite the presence of the coactivator CBP, yet activated ERα. Furthermore the inhibition of ERβ was attributed to a specific serine residue located within the hinge region, not present in ERα. Additional studies of ErbB2/ErbB3-initiated signaling revealed that it triggered the activation of the PI3K/Akt pathway which targeted the serine residue within the hinge region of ERβ. In fact, phosphorylation of ERβ by the PI3K/Akt pathway led to an increase in receptor ubiquitination which promoted its degradation by the ubiquitin-proteasome system which was subtype specific. Interestingly, proteasomal degradation required the presence of the coactivator CBP, which is normally involved in assisting nuclear receptor transcriptional activity. Although the activation of the PI3K/Akt pathway correlated with a decrease in the expression of ERβ target genes it led to an increase in the proliferation of breast cancer cells. Inhibiting the degradation of ERβ reduced the enhanced proliferation of breast cancer cells brought about by the treatment of ErbB3’s ligand, Hrgβ1. Increasing evidence indicates that growth factor signaling pathways can selectively regulate the transcriptional activity of ER subtypes, and the ratio of ERα/ERβ expression in breast tumours is becoming a popular prognostic factor to evaluate the severity of the tumour. Therefore the molecular characterization of the coupling between growth factor signaling and ER function should provide improved therapeutical approaches to overcome or delay the onset of resistance to endocrine therapy in hormone-dependent cancers.
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Wen, Hui-Chun, and 溫慧鈞. "Part I ：A study of Ro 31-8220, bisindolylmaleimide VIII and LY 379196 -induced PI3-kinase/Akt activation in epithelial cells. Part II：Regulatory roles of Ca2+ and ERK-mediated cPLA2 phosphorylation in arachidonic acid release in epithelial cells." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/60116603379290169759.
Full textAbstract:
碩士國立臺灣大學
藥理學研究所
88
Part I ：A study of Ro 31-8220, bisindolylmaleimide VIII and LY 379196 -induced PI3-kinase/Akt activation in epithelial cells. The lipid kinase phosphoinositide 3-kinase (PI3K) and its target kinase Akt are involved in a variety of signal transduction and diverse cellular functions. Although substantial studies have begun to explore the regulation of PI3K/Akt cascade by different signaling pathways, whether protein kinase C (PKC) activity plays a crucial role remains as yet unclear. In this study, we found that PKC inhibitors Ro 31-8220, bisindolylmaleimide VIII and PKCβ selective inhibitor LY 379196 consistently caused a concentration-dependent Akt phosphorylation at Ser 473. The effects were blocked by the PI3K inhibitors wortmannin and LY 294002, and the PDK inhibitor SB 203580, suggesting that Akt phosphorylation by PKC inhibitors was mediated by the PI3K pathway. Accompanied with these findings, all these three PKC inhibitors increased Akt and the upstream kinase ILK activities. However, some PKC inhibitors tested, such as staurosporine, calphostin C, rottlerin and Go 6976 had no effects on Akt phosphorylation. Interestingly, although PMA can slightly attenuated Akt phosphorylation, its short term or long term treatment cannot alter the response of Akt phosphorylation caused by Ro 31-8220, bisindolylmaleimide VIII and LY 379196. Thus, we suggested that the stimulating responses of these three PKC inhibitors might be PKC-independent. Both the stimulatory effects of the three PKC inhibitors and the inhibitory effect of PMA on Akt phosphorylation were all evidenced in Akt-overexpressed HEK293 cells. Moreover, we also found that these stimulating effects could not result from phosphatase inhibition, because after treatment with protein phosphatase 2A (PP2A) inhibitor okadaic acid, tyrosine phosphatase inhibitor sodium orthovanadate or protein phosphatase 2B (PP2B) inhibitor FK506, the responses of Ro 31-8220, bisindolylmaleimide VIII and LY 379196 still existed. Finally, we suggested the possible involvement of ROS generation in this event, as H2O2 dramatically increased Akt phosphorylation and antioxidants GSH and NAC can attenuate the responses of Ro 31-8220 and bisindolylmaleimide VIII. Also we found that Ro 31-8220 and bisindolylmaleimide VIII can increase ROS production. Taken together, we found a novel and PKC-independent stimulatory action of Ro 31-8220, bisindolylmaleimide VIII on PI3K/Akt signaling pathway, and this response might be mediated by the production of reactive oxygen species. Part II：Regulatory roles of Ca2+ and ERK-mediated cPLA2 phosphorylation in arachidonic acid release in epithelial cells. The release of arachidonic acid (AA) and its connection with the triggering of the MAP kinase cascade were studied in the human A549 epithelial cell line upon stimulation with thapsigargin. Thapsigargin can increase AA release along with the increase of intracellular calcium concentration, phosphorylation and activation of extracellular signal-regulated kinase (ERK) and cytosolic phospholipase A2 (cPLA2). Both ERK and cPLA2 phosphorylation in response to thapsigargin were inhibited by PD 98059, a specific inhibitor of MAP kinase kinase of the ERK group (MEK), and EGTA. cPLA2 phosphorylation was not affected by Ro 31-8220 (an inhibitor of all PKC isoforms) or LY 379196 (a PKCb selective inhibitor), while both of them indeed attenuated ERK activation. On the other hand, rottlerin (the selective PKCd inhibitor), SB 203580 (the selective p38 MAPK inhibitor) and wortmannin (the PI 3-kinase inhibitor) can affect neither cPLA2 nor ERK phosphorylation. In A549 cells, PKC activator PMA cannot increase either the basal or thapsigargin-induced AA release, while it can induce the phosphorylation of ERK and cPLA2. The PMA-induced ERK phosphorylation was inhibited by Ro 31-8220, LY 379196, rottlerin and PD 98059, but unaffected by SB 203580 and wortmannin. Moreover the ERK and cPLA2 phosphorylation induced by PMA were non-additive with those of thapsigargin. This implies that intracellular Ca2+ level is the key factor for induction of cPLA2 activity and thapsigargin-elicited ERK activation itself is substantially sufficient for cPLA2 activation upon intracellular Ca2+ increase.
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Wang, Cheng-Yi, and 汪政毅. "Phosphorylations of Thr308 and Ser473 Induce Proteasome-Dependent Degradation of Akt." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/wyqzc8.
Full textAbstract:
碩士國立陽明大學
生化暨分子生物研究所
97
The serine/threonine protein kinase Akt, also known as PKB, is a downstream effector molecule of phosphoinositide 3-kinase (PI3K) and is thought to mediate many biological functions including cell survival, proliferation, and carcinogenesis. Upon stimulation with a growth factor, phosphorylations of Akt at Thr308 and Ser473 is required for full activation of the kinase. Heat shock protein 90 (Hsp90) binds to Thr308 and Ser473-phosphorylated Akt, by which its kinase activity is stabilized. Inhibition of Hsp90 ATPase activity by Hsp90 inhibitor contributes to Akt ubiquitination and proteasome-dependent degradation; however, the mechanism of degradation and the involvement of a potential E3 ubiquitin ligase in this process remain uncertain. In our study we show that phosphorylations of Thr308 and Ser473 induced by Hsp90 specific inhibitor geldanamycin (GA) or by growth factor insulin promote Akt to enter the ubiquitin-proteasome pathway. Mutations at either Thr308 or Ser473 or both sites result in decreased rate of ubiquitination. We also show that CHIP (C-terminus of Hsc70 Interacting Protein) is a major E3 ubiquitin ligase that contributes to Akt ubiquitination and degradation. Mutations of Akt at either Thr308 or Ser473 or both sites weaken its ability to bind to CHIP, which is correlated with ubiquitin phenomenon. Our study unveils that the well-known phosphorylations, in addition to responsible for Akt activation, also turn out to transduce recognition signals for CHIP-mediated ubiquitination and proteasomal degradation.
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Busch, F., A. Mobasheri, P. Shayan, C. Lueders, R. Stahlmann, and M. Shakibaei. "Resveratrol modulates interleukin-1beta-induced phosphatidylinositol 3-kinase and nuclear factor kappaB signaling pathways in human tenocytes." 2012. http://hdl.handle.net/10454/5903.
Full textAbstract:
Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1beta-mediated inflammatory signaling. Resveratrol suppressed IL-1beta-induced activation of NF-kappaB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1beta-induced NF-kappaB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IkappaB kinase, IkappaBalpha phosphorylation, and inhibition of nuclear translocation of NF-kappaB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-kappaB activation. Inhibition of PI3K by wortmannin attenuated IL-1beta-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1beta-induced activation of NF-kappaB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-kappaB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-kappaB.
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木村, 薫., and Kaoru Kimura. "β-Hydroxy-β-methylbutyrate facilitates PI3K/Akt-dependent mammalian target of rapamycin and FoxO1/3a phosphorylations and alleviates tumor necrosis factor α/interferon γ-induced MuRF-1 expression in C2C12 cells." Thesis, 2014. http://hdl.handle.net/2237/20560.
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