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RACK1 (Receptor for activated protein C kinase 1) est une protéine ribosomale associée à de nombreuses voies de signalisation. RACK1 est nécessaire à la traduction sélective de virus contenant des sites d’entrée interne du ribosome (IRES). En outre, l’expression de RACK1 est nécessaire au cours du développement, suggérant que ce facteur participe à la traduction de certains ARNm cellulaires. Dans le but de mieux comprendre la fonction de RACK1 chez la drosophile, j’ai au cours de ma thèse caractérisé l’interactome de RACK1 et un IRES viral régulé par ce facteur. J’ai également essayé d’établir un lien entre signalisation cellulaire et traduction, et montré que la région du knob est importante pour la fonction de RACK1 au ribosome. Enfin, j’ai établi que RACK1 est nécessaire à la réponse à des stress abiotiques, et identifié les gènes cellulaires régulés par RACK1 dans ce contexte. J’ai en particulier découvert que RACK1 était un régulateur négatif de l’expression de plusieurs gènes de l’immunité innée. Mes résultats suggèrent que RACK1 joue un rôle pivot au sein du ribosome, régulant la traduction de façon positive ou négative selon l’ARNm et le contexte cellulaire. / RACK1 (Receptor for activated protein C kinase 1) is a ribosomal protein associated to many signaling pathways. RACK1 is required for the selective translation of viruses containing internal ribosome entry sites (IRES). In addition, expression of RACK1 is necessary during development, suggesting that it regulates the translation of cellular mRNAs. In order to better understand the function of RACK1 in Drosophila, I have participated in the characterization of the RACK1 interactome and of a RACK1-dependent viral IRES. I have also attempted to establish a connection between the function of RACK1 in signaling and in translation, and I have shown that the knob domain of RACK1 is important for IRES-dependent translation. Finally, I have established that RACK1 is required for the response to abiotic stresses, and I have identified cellular genes regulated by RACK1 in this context. In particular, I discovered that RACK1 is a negative regulator of several innate immunity genes. My results suggest that RACK1 plays a pivotal role within the ribosome, regulating translation positively or negatively in an mRNA- and possibly context-specific manner.

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Le virus de l’hépatite C (VHC) dépend de facteurs cellulaires pour accomplir son cycle viral et persister dans l’hôte. L’une des stratégies de notre laboratoire consiste à étudier de manière approfondie le réseau d’interactions virus-hôte, afin d’identifier de nouvelles cibles thérapeutiques cellulaires et de développer des antiviraux plus efficaces pour vaincre la résistance virale. Durant ma thèse j’ai étudié deux facteurs cellulaires importants pour le VHC. Le premier est la protéine ribosomale RACK1. Nous avons montré que cette protéine est spécifiquement requise pour la traduction IRES-dépendante du VHC, et non pour la traduction coiffe-dépendante. Le deuxième facteur est une protéine de surface des gouttelettes lipidiques appelée TIP47. Nous avons montré que cette protéine est importante à la fois pour l’assemblage et pour l’export des particules virales. L’ensemble de ces travaux montre que de nouvelles cibles thérapeutiques pourraient être envisagées pour lutter contre le VHC. / The hepatitis C virus (HCV) relies on cellular factors to complete its life cycle and persist in its host. One of the strategies employed by our laboratory is the in-depth study of the network of virus-host interactions to identify new therapeutic cellular targets and develop more effective antivirals to overcome viral resistance.During my PhD, I studied two cellular factors involved in the HCV life cycle. The first factor is the ribosomal protein RACK1. We have shown that this protein is specifically required for the HCV IRES-mediated translation but not for the cap-mediated translation. The second factor is the lipid droplets binding protein TIP47. We have shown that this protein is important for both assembly and export of viral particles. This work shows that new therapeutic targets could be considered in the fight against HCV.

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Ras genes are frequently mutated in human cancers and present compelling targets for therapeutic intervention. While previous attempts to directly inhibit oncogenic Ras function have largely been unsuccessful use of targeted agents to inhibit the three primary oncogenic pathways activated by mutated Ras: RalGEF-Ral, PI3K-Akt and Raf- MEK-Erk, is an area of intense investigation. Here, we describe the ability of a novel pharmacological inhibitor of geranylgeranyltransferase I, GGTI-2417, to inhibit Ral prenylation and localization. We further used a Ral rescue system to selectively preserve RalA and RalB function and localization during GGTI-2417 treatment and determine the precise roles for inhibition of Ral prenylation in the GGTI anti-cancer response. Specifically, we determined inhibition of RalA is required for GGTI-attenuation of anchorage independent growth whereas inhibition of RalB is required for inhibition of proliferation, induction of apoptosis, suppression of survivin and induction of p27Kip1. We next determined the role of RalGEF-Ral signaling as well as PI3K-Akt and Raf-MEKErk signal transduction pathways in an in vitro model of human ovarian surface epithelial (T80 HOSE) cell Ras-dependent transformation. Using both small interfering RNA (siRNA) and pharmacological inhibitors of Ral, PI3K and MEK we determined that Ras signaling via Ral and PI3K but not MEK is required for ovarian oncogenesis. Furthermore, stable expression of Ras mutants unable to activate Raf-MEK-Erk signaling were able to robustly transform T80 cells. Since we had confirmed the importance of Ral proteins to human epithelial malignancies we next sought to explore the molecular interactions governing Ral transformation using a proteomics approach to rapidly identify proposed Ral interacting partners. Using immunoprecipition of transiently overexpressed FLAG-tagged RalA and RalB followed by 1D-gel separation and tandem MS/MS analysis we determined a database of proposed Ral interacting proteins. One of these, RACK1, is a validated RalA and RalB interacting protein which is at least partially required for Ras and Ral transformation. These results provide both a strong impetus and a solid basis for future studies into the mechanisms of RalA- and RalB- dependent transformation.

No description available.

RACK1 (Receptor for activated C kinase 1) is an evolutionary conserved protein which has essential role in most studied eukaryotic organisms, except for yeast. Although RACK1 was originally described as a binding partner of protein kinase C, later studies re- vealed its significant role in other cellular signalizations such as MAPK, Src or FAK. Thanks to this, RACK1 participates in the regulation of key cellular processes including migration, apoptosis or translation. As a binding partner of a small ribosomal subunit, RACK1 contributes to transla- tion regulation by integrating signals from different cellular pathways and several transla- tional components such as PKC and eIF6. Moreover, RACK1 has a role in translation regu- lation during stress. Under stress conditions there is a global reduction of translation, in- creased expression of specific mRNAs important for cellular stress response and formation of cytosolic foci called stress granules (SGs). SGs play an important role in protection of mRNAs and translation components against degradation. SGs also function in prevention of apoptosis. RACK1 has been identified as one of many components of SGs and its localization into SGs leads to inhibition of RACK1-mediated pro-apoptotic pathways. Aim of this diploma thesis was to elucidate the role of...

Yes / Background: The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes. Results: We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gβ function in D. discoideum as developmental and other defects were not rescued in Gβ null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development. Conclusion: DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins. / This work was supported by the DFG and SFB670. TYR acknowledges support from the Professorinnen Program of the University of Cologne.

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