RAS is one of the most commonly mutated genes in human cancer; its aberrant activation drives tumor cell proliferation and survival. However, RAS mutations are rare in some cancers, including breast cancer, even though the Ras pathway is hyperactivated, suggesting that alternative mechanisms deregulate Ras signaling in these settings. The RasGAPs are negative regulators of Ras and, as such, are poised to function as tumor suppressors whose loss might contribute to Ras pathway hyperactivation in cancer. However, the RasGAPs remain an understudied family of genes whose role in cancer has not been fully explored. In this Dissertation I identify a previously uncharacterized RasGAP, RASAL2, as the newest tumor suppressor in this gene family.

No description available.

G3BP1 and G3BP2 are members of a highly conserved family of multi-functional RNA binding proteins, which appear to co-ordinate signal transduction and post-transcriptional gene regulation. Both proteins are over-expressed in cancer, and G3BP1 promotes cell proliferation and survival. Aberrant expression of various RNA binding proteins is common in cancer, and several of these proteins influence tumorigenesis. Therefore, detailed examination of RNA binding proteins, such as G3BPs, may provide insights into the post-transcriptional mechanisms underlying tumorigenesis. Tumours arise as a consequence of genetic mutation or alteration, which often result from stress-induced DNA damage. Cancer progression is facilitated by various epigenetic stress adaptation mechanisms. Stressful stimuli induce transitory translational shut-off, mediated by phosphorylation of eukaryotic initiation factor alpha;(eIF2alpha;). This phosphorylation event leads to formation of discrete cytoplasmic foci known as stress granules (SGs), which are translationally-silent sites of mRNA sorting. It was initially thought that an RNA-binding protein, T-cell internal antigen 1 (TIA-1), was instrumental in both the formation and functioning of SGs, because over-expression of TIA-1 induces spontaneous SGs and concomitantly causes a decrease in reporter gene expression. It is now clear that SG content can change depending on the type of stress, and that various proteins, including G3BP1, can induce spontaneous SGs. In vitro evidence previously implicated both G3BP1 and G3BP2 as endoribonucleases, so it was suggested that G3BPs act to target mRNA for decay at the SG. This project sought to further investigate this proposal, and in this way gain insight into the specific function of G3BPs in post-transcriptional regulation during tumorigenesis. Characterisation of G3BP1 and G3BP2 expression and localisation patterns in human cells and cancer was necessary before functional analyses in human cell systems could be undertaken. Both proteins were found to be over-expressed in breast cancer, irrespective of cancer stage or grade. G3BP1 and G3BP2 were also expressed in all human cell lines tested, despite previously observed tissue-specific expression. These results support the notion that G3BP expression is switched on in parallel with cell proliferation, and as such, may influence tumorigenesis. The results of further analyses suggested that the diverse functions attributed to G3BP1 and G3BP2 may be facilitated by isoform-specific expression, various post-translational modifications and sub-cellular localisation. Despite the absence of a canonical endoribonuclease domain, it was previously reported that site-specific phosphorylation of G3BP1 enables the protein to degrade a synthetic c-myc RNA substrate in vitro. This finding implicated G3BP in the specific regulation of a proto-oncogene. Tailored reporter assays were thus designed in order to address the in vivo consequences of G3BP's putative endoribonuclease activity. Contrary to expectations, all G3BP family members increased or maintained the expression of a range of reporters, at both the mRNA and protein level, irrespective of the presence of any particular cis-acting element, coding sequence or promoter. These results support the emerging notion that G3BPs positively affect the expression of at least some of their target mRNAs, and may also indirectly promote transcription. In contrast to the theory that G3BPs degrade proto-oncogenic mRNA/s, these findings are consistent with a role for G3BP in promoting cell proliferation and survival. Further analyses showed that G3BP1 and G3BP2 simultaneously increased reporter gene expression and induced SG formation. These findings highlighted the fact that SGs are dynamic sorting stations for mRNAs, and not merely sites of stalled translation. This result also supports the notion that a variety of proteins may be recruited to the SG to facilitate a multitude of mRNA fates. Although the precise role of the SG in stress adapation is not known, it is clear that an appropriate integrated stress response (ISR) is required for cells to survive in sub-optimal conditions. It was found that specific G3BP1 knockdown inhibited SG formation and cell survival, and this appeared to occur downstream of eIF2alpha; phosphorylation. The phosphorylation of eIFalpha; is the only factor known to be necessary for SG formation and cell survival. This data is the first to implicate SG formation itself, downstream of eIF2alpha; phosphorylation, in the survival phase of the ISR. The results also suggest that G3BP1 plays a pivotal role in the post-transcriptional mechanisms underlying stress adaptation. To facilitate future analysis of G3BP roles in the regulation of specific transcripts and in SG biology, a pilot study to identify G3BP RNA ligands was undertaken. Immunoprecipitation of epitope-tagged G3BP1 from stable cell lines facilitated purification and isolation of RNA in association with G3BP1. Specific RNA transcripts were subsequently detected and identified by microarray. Many genes were enriched in the G3BP1 immunoprecipitate. Transcript enrichment in the control immunoprecipitate was comparatively weak and seemingly random, suggesting that several replicates will enable generation of a reliable target list. This work forms a promising basis for further investigations into G3BP functionality, and also provides a platform for broader and more large-scale analyses of the mechanisms of post-transcriptional gene regulation. The work presented in this thesis addressed the potential post-transcriptional mechanisms by which the G3BP family of proteins mediate cell proliferation and survival. Both G3BP1 and G3BP2 were shown to be over-expressed in tumours and each appeared to promote reporter gene expression. G3BP1 was also found to play a pivotal role in stress adaptation. A technique to identify novel RNA ligands was assessed, and it was found that G3BP1 may interact with various mRNA transcripts. It is hypothesised that the G3BP family of proteins, and in particular G3BP1, function to determine the fate of specific RNAs in response to cellular stress and other stimuli. In this way, G3BP proteins may facilitate appropriate responses to extra-cellular stimuli which allow for cell proliferation and survival.

RasGAP is a GTPase activating protein (GAP) that deactivates Ras by promoting Ras-GTP hydrolysis to Ras-GDP. In Drosophila melanogaster, RasGAP is required for the long-term survival of neurons in the adult brain because mutants in the RasGAP gene (vap) show an age-related neurodegenerative phenotype, with dying neurons showing morphological features of autophagy. RasGAP was shown to have a GAP-independent role within fly neurons that is dependent on its SH2 domains. The aim of this study was to identify proteins that interact with the SH2 domains of RasGAP and to understand the roles of these proteins in neuronal survival. By using tagged RasGAP affinity purification and mass spectrometry of RasGAP protein complexes from S2 cells, Sprint, a Ras effector and putative activator of the endocytic GTPase Rab5, was identified as a novel SH2-dependent RasGAP interacting protein. The interaction between Sprint and RasGAP is phosphotyrosine-dependent, since it requires tyrosine phosphorylation of Sprint. In addition, Sprint and RasGAP interaction requires the SH2 domains of RasGAP but not Sprint or the conserved site of RasGAP tyrosine phosphorylation (pTyr363), indicating an association between these two molecules. RasGAP and Sprint co-localised with Rab5-positive early endosomes and this co-localisation depended on the SH2 domains of both RasGAP and Sprint. This study demonstrates a key role for this interaction in neurodegeneration: mutation of Sprint (or Rab5) suppressed the autophagic neuronal cell death caused by the loss of RasGAP. These results indicate that the long-term survival of adult neurons in Drosophila depends on a critical balance between Ras activation and endocytosis, and that this balance is maintained by the interplay between RasGAP and Sprint.

L'objectif de cette thèse consiste à inhiber la voie de signalisation liée aux protéines Ras, très fréquemment impliquée dans les tumeurs humaines, en concevant des inhibiteurs d'interactions protéine-protéine. Au sein de cette voie, la protéine RasGAP joue un rôle particulier, à la fois régulateur négatif et effecteur de Ras. La cible thérapeutique constituée par le domaine SH3 de la protéine RasGAP avait déjà été identifiée par plusieurs équipes (Tocqué et al., 1997). Plus récemment, notre laboratoire a complété ces travaux par l'identification des protéines Aurora comme partenaires de RasGAP-SH3. En collaboration avec Aptanomics, en mettant en oeuvre la technique des aptamères peptidiques, nous avons apporté une nouvelle validation de cette cible : nous avons obtenu par un crible double-hybride de nouvelles protéines synthétiques (aptamères) interagissant spécifiquement avec le domaine SH3 de RasGAP, et dont l'expression dans des cellules tumorales provoque une diminution de la capacité à former des colonies et de la viabilité cellulaire. Afin d'amorcer une démarche de conception rationnelle d'inhibiteurs de ce SH3, nous avons synthétisé les peptides exposés à la surface de ces aptamères et responsables de leur interaction avec RasGAP-SH3. Nous avons ensuite mesuré l'affinité de ces peptides pour RasGAP-SH3 par anisotropie de fluorescence. Nous avons ainsi obtenu un peptide cyclique dont l'affinité pour le domaine SH3 est de l'ordre de quelques centaines de nanomolaire, et dont nous avons déterminé l'empreinte sur ce domaine enrichi en 15N par RMN, en nous appuyant sur la structure du domaine, déjà résolue au laboratoire (Yang et al., 1994). Les données structurales que nous avons obtenues devraient permettre, dans un court délai, de proposer des modifications de ces peptides, afin d'augmenter l'affinité de nos inhibiteurs et de les vectoriser pour leur conférer une activité sur cellules tumorales en culture. Enfin, ces peptides, rendus fluorescents par leur couplage à un fluorophore, vont être utilisés comme ligands de référence dans un crible de chimiothèque à haut débit, afin de découvrir de petites molécules inhibitrices du domaine SH3 de RasGAP.