Melanoma, the most lethal form of skin cancer, arises from altered cells in the melanocyte lineage, but the mechanisms by which these cells progress to melanoma are unknown. To understand the early cellular events that contribute to melanoma formation, we examined melanocytes in melanoma-prone zebrafish strains expressing BRAFV600E, the most common oncogenic form of the BRAF kinase that is mutated in nearly 50% of human melanomas. We found that, unlike wild-type melanocytes, melanocytes in transgenic BRAFV600Eanimals were binucleate and tetraploid. Furthermore, melanocytes in p53-deficient transgenic BRAFV600Eanimals exhibited 8N and greater DNA content, suggesting bypass of a p53-dependent arrest that stops cell cycle progression of tetraploid melanocytes. These data implicate tetraploids generated by increased BRAF pathway activity as contributors to melanoma initiation. Previous studies have used artificial means of generating tetraploids, raising the question of how these cells arise during actual tumor development. To gain insight into the mechanism by which BRAFV600E generates binucleate, tetraploid cells, we established an in vitro model by which such cells are generated following BRAFV600E expression. We demonstrate thatBRAFV600E-generated tetraploids arise via cytokinesis failure during mitosis due to reduced activity of the small GTPase RhoA. We also establish that oncogene-induced centrosome amplification in the G1/S phase of the cell cycle and subsequent increase in the activity of the small GTPase Rac1, partially contribute to this phenotype. These data are of significance as recent studies have shown that aneuploid progeny of tetraploid cells can be intermediates in tumor development, and deep sequencing data suggest that at least one third of melanomas and other solid tumors have undergone a whole genome doubling event during their progression. Taken together, our melanoma-prone zebrafish model and in vitro data suggest a role for BRAFV600E-inducedtetraploidy in the genesis of melanomas. To our knowledge, this is the first in vivo model showing spontaneous rise of tetraploid cells that can give rise to tumors. This novel role of the BRAF oncogene may contribute to tumorigenesis in a broader context.

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Emerging studies revealed that the Aryl hydrocarbon receptor (AhR), a receptor sensing environmental contaminants, is executing an immunomodulatory function. However, it is an open question to which extent this is achieved by its role as a transcription factor or via non-genomic signaling. We utilized a multi-post-translational modification-omics approach to examine non-genomic AhR-signaling after activation with endogenous (FICZ) or exogenous (BaP) ligand in endotoxin-activated (LPS) monocyte-derived macrophages. While AhR activation affected abundances of few proteins, regulation of ubiquitination and phosphorylation were highly pronounced. Although the number and strength of effects depended on the applied AhR-ligand, both ligands increased ubiquitination of Rac1, which participates in PI3K/AKT-pathway-dependent macrophage activation, resulting in a pro-inflammatory phenotype. In contrast, cotreatment with ligand and LPS revealed a decreased AKT activity mediating an antiinflammatory effect. Thus, our data show an immunomodulatory effect of AhR activation through a Rac1ubiquitination-dependent mechanism that attenuated AKT-signaling, resulting in a mitigated inflammatory response.

Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms. We show that human embryonic kidney 293 cells expressing DDR1b migrate more than DDR1a expressing cells on DDR selective substrata as well as on collagen I in vitro. In addition, DDR1b expressing cells show increased lung colonization after tail vein injection in nude mice. DDR1a and DDR1b differ from each other by an extra 37 amino acids in the DDR1b cytoplasmic domain. Interestingly, these 37 amino acids contain an NPxY motif which is a central control module within the cytoplasmic domain of β integrins and acts by binding scaffold proteins, including talin. Using purified recombinant DDR1 cytoplasmic tail proteins, we show that DDR1b directly binds talin with higher affinity than DDR1a. In cells, DDR1b, but not DDR1a, colocalizes with talin and integrin β1 to focal adhesions and enhances integrin β1-mediated cell migration. Moreover, we show that DDR1b promotes cell migration by enhancing Rac1 activation. Mechanistically DDR1b interacts with the GTPase-activating protein (GAP) Breakpoint cluster region protein (BCR) thus reducing its GAP activity and enhancing Rac activation. Our study identifies DDR1b as a major driver of cell migration and talin and BCR as key players in the interplay between integrins and DDR1b in regulating cell migration.

Le facteur de l’ADP-ribosylation 6 (ARF6) et Rac1 sont des petites protéines liant le GTP qui régulent plusieurs voies de signalisation comprenant le trafic de vésicules, la modification des lipides membranaires et la réorganisation du cytosquelette d’actine. Cependant, les mécanismes moléculaires par lesquels ARF6 et Rac1 agissent de concert afin de contrôler ces différents processus cellulaires restent méconnus. Dans cette étude, nous montrons que, dans les cellules HEK293, ARF6 et Rac1 sont retrouvées en complexe suite à la stimulation du récepteur à l’angiotensine. Des expériences réalisées in vitro nous indiquent que ces deux GTPases interagissent ensemble directement, et que ARF6 s’associe préférentiellement avec la forme inactive de Rac1. L’inhibition de l’expression de ARF6 par interférence à l’ARN entraîne une activation marquée en cellule de Rac1 via le facteur PIX, indépendamment de la stimulation d’un récepteur, ce qui provoque la migration non contrôlée des cellules. Les arrestines, protéines de régulation de la désensibilisation des récepteurs couplés aux protéines G, servent de protéines d’échafaudage pour Rac1 et ARF6, en interagissant directement avec les GTPases et en augmentant leur association stimulée par l’angiotensine. De plus, les arrestines permettent l’activation, en s’en dissociant, de la MAP Kinase p38 qui régule l’activité de ARF6 et son interaction précoce avec les arrestines. Mis ensemble, ces résultats montrent que les arrestines contrôlent l’activité de ARF6, en influençant p38. ARF6 joue un rôle inhibiteur sur l’activation basale de Rac1 pour permettre ensuite son recrutement et son activation dépendante de l’angiotensine. Cette étude nous a permis de préciser le mode de régulation mis en jeu dans l’initiation de la migration cellulaire, suite à l’activation d’un récepteur couplé aux protéines G. Par le fait même, nous avons identifié certains des acteurs impliqués dans ce processus, offrant ainsi de nouvelles cibles pour le traitement des déséquilibres pathophysiologiques de la migration cellulaire. / The ADP-ribosylation factor 6 (ARF6) and Rac1 are small GTP-binding proteins that regulate several signaling events ranging from vesicle trafficking, to modification of membrane lipids and reorganization of the actin cytoskeleton. However, the molecular mechanisms by which ARF6 and Rac1 act in concert to control these different cellular processes remain unclear. Here, we show that in HEK 293 cells, ARF6 and Rac1 can be found in complex upon stimulation of the angiotensin receptor (ATR). In vitro experiments indicate that these two small G proteins can directly interact together, and that ARF6 preferentially interacts with the GDP-bound form of Rac1. Depletion of ARF6 by RNA interference leads to a marked PIX-dependent Rac1 activation in cells, independently of receptor stimulation, leading to uncontrolled cell migration. Arrestins, which are known for their role in G protein-coupled receptor desensitization, act as scaffold proteins toward Rac1 and ARF6, by directly interacting with the GTPases and by increasing their agonist-promoted association. Besides, arrestins allow p38 MAP Kinase activation, by releasing it, which regulates ARF6 activity and early association occurring between arrestins and ARF6. Taken together, this study shows that arrestins control ARF6 activity, by managing p38. ARF6 is an inhibitor of basal Rac1 activation to further allow the protein to be recruited and activated following angiotensin treatment. This study allowed us to precise how cell migration induction is regulated following G protein-coupled receptor activation. As a result, we identified some of the key players implicated in this process, providing new targets in the treatment of patho-physiological inbalance in cell migration.

Thesis (M.Sc.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

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Rho-GTPases are essential signalling proteins which regulate a multitude of central cellular processes that are vital for organisms to thrive and adapt to changing environments. Many regulatory networks involving Rho proteins have first been elucidated in the model yeast Saccharomyces cerevisiae, in which Rho5 emerges as a central hub connecting different signalling pathways, such as the responses to cell wall stress, high medium osmolarity, and oxidative stress. In this work, the rapid translocation of Rho5 to mitochondria as reaction to oxidants and glucose starvation was thoroughly investigated. The studies on structure-function relationships was focussed on the C-terminal region of the Rho5 which in other Rho-type GTPases determines their spatio-temporal distribution and contributes to their physiological function. The C-terminal end of these GTPases is considered to be a hypervariable region (HPR) that consists of a polybasic region (PBR) and its preceding amino acid residues, followed by the CAAX motif which becomes prenylated at its cysteine residue. These motifs are conserved in the yeast Rho5 where the PBR contains a serine residue as a putative phosphorylation target. Moreover, Rho5 of S. cerevisiae is characterized by an extension preceding the PBR that comprises 98 amino acid residues. While substitutions of the serine residue within the PBR for either phosphomimetic or non-phosphorylatable residues indicate that it is of minor physiological importance, deletion analyses of the yeast-specific extension showed that it is required for proper localization of Rho5 to the plasma membrane. As expected, substitution of the cysteine residue within the CAAX motif also prevented proper plasma membrane localization, accompanied by a loss of function both with respect to oxidative stress response and glucose starvation. Results from studies employing a trapping-device of GFP-Rho5 to the mitochondrial surface indicate that the GTPase needs to be activated at the plasma membrane by its dimeric GDP/GTP exchange factor (GEF) which is composed of Dck1 and Lmo1, in response to stress conditions. The trimeric DLR complex is then capable of rapidly translocate to mitochondria and fulfil its functions at the organelle. This view was supported by the finding that a constitutively active Rho5 variant restored function when trapped to mitochondria. Interestingly, Rho5 requires the dimeric GEF for the translocation process under oxidative stress while Dck1 and Lmo1 can reach the mitochondria independent from each other. Finally, the human Rho5 homolog Rac1 cannot complement the defects of a rho5 deletion and does not show a proper intracellular distribution, unless its C-terminal end is equipped with the yeast-specific extension. Taken together, the results of this thesis contributed to a better understanding of the structure-function relationships of Rho5 and its human homolog Rac1.