Les cellules NK sont des lymphocytes ayant un rôle majeur dans les réponses immunes innées, particulièrement contre les cellules tumorales ou infectées par un agent pathogène. L'activation des cellules NK déclenche leur cytotoxicité cellulaire et/ou sécrétion de cytokines, telle que l'IFN-gamma. L'activité des cellules NK est régulée par une variété de facteurs intrinsèques et extrinsèques qui assurent leur tolérance et efficacité. L'analyse de la signalisation NK permettrait de démontrer leur potentiel clinique. Parmi les protéines impliquées dans la signalisation NK, les protéines adaptatrices ont été très peu analysées. Dans ce projet, je décris les adaptateurs Dok-1/Dok-2 (Downstream of kinase), lesquels sont fortement exprimés dans des cellules hématopoïétiques, notamment dans le lignage myéloïde et les cellules NK. La structure de Dok-1 et Dok-2 est pourvue de différents domaines d'interaction protéine-protéine leur permettant de recruter différentes molécules de signalisation intracellulaire. Au cours de mon travail de thèse, je démontre que Dok-1 et Dok-2 sont phosphorylées sur tyrosine suite à l'activation des NK. La surexpression de Dok-1/Dok-2 dans les cellules NK induit un frein à leur activation déclenchée par des récepteurs activateurs. En revanche, l'invalidation d'expression de Dok1 et Dok2 chez la souris entraine un défaut de développement/maturation NK, mais également une augmentation de leur sécrétion d'IFN-gamma induite par l'engagement de récepteurs activateurs. Ces résultats dévoilent ainsi que Dok-l et Dok-2 sont impliquées dans une boucle de rétrocontrôle négatif en aval des récepteurs activateurs des cellules NK chez l'homme et chez la souris. / NK cells are an important component of innate immunity by virtue of their ability to recognize microbe-infected, transformed (tumors) and allogenic cells, while sparing most autologous healthy cell. NK cell activation can elicit two different effector functions: cytotoxicity of target cell and/or secretion of a large array of cytokines and chemokines. NK cell activation is regulated by intrinsic and extrinsic mechanisms that ensure NK tolerance and efficacy. Determining signaling events downstream of the NK receptors is in this line of establishing the clinical potential of NK cells. Among the proteins involved in NK signaling, the adaptor molecules are poorly understood. Here, I described two Downstream of tyrosine kinase (Dok) adaptors: Dok-1 and Dok-2; these two proteins are mainly expressed in hematopoietic cells, especially in myeloid lineage and NK cells. Dok-1 and Dok-2 adaptors contain a variety of protein-protein interaction motifs enabling them to recruit various intracellular signaling molecules. During my PhD work, I demonstrate that the cytoplasmic signaling molecules Dok1 and Dok2 are tyrosine phosphorylated upon NK cell activation. Overexpression of Dok proteins in human NK cells reduces cell activation induced by NK cell activating receptors. Dok1 and Dok2 gene ablation in mice induces an NK cell developmental/maturation defect and leads to increased IFN-gamma production induced by activating receptors. Taken together, these results reveal that Dok-1 and Dok-2 adaptors are involved in an intrinsic negative feedback loop downstream of NK cell activating receptors in mouse and human.

The Rip2 kinase contains a caspase recruitment domain (CARD) and has been implicated in the activation of the transcriptional factor NF-кB downstream of Nod-like receptors. However, how Rip2 mediates innate immune responses is still largely unclear. We show that Rip2 and IKK-γ become stably polyubiquitinated upon treatment of cells with the Nod2 ligand, muramyl dipeptide. We demonstrate a requirement for the E2 conjugating enzyme Ubc13, the E3 ubiquitin ligase Traf6 and the ubiquitin activated kinase Tak1 in Nod2-mediated NF-кB activation. We also show that M. tuberculosisinfection stimulates Rip2 polyubiquitination. Collectively, this study revealed that the Nod2 pathway is ubiquitin regulated and that Rip2 employs a ubiquitin-dependent mechanism to achieve NF-кB activation. We also demonstrate that intraphagosomal M. tuberculosis stimulates the cytosolic Nod2 pathway. We show that upon Mtb infection, Nod2 recognition triggers the expression of type I interferons in a Tbk1- and Irf5-dependent manner. This response is only partially impaired by the loss of Irf3 and therefore, differs fundamentally from those stimulated by bacterial DNA, which depends entirely on this transcription factor. This difference appears to result from the unusual peptidoglycan produced by mycobacteria, which we show is a uniquely potent agonist of the Nod2/Rip2/Irf5 pathway. Thus, the Nod2 system is specialized to recognize bacteria that actively perturb host membranes and is remarkably sensitive to Mycobacteria, perhaps reflecting the strong evolutionary pressure exerted by these pathogens on the mammalian immune system.

Crucial to a cell’s ability to migrate is the organization of its plasma membrane and associated proteins in a polarized manner to interact with and respond to its surrounding environment. Cells interact with the extracellular matrix (ECM) through specialized contact sites, including podosomes and invadopodia. Tumor cells use F-actin-rich invadopodia to degrade ECM and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction and degradation. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II,reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV increases the number of F-actin punctae, which are highly dynamic and co-localize with markers of podosomes and invadopodia. Endogenous SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases the average amount of matrix degradation; RNAi-mediated downregulation of SV decreases degradation. Cortactin, an essential component of both podosomes and invadopodia, binds SV sequences in vitro and contributes to the formation of EGFP-SV induced punctae. Additionally, SV affects cortactin localization,which could provide a mechanism for SV action at invadopodia. The formation of cholesterol-rich membrane rafts is one method of plasma membrane organization. A property of membrane rafts is resistance to extraction with cold Triton X-100 and subsequent flotation to low buoyant densities. The actin cytoskeleton has been implicated in many signaling events localized to membrane rafts, but interactions between actin and raft components are not well characterized. Our laboratory isolated a heavy detergent resistant membrane fraction from neutrophils, called DRM-H, that contains at least 23 plasma membrane proteins. DRM-H is rich in cytoskeletal proteins, including fodrin, actin, myosin II, as well as supervillin. DRM-H also contains proteins implicated in both raft organization and membrane-mediated signaling. DRM-H complexes exhibit a higher buoyant density than do most DRMs (referred to as DRM-L), which are deficient in cytoskeletal proteins. By using similar purification methods, I find that COS-7 cells also contain cytoskeleton-associated DRMs. In addition, when transfected into COS-7 cells, estrogen receptor (ER)α associates with DRM-H, while ERβ is seen in both DRM-L and DRM-H populations, suggesting a role for DRM-H in nongenomic estrogen signaling. Thus, the cytoskeleton-associated DRM-H not limited to hematopoietic cells and could constitute a scaffold for membrane raftcytoskeleton signaling events in many cells. Taken together, our results show that SV is a component of cytoskeleton-associated membrane rafts as well as podosomes and invadopodia, and that SV plays a role in invadopodial function. SV, with its connections to both membrane rafts and the cytoskeleton, is well situated to mediate cortactin localization, activation state, and/or dynamics of matrix metalloproteases at the ventral cell surface for proper matrix degradation through invadopodia. The molecular dissection of invadopodia formation and function may contribute to a greater understanding of in vivo invasion, and thus, tumor cell metastasis.

Innate immunity is the first line of defense against invading pathogens. It functions to eliminate pathogens and also to control infections. The innate immune response is also important for the development of pathogen-specific adaptive immune responses. As a result, the study of innate immune signaling pathways is crucial for understanding the interactions between host and pathogen. Unlike mammals, insects lack a classical adaptive immune response and rely mostly on innate immune responses. Innate immune mechanisms have been widely studied in the fruit fly, Drosophila melanogaster. The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophila show strong homology to those of vertebrates making them ideal for studying these pathways. Drosophila immunity relies on cellular and humoral innate immune responses to fight pathogens. The hallmark of the Drosophilahumoral immune response is the rapid induction of antimicrobial peptide genes in the fat body. The production of these antimicrobial peptides is regulated by two immune signaling pathways-Toll and Immune Deficency (IMD) pathways. The Toll pathway responds to many Gram-positive bacterial and fungal infections , while the IMD pathway is potently activated by DAP-type peptidoglycan (PGN) from Gram-negative bacteria and certain Gram-positive bacteria. Two receptors, PGRP-LC and PGRP-LE, are able to recognize DAP-type PGN at the cell surface or in the cytosol, respectively, and trigger the IMD pathway. Upon binding DAP-type PGN, both PGRP-LC and PGRP-LE dimerize/ multimerize and signal to the downstream components of IMD pathway. It is unclear how the receptor activates its downstream components. My work has focused on understanding the molecular events that take place at the receptors following there activation. In these studies I have identified a common motif in the N-terminal domains of both the receptors, known as the RHIM-like domain. The RHIM-like domain is critical for signaling by either receptor, but the mechanism(s) involved remain unclear. IMD, a downstream component of the pathway, associates with both PGRP-LC and -LE but the interaction of PGRP-LC with IMD is not mediated through its RHIM-like domain. Also, mutations affecting the PGRP-LC RHIM-like motif are defective in all known downstream signaling events. However, the RHIM-like mutant receptors are capable of serving as a platform for the assembly of all known components of a receptor proximal signaling complex. These results suggest that another, unidentified component of the IMD signaling pathway may function to mediate interaction with the RHIM-like motif. I performed a yeast two-hybrid screen to identify proteins that might interact with the receptor PGRP-LC through its RHIM- like domain. With this approach, two new components of the IMD pathway were identified. The first component I characterized is called Rudra and it is a critical feedback inhibitor of peptidoglycan receptor signaling. The other factor is known as RYBP, it includes a highly conserved ubiquitin binding motif (NZF), and RNAi studies suggest it is a critical component of the IMD pathway. The identification and characterization of these two new components of the IMD pathway has provided a new insight into the molecular events that take place proximal to the receptor.

ErbB2/HER2 est un récepteur tyrosine kinase de la famille d'EGFR (ErbB1) surexprimé dans plus de 20% des cancers du sein et associé à une forme particulièrement agressive de la maladie. Les récepteurs ErbBs sont actifs seulement sous forme de dimères, permettant la phosphorylation de leur queue C-terminale par leur domaine tyrosine kinase. La phosphorylation entraine l'interaction avec des protéines adaptatrices et l'activation de voies de signalisation, Ras/MAPK et PI3K/Akt principalement. Ces voies contrôlent la prolifération, la motilité cellulaire et la résistance à l'apoptose. Contrairement à ErbB1/3/4, ErbB2 dimérise en l'absence de ligand. Comprendre les autres mécanismes de régulation de la phosphorylation de ses tyrosines et de ses interactions est donc particulièrement intéressant.ErbB2 a fait l'objet de nombreuses études structurales et fonctionnelles. Elles ont permis la mise au point de traitements ciblés efficaces mais sujets à l'apparition de résistance, dont l'anticorps Trastuzumab, ciblant sa partie extracellulaire. La queue C-terminale d'ErbB2 (CtErbB2) a été très souvent ignorée dans ces études. Cette partie étant intrinsèquement désordonnée, il a fallu attendre ces dernières années pour que les concepts et les outils permettant de l'étudier émergent.Dans cette thèse, j'ai d'abord effectué la caractérisation structurale et dynamique de CtErbB2. J'ai montré que bien qu'étant dépourvue de toute structure stable, cette région riche en prolines possède plusieurs structures secondaires transitoires et un contact longue-distance participant très probablement à la régulation de ses interactions intra- et inter-moléculaires. Dans une deuxième partie je me suis intéressée à la caractérisation de la protéine adaptatrice Grb2, partenaire essentiel de ErbB2 pour l'activation de la voie des MAP kinases. L'organisation en solution des domaines de cette protéine modulaire dans sa forme libre était jusque là inconnue. J'ai ensuite étudié l'interaction entre Grb2 et CtErbB2, et montré que CtErbB2 interagit non seulement avec le domaine SH2 de Grb2 (par l'intermédiaire d'une phosphotyrosine), mais aussi avec son domaine SH3 N-terminal (grâce à un motif polyproline). Enfin, j'ai mis en place plusieurs stratégies de phosphorylation des tyrosines de CtErbB2, dans le but d'étudier plus largement l'effet des phosphorylations sur l'ensemble de cette région. / ErbB2/HER2 is a receptor tyrosine kinase of the EGFR (ErbB1) family overexpressed in 20% of breast cancers and associated to a particularly aggressive form of the disease. ErbB receptors are only active upon dimerization that enables phosphorylation of their C-terminal tail by their tyrosine kinase domain. Phosphorylation then triggers interaction with adaptor proteins and activation of signaling pathways, mainly Ras/MAPK and Akt/PI3K. Those pathways control cell proliferation, motility and resistance to apoptosis. Contrary to ErbB1/3/4, ErbB2 can dimerize without any ligand. Understanding other mechanisms of regulation of its tyrosine phosphorylation and of its interactions is thus particularly interesting.ErbB2 structure and function have been extensively studied. This has led to the development of several FDA-approved targeted drugs, that are effective but to which resistance occurs, amongst which the Trastuzumab antibody that targets ErbB2 extracellular domain. The C-terminal tail of ErbB2 (CtErbB2) has been widely ignored in these studies. Since it is intrinsically disordered, the concepts and tools to study it have only emerged in the last few years.In the present work, I have performed the structural and dynamic study of CtErbB2. I showed that despite its lack of any stable structure, this proline-rich region exhibits several transient secondary structures and a long-range contact that might participate in the regulation of its intra- and inter-molecular interactions. Then, I characterized the adaptor protein Grb2, which is a partner of ErbB2 that is essential for the activation of the MAPK pathway. The solution organization of the domains of this modular protein in its apo-form was unknown so far. I also studied the interaction between Grb2 and CtErbB2, showing that in addition to the known SH2-phosphotyrosine interaction, a polyproline motif of CtErbB2 binds to the N-terminal SH3 domain of Grb2. Finally, I implemented several strategies to phosphorylate CtErbB2 tyrosines, to study more extensively the effect of phosphorylation on the whole tail.

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G protein-coupled receptors (GPCRs) form the biggest family of membrane receptors and are involved in numerous physiological processes. Collectively, these receptors are also prominently targeted by the pharmaceutical industry due to their implications in multiple diseases and disorders. GPCR signaling is tightly regulated. Several kinases, activated downstream of the receptor, initiate negative feedback loops; and arrestins play a crucial role in these regulatory processes by desensitizing the ligand–activated receptor and promoting its endocytosis. By doing so, arrestins control the duration and the amplitude of signal transduction at the cell surface. In the last few years, several non-canonical roles have also been attributed to arrestins, such as the post-endocytic activation of several signalling pathways, or the regulation of crosstalks between GPCRs and various other signalling events. My thesis project was aimed at providing a better understanding of the non-canonical functions of arrestins. The first objective of my research work was to investigate a possible reciprocal effect of the activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) on GPCR signaling. We demonstrated that stimulation of ERK1/2, either by a cell surface receptor or a constitutively active mutant, leads to a reduction in steady-state expression levels of many GPCRs at the cell surface. This receptor redistribution mechanism is dependent on beta-arrestins phosphorylation. In vitro kinase assays combined with complementation experiments in mouse embryonic fibroblasts (MEFs) lacking beta-arrestins, revealed that beta-arrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This ERK1/2- and arrestins mediated regulatory process was found to result in a global dampening of cell responsiveness. The second objective of my research work was to identify and develop a small organic compound that inhibits the interaction between arrestins and the adaptor protein AP-2, without interfering with the recruitment of arrestin to the receptor. This inhibitor, named Barbadin, was found to specifically block endocytic processes that are dependent on the interaction between arrestins and the appendage domain of the b-subunit of AP-2. We demonstrated its value as an analytical tool in studying the role of the arrestins in GPCR signaling, such as cAMP production and ERK1/2 activation. These results support the concept that beta-arrestin/AP-2-dependent signaling is important to both G protein-dependent and -independent pathways. The third objective of my research work was to develop a BRET-based biosensor able to detect signal-dependent PTEN conformational changes. This biosensor was validated by monitoring PTEN activation induced by targeted mutations affecting key intramolecular interactions or by modulating signalling pathways that impact PTEN function. We also demonstrated the value of this biosensor in studying PTEN/protein interactions using two known interactors that activate PTEN, beta-arrestin-2 and RhoA. Finally, we uncovered PTEN activation by several GPCRs, previously unknown as PTEN regulators. Given the central role of the tumor suppressor PTEN in oncogenesis, this biosensor could also provide a precious tool for anti-cancer drug research. To conclude, my research work highlighted non-canonical mechanisms for arrestins to activate GPCR-dependent signaling pathways, such as cAMP, ERK1/2 and PTEN, as well as negatively regulate GPCR signaling upon phosphorylation by ERK1/2. This work was made possible by the development of new tools: a beta-arrestin inhibitor named Barbadin and a PTEN BRET-based biosensor that have both shown their usefulness in studying beta-arrestin noncanonical signaling. / Les récepteurs couplés aux protéines G (RCPG) représentent la plus grande famille de récepteurs membranaires et sont impliqués dans un grand nombre de processus physiologiques. Cette famille de récepteurs constitue aussi une cible majeure dans la recherche pharmaceutique au vu de son importance dans de nombreuses pathologies. La signalisation des RCPG est étroitement régulée. Plusieurs kinases activées en aval du récepteur initient des boucles de régulation négative. Les arrestines jouent un rôle clé dans ces processus de régulation en favorisant la désensibilisation du récepteur activé par le ligand, suivie de son endocytose. Ainsi, les arrestines contrôlent la durée et l’amplitude de la transmission du signal à la surface de la cellule. Ces dernières années, plusieurs rôles non-canoniques ont été attribués aux arrestines comme l’activation de voies de signalisation post-endocytiques, ou la modulation de la régulation croisée entre les RCPG et d’autres acteurs de la signalisation cellulaire. Le premier objectif de mon travail de recherche est d’examiner l’effet réciproque de l’activation des kinases ERK1/2 (extracellular signal-regulated kinases 1/2) sur la signalisation des RCPG. Nous avons démontré que la stimulation de ERK1/2, soit par un récepteur de surface soit par l’utilisation d’un mutant constitutivement actif, conduit à la baisse de l’expression de surface basale de nombreux RCPG. Des essais kinases in vitro, combinés à des expériences de complémentation dans des fibroblastes embryonnaires de souris (MEF), où les gènes beta-arrestine-1/2 ont été supprimés, démontrent l’importance de la phosphorylation par ERK1/2 des résidus Ser14 et Thr276 dans ce mécanisme de séquestration des RCPG. Cette régulation, contrôlée par ERK1/2 et arrestine, conduit à une baisse globale de la capacité de réponse de la cellule aux stimuli extracellulaires. Le deuxième objectif de mon travail de recherche est d’identifier et de développer une petite molécule organique qui inhibe l’interaction entre l’arrestine et la protéine adaptatrice du complexe d’endocytose AP-2, sans toutefois empêcher la formation du complexe arrestine/récepteur. Cet inhibiteur, nommé Barbadin, bloque sélectivement les processus d’internalisation dépendants de l’interaction entre arrestine- et la sous-unité beta2 de la protéine adaptatrice AP-2. Barbadin représente le premier inhibiteur des fonctions d’arrestine, et nous avons démontré son utilité comme outil analytique pour déterminer la contribution des arrestines dans l’activation de plusieurs voies de signalisation en aval des RCPG, telles que la production d’AMP cyclique (AMPc) ou l’activation des kinases ERK1/2. Nos résultats démontrent l’importance du complexe arrestine/AP-2 dans la signalisation dépendante et indépendante des protéines G. Le troisième objectif de mon travail de recherche est de développer un biosenseur BRET capable de mesurer les changements de conformation du suppresseur de tumeur PTEN. Nous avons validé ce biosenseur en mesurant l’activation de PTEN suite à des mutations ciblées déstabilisant les interactions intramoléculaires au sein de cette protéine ou en modulant différentes voies de signalisation qui affectent sa fonction. Nous avons démontré l’intérêt de ce nouvel outil dans l’étude des interactions entre PTEN et des partenaires protéiques, en utilisant deux interacteurs connus pour activer PTEN : b-arrestine-2 et RhoA. Finalement, en utilisant ce biosenseur, nous avons démontré pour la première fois la capacité de plusieurs RCPG à induire l’activation de PTEN. Étant donné le rôle central de PTEN dans le développement tumoral, ce biosenseur constitue aussi un outil précieux pour la recherche de nouveaux médicaments anticancer. Ainsi, au travers de ces trois lignes directrices, nous avons pu mettre en lumière de nouveaux rôles non-canoniques des arrestines, soit dans l’activation de voies de signalisation, (comme la production d’AMPc, l’activation de ERK1/2 ou de PTEN), soit comme régulateur négatif de la signalisation des RCPG après phosphorylation par ERK1/2. Ce travail a été rendu possible par le développement de nouveaux outils pour l’étude des RCPG : un inhibiteur de beta-arrestine, Barbadin, et un biosenseur BRET de PTEN ; tous deux ayant démontré leur utilité dans l’étude des voies de signalisation non-canoniques des arrestines.