The Src homology 3 domain (SH3) and cysteine rich domain (C1) 3 (Stac3) gene is a novel gene copiously expressed in skeletal muscle. The objective of this research was to determine the role of Stac3 in development, specifically in skeletal muscle. We achieved this objective by evaluating the phenotypic effects of Stac3 gene inactivation on development in mice. At birth homozygous Stac3 null (Stac3-/-) mice died perinatally and remained in fetal position with limp limbs, but possessed otherwise normal organs based on gross and histological evaluations. The primary phenotypes displayed at term in Stac3-/- mice were reduced late gestational body weights, increased prevalence of myotubes with centrally located nuclei and severe deformities throughout all skeletal muscles. At embryonic day 18.5 (E18.5) Stac3-/- mice displayed a 12.7% reduction (P < 0.001) in weight compared to wild type (Stac3+/+) or heterozygous (Stac3+/-) littermates while at E15.5 body weights and morphology were similar. At birth (P0) and at E17.5, Stac3-/- mice had 59% and 24% (P < 0.001) more myotubes with centrally located nuclei, respectively, than Stac3+/- or Stac3+/+ littermates. Stac3-/- mice also displayed increased myotube and myofiber cross sectional area at P0 (P < 0.001) and E17.5 (P < 0.05) with disorganized fiber bundling. Overall, these data show Stac3 is necessary for development of viable offspring and suggest Stac3 plays a critical role in fetal development where its primary phenotype is exhibited in skeletal muscle. / Master of Science

Les spectrines, protéines liant l'actine, sont des constituants majeurs du squelette membranaire, réseau multiprotéique localisé sous la membrane plasmique. Érythroïdes ou non érythroïdes (dans ce cas, on les appelle fodrines), les spectrines ont un rôle structural important dans la membrane comme cela a déjà été démontré, pour la spectrine, dans le globule rouge. Elles sont constituées de deux longues chaînes a et ß associées côte à côte en tétramères (aß)2 qui forment les longs filaments du réseau. Chacune des chaînes est composée par la répétition de segments homologues, 22 pour a et 17 pour ß. Ces segments sont constitués de trois hélices a (hélices A, B et C) repliées sur elles-mêmes. Cette succession de structures trihélicoïdales est parfois interrompue par un domaine particulier comme le domaine SH3 (Src Homology 3) présent au milieu de la chaîne a. Les tétramères (aß)2 de spectrine constituent les filaments du squelette membranaire. L'interaction tête-à-tête de deux dimères aß implique les extrémités NH2 de la chaîne a et COOH de la chaîne ß. D'une part la sévérité du défaut d'auto-association et la localisation des mutations associées à celui-ci et d'autre part, les séquences en acides aminés des extrémités impliquées dans le site d'auto-association ont permis de proposer un modèle : la première hélice C isolée de la chaîne a pourrait s'associer aux deux dernières hélices A et B de la chaîne ß pour reconstituer une unité conformationnelle trihélicoïdale semblable à celles observées le long de la molécule. Un défaut dans la formation du tétramère est le support moléculaire le plus fréquemment observé dans les elliptocytoses héréditaires (HE). À l'aide de peptides recombinants, nous avons défini, sur les deux chaînes, les régions nécessaires et suffisantes possédant les caractéristiques pleinement fonctionnelles du site de tétramérisation. Nous avons ensuite, par mutagenèse dirigée, reproduit le lien entre la présence de mutations HE localisées dans les hélices A ou B et le défaut d'auto-association observé dans les globules rouges de patients HE. La présence d'un domaine SH3 localisé au milieu de la chaîne a confère probablement aux spectrine des fonctions autres que le maintien et la stabilité de la membrane. Les SH3, petits domaines protéiques, participent aux interactions protéine/protéine. Le seul partenaire connu du domaine SH3 de la fodrine était la sous-unité a du canal sodium sensible à l'amiloride (ENaC) mais la fonction de ce complexe n'était pas encore caractérisée. À l'aide de différentes méthodes, nous avons remis en cause l'interaction directe entre ENaC et le SH3 de la fodrine. La fonction de ce domaine SH3 étant liée à la nature de son ligand, nous avons donc recherché les partenaires putatifs du domaine SH3 de la fodrine par la technique du double-hybride. 29 partenaires ont été identifiés, regroupés en 19 familles et 10 clones isolés. La spécificité des interactions a été étudiée à la fois par double-hybride, à l'aide de mutants du domaine SH3 de la fodrine produits par mutagenèse dirigée. Les interactions vis-à-vis d'autres domaines SH3 (spectrine ou une protéine de levure non relatée Scd2) ont été également analysées. Enfin, la spécificité de certains partenaires a été confirmée par des études d'interactions in vitro. Deux des protéines les plus spécifiques du domaine SH3 de la fodrine sont des protéines tyrosine-phosphatase PTP. La première est l'isoforme A de PTP de faible poids moléculaire (LMPTPA) mais l'interaction n'a pas été confirmée in vitro. La deuxième, TD14, est une nouvelle PTP dont la seule fonction connue est d'inhiber la formation des foyers tumoraux des cellules surexprimant l'oncogène Ha-ras. Ces PTP pourraient soit déphosphoryler la fodrine, soit être recrutées sous la membrane pour déphosphoryler une autre cible. Nous avons également identifié trois partenaires (N-WASP, Evl et une protéine de la famille des formines) suggérant que les domaines SH3 des spectrines pourraient être impliqués dans les processus de polymérisation de l'actine liés à la mobilité ou la différenciation cellulaire. Mot clés : spectrine, fodrine, tétramérisation, SH3, double-hybride, polymérisation de l'actine

SH3 domains are excellent models for probing folding and protein interactions. This thesis describes NMR studies of several SH3 domains, including the N-terminal SH3 domain of the Drosophila adaptor protein Drk (drkN SH3 domain), the SH3 domain of the proto-oncogene tyrosine-kinase Fyn, and the SH3 domains of the human adaptor protein CIN85, involved in Cbl-mediated downregulation of epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs). The drkN SH3 domain is an ideal system for studying disordered states. The unique quality of this isolated domain is that it exists in an approximately 50/50 equilibrium between its folded and unfolded states under non-denaturating buffer conditions. Interestingly, the single T22G mutation dramatically stabilizes the domain. Here the NMR structures of the drkN SH3 domain and its T22G mutant are determined and compared in order to illuminate the causes of the marginal stability of the domain. Solvent exposure of the folded and the unfolded drkN SH3 domains are probed and compared with a novel NMR technique using molecular oxygen dissolved in solution as a paramagnetic probe. The changes in partial molar volume along the folding trajectories of the drkN SH3 and Fyn SH3 domains are also studied and analyzed here in terms of changes in protein hydration and packing accompanying folding. Finally, the interactions between the SH3 domains of CIN85 and ubiquitin are discussed. All three are shown to bind ubiquitin. The structure of the SH3-C domain in complex with ubiquitin is presented and the effect of disruption of ubiquitin binding on ubiquitination of CIN85 and EGFR in vivo is discussed. SH3 domains are easily amendable to a wide range of NMR approaches and provide a good system for development and testing of novel methods. Through the use of these approaches significant insights into details of SH3 domain structure, stability, mechanisms of folding and cellular function have been gained.

Grb2, a modular protein comprised of a central SH2 domain flanked between a N-terminal SH3 (nSH3) domain and a C-terminal SH3 (cSH3) domain, is a component of cell signaling networks involved in the transmission of extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. The Grb2-Sos1 interaction is mediated through the combinatorial binding of nSH3 and cSH3 domains of Grb2 to various sites - designated S1, S2, S3, and S4 - containing PXpsiPXR motifs within Sos1. Here, using a diverse array of biophysical techniques, including in particular isothermal titration calorimetry coupled with molecular modeling and semi-empirical analysis, I provide new insights into the Grb2-Sos1 interaction in thermodynamic and structural terms. My data show that Grb2 exists in monomer-dimer equilibrium in solution and that the dissociation of dimer into monomers is entropically-driven. The heat capacity change observed was much smaller than that expected from the rather large molecular surfaces becoming solvent-occluded upon dimerization, implying that monomers undergo conformational rearrangement upon dimerization. 3D structural models suggest strongly that such conformational rearrangement may arise from domain swapping. I further show that the nSH3 domain of Grb2 binds to the S1 site containing the proline-rich consensus motif PXpsiPXR with an affinity that is nearly three-fold greater than that observed for the binding of the cSH3 domain. It is also demonstrated that such differential binding of the nSH3 domain relative to the cSH3 domain is largely due to the requirement of a specific acidic residue, in the RT loop, to engage in the formation of a salt bridge with the arginine residue in the consensus motif PXpsiPXR. The data further reveal that, while binding of both SH3 domains to Sos1 is under enthalpic control, the nSH3 binding suffers from entropic penalty in contrast to entropic gain accompanying the binding of cSH3, implying that the two domains employ differential thermodynamic mechanisms for Sos1 recognition. Additionally, my data reveal that while the nSH3 domain of Grb2 binds with affinities in the physiological range to all four sites S1-S4, the cSH3 domain can only do so at the S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXpsiPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXpsiPXR motifs at S2, S3 and S4 sites, the PXpsiPXR motif at S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of the cSH3 domain. In contrast, these two additional arginine residues augment the binding of the nSH3 domain to the S1 site but their role is not critical for the recognition of S2, S3 and S4 sites. Molecular modeling is employed to rationalize my new findings in structural terms. Taken together, this thesis provides novel insights into the physicochemical basis of a key protein-protein interaction pertinent to cellular signaling and cancer. My studies bear the potential for the development of novel therapies with less toxicity but more effectiveness for the treatment of disease.

Due to their simplicity, viruses require the assistance of host factors for various aspects of their replication cycle. This study investigated the interaction of one of the two non-structural replicase proteins of rubella virus (RUBV), P150, with cell proteins. RUBV forms replication complexes for replicating its RNA in association with membranes of endosomes and lysosomes; the thusly modified endosomes/lysosomes are termed cytopathic vacuoles or CPVs. In the first study, a RUBV expressing a FLAG epitope-tagged P150 was used to co-immunoprecipitate putative interacting cell proteins from an infected cell lysate fraction enriched for CPVs using differential centrifugation. However, the only interacting protein identified was the companion RUBV replicase protein P90. Thus, cell proteins do not bind with either sufficient affinity or in stoichiometric amounts to be detected by this method and may not be a component of the virus holoenzyme. In the second study, a proline-rich region within P150 with three PxxPxR consensus SH3 domain-binding motifs was investigated for its ability to bind cell proteins. Substitution mutations (to alanine) of the two prolines were made in each of these motifs with the finding that mutations in the first two motifs led to lower viral titers and a small plaque phenotype with reversion to the wt sequence within one passage. Mutations in the third motif had a wt phenotype and did not revert. However, these mutations did not affect viral RNA synthesis, suggesting that the importance of these motifs is in a later stage of viral life cycle, e.g. virion assembly and release. To extend these findings, the proline hinge region with either the wt or mutant sequence was expressed as a GST-fusion in human cells. Pulldown experiments revealed specific binding with human p32 protein (gC1qR), which was previously shown to interact with the RUBV capsid protein. Binding of p32 with P150 was confirmed. The function of p32 in the RUBV replication cycle is unclear, but could involve virion assembly and release or induction of apoptosis.

SRMS (Src-Related tyrosine kinases lacking C-terminal Regulatory tyrosine and N terminal Myristoylation Sites) belongs to a family of non-receptor tyrosine kinases, which also includes breast tumor kinase (BRK). SRMS was first identified in 1994 in a screen for the genes that regulate the growth and differentiation of neuroepithelial cells. This 54 kDa protein spanning 488 amino acids, consists of the prototypical Src homology 3 (SH3), Src homology 2 (SH2) and a tyrosine kinase domain. While BRK has been documented for its expression in over 60 % of breast carcinomas, information on SRMS on similar grounds remains absent from the literature. Furthermore, unlike BRK, knowledge of how SRMS regulates its enzymatic activity as well as the identification of its substrates remains unknown. The work in this thesis demonstrates that SRMS is potentially expressed in the majority of breast carcinomas. To understand the biochemical and cellular functions of SRMS, a series of mutants comprising point mutations as well as the deletion of the N-terminal region and the functional, SH3 and SH2 domains, were generated and assessed for enzymatic activity in cells. This study demonstrates for the first time that the wild type protein is apparently constitutively active and that its N-terminal region regulates its enzymatic activity. As well, three critical amino acid residues in the protein namely, lysine 258 (ATP binding site), tyrosine 380 (auto-phosphorylation site) and tryptophan 223 (intramolecular interaction) have been characterized. All three residues have been determined to be essential for the enzymatic activity of SRMS. Finally, the adapter protein Dok1 has been characterized as a novel substrate of SRMS. The results from the present study underscore the potential significance of the catalytically active non-receptor tyrosine kinase, SRMS that should serve as a foundation upon which further research may ensue in the context of breast tumorigenesis.

SH3 domains are excellent models for probing folding and protein interactions. This thesis describes NMR studies of several SH3 domains, including the N-terminal SH3 domain of the Drosophila adaptor protein Drk (drkN SH3 domain), the SH3 domain of the proto-oncogene tyrosine-kinase Fyn, and the SH3 domains of the human adaptor protein CIN85, involved in Cbl-mediated downregulation of epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs). The drkN SH3 domain is an ideal system for studying disordered states. The unique quality of this isolated domain is that it exists in an approximately 50/50 equilibrium between its folded and unfolded states under non-denaturating buffer conditions. Interestingly, the single T22G mutation dramatically stabilizes the domain. Here the NMR structures of the drkN SH3 domain and its T22G mutant are determined and compared in order to illuminate the causes of the marginal stability of the domain. Solvent exposure of the folded and the unfolded drkN SH3 domains are probed and compared with a novel NMR technique using molecular oxygen dissolved in solution as a paramagnetic probe. The changes in partial molar volume along the folding trajectories of the drkN SH3 and Fyn SH3 domains are also studied and analyzed here in terms of changes in protein hydration and packing accompanying folding. Finally, the interactions between the SH3 domains of CIN85 and ubiquitin are discussed. All three are shown to bind ubiquitin. The structure of the SH3-C domain in complex with ubiquitin is presented and the effect of disruption of ubiquitin binding on ubiquitination of CIN85 and EGFR in vivo is discussed. SH3 domains are easily amendable to a wide range of NMR approaches and provide a good system for development and testing of novel methods. Through the use of these approaches significant insights into details of SH3 domain structure, stability, mechanisms of folding and cellular function have been gained.

In the budding yeast Saccharomyces cerevisiae, endocytosis comprises bulk uptake (fluids and membranes) and receptor-mediated internalisation (membrane proteins). Both processes require efficient actin filament assembly. Key factors that nucleate the assembly of actin filaments are the Arp2/3 complex and a number of NPFs (Nucleation Promoting Factors), which are responsible for temporal and spatial regulation of Arp2/3 activity. In yeast, in addition to Las17p, the orthologue of WASP (Wiskott-Aldrich syndrome protein), one type I unconventional myosin (Myo5p) exhibits strong NPF activity through coordination with the WH2 (WASP Homology 2) domain containing protein Vrp1p, the yeast orthologue of WIP (WASP Interacting Protein). Here, we identified another key Vrp1p domain (Hof One Trap/HOT), which binds directly to the SH3 domain of the cytokinesis protein Hof1p, is important for Vrp1p function in vivo. The key function of the Vrp1p HOT domain is to counteract the inhibitory effect of the Hof1p SH3 domain in Myo5p-stimulated actin assembly and endocytosis. We have also revealed a novel actin monomer binding domain (VH2) in Vrp1p, which is functionally redundant with the WH2 domain. Receptormediated endocytosis requires stable interaction of Vrp1p with Las17p. However, we find that bulk uptake of fluid and membrane takes place without Vrp1p-Las17p association and requires only functional WH2 and HOT domains of Vrp1p. Finally, we identified a number of other potential Hof1p SH3 domain interactors using an affinity isolation approach and compared this interaction profile with those of several other yeast SH3 domains. The unique Vrp1p-Hof1p interaction pattern allows us to gain insight into the pathology of Wiskott-Aldrich syndrome.

Ce projet de recherche explore un nouveau mécanisme de régulation de l’activité du domaine HECT de la ligase Itch. Ce domaine est responsable de la polyubiquitylation des protéines impliquant le plus souvent leur dégradation par le protéasome. Itch est une ligase de l’ubiquitine de la famille CWH contenant un domaine HECT catalytique en C-terminal, quatre domaines WW, et un domaine C2 N-terminal qui est important pour sa localisation cellulaire. Les ligases CWH interagissent par leur domaine WW avec leurs ligands. Un mécanisme proposé pour ces ligases est que la première molécule d’ubiquitine liée au substrat active le domaine HECT de manière à former une chaine d’ubiquitine sur le substrat. Itch a une particularité dans la famille CWH, car elle possède un domaine riche en proline qui lui permet d’interagir avec plusieurs protéines à domaine SH3. Dans cette étude, nous avons déterminé l’effet de l’ubiquitylation initiale des protéines SH3 sur l’activité du domaine HECT de la ligase Itch, et sur la régulation de ces substrats. / The subject of this research is to reveal a new mechanism of regulation of the Itch ubiquitin ligase through its catalytic HECT domain. The HECT domain is in charge of polyubiquitylating proteins, which is often responsible for their degradation by the proteasome. Itch belongs to the CWH subfamily of ubiquitin ligases, characterized by the presence of C-terminal catalytic HECT domain, four WW domains for ligand binding and a N-terminal C2 domain important for the subcellular localization of the ligase. Attachment of a first ubiquitin moïties to the substrates is proposed to stimulate the ligase activity, promoting the creation of a polyubiquitin chain. Itch is the only member of its subfamily that has been shown to interact and promote the ubiquitylation of SH3 domain-containing proteins through a conserved proline-rich region located upstream of the four WW domains. In this work, we have determined the impact of the addition of a single ubiquitin to SH3 domain-containing proteins on the processivity of the ubiquitylation reaction catalysed by Itch and on the regulation of this class of substrates.

Kinase Src plays an essential role in signal transduction from activated surface receptors. Src is involved in signal pathways that participate in the control of cell proliferation, differentiation or motility. That is why Src activation undergoes strict and complex regulation. Inactive conformation is maintained by intramolecular inhibitory interactions. SH3 domain associates with a polyprolin helix in CD linker whereas SH2 domain binds phosphorylated C-terminal tyrosine 527. Both regulatory domains maintain contacts with the lobes of a kinase domain thereby stabilizing an inactive conformation of the catalytic domain. Transition to an active state is accompanied by a disruption of these inhibitory interactions. Conformation changes are substantially influenced by the phosphorylation status of key tyrosines 416 and 527. Phosphoproteomic analysis revealed new Src tyrosine residue, which can be phosphorylated in vivo. It has been found, that tyrosine works as an additional regulator of Src activity. This is Tyr 90, which forms one of the hydrophobic pockets in the binding surface of Src SH3 domain. Based on the expression of phosphomimic mutant Src 90E in S. pombe or in SYF lineage, it has been observed, that Tyr 90 phosphorylation elevates Src kinase activity. The reason is that the phosphate...