Exploring Rac GTPase regulation : the molecular mechanisms governing the DOCK180 and ELMO interaction and the role of this complex in Rac-mediated cell migration

Les protéines DOCK180 et ELMO coopèrent ensemble biochimiquement et génétiquement afin d’activer la GTPase Rac1 lors de plusieurs évènements biologiques. Toutefois, le rôle que jouent ces protéines dans la signalisation par Rac est encore mal compris. Nous émettons l’hypothèse que Dock180 agit comme activateur de Rac, alors que ELMO est requis pour l’intégration de la signalisation de Rac plutôt que son activation per se. Nous postulons que ELMO agit comme signal de localisation intracellulaire afin de restreindre de façon spatio-temporelle la signalisation de Rac en aval de Dock180, et/ou que ELMO agit comme protéine d’échafaudage entre Rac et ses effecteurs pour amplifier la migration cellulaire. Dans l’objectif nº 1, nous démontrons que le domaine PH atypique de ELMO1 est le site d’interaction principal entre cette protéine et DOCK180. De plus, nous démontrons que la liaison entre ELMO et DOCK180 n’est pas nécessaire pour l’activation de Rac, mais est plutôt essentielle pour faciliter la réorganisation du cytosquelette induite par l’activation de Rac en aval de Dock180. Ces résultats impliquent que ELMO pourrait jouer des rôles additionnels dans la signalisation par Rac. Dans l’objectif nº 2, nous avons découvert l’existence d’une homologie structurelle entre ELMO et un module d’autorégulation de la formine Dia1, et avons identifié trois nouveaux domaines dans la protéine ELMO : les domaines RBD, EID et EAD. De façon analogue à Dia1, nous avons découvert que ELMO à l’état basal est autoinhibé grâce à des intéractions intramoléculaires. Nous proposons que l’état d’activation des protéines ELMO est régulé de façon similaire aux formines de la famille Dia, c’est-à-dire grâce à des interactions avec d’autres protéines. Dans l’objectif nº 3, nous identifions un domaine RBD polyvalent chez ELMO. Ce domaine possède une double spécificité pour les GTPases de la famille Rho et Arf. Nous avons découvert que Arl4A agit comme signal de recrutement membranaire pour le module ELMO/DOCK180/Rac. Nos résultats nous permettent de supposer que d’autres GTPases pourraient être impliquées dans l’activation et la localisation de cette voie de signalisation. Nous concluons qu’à l’état basal, ELMO et DOCK180 forment un complexe dans lequel ELMO est dans sa conformation autoinhibée. Bien que le mécanisme d’activation de ELMO ne soit pas encore bien compris, nous avons découvert que, lorsqu’il y a stimulation cellulaire, certaines GTPases liées au GTP peuvent intéragir avec le domaine RBD de ELMO pour relâcher les contacts intramoléculaires et/ou localiser le complexe à la membrane. Ainsi, les GTPases peuvent servir d’ancrage au complexe ELMO/DOCK180 pour assurer une regulation spatiotemporelle adequate de l’activation et de la signalisation de Rac.DOCK180 and ELMO cooperate biochemically and genetically to activate Rac in several biological events. However, the role of these proteins in Rac signaling is still poorly understood. We hypothesize that DOCK180 functions as a RacGEF, with ELMO binding to DOCK180 being required for integration of proper Rac signaling rather than Rac activation per se. We postulate that ELMO acts as a subcellular targeting signal for spatio-temporal restriction of DOCK180-mediated Rac signaling and/or as a scaffold for Rac effectors to enforce cell migration. In Aim #1, we elucidate that the atypical ELMO1 PH is the major DOCK180 binding site. We demonstrate that the binding of ELMO1 to DOCK180 is not necessary for Rac GTP-loading, but is instead required to facilitate Rac-GTP induced cytoskeletal changes following DOCK180 activation. These results imply additional roles for ELMO in mediating Rac signaling. In Aim #2, we reveal structural homology between ELMO and an autoregulatory module in the formin, Dia1, and identify three novel domains in ELMOs: the RBD, EID and EAD. Analogous to Dia1, we uncovered that ELMO is autoinhibited via intramolecular interactions at basal state. We propose that the activation state of ELMO proteins is regulated, much like in Dia-family formins, via interaction with other proteins. Aim #3 identifies a polyvalent RBD in ELMO with dual specificity for Rho and Arf family GTPases. We found Arl4A as a novel membrane recruitment signal for the ELMO/DOCK180/Rac module. Our results may have broad implications in the activation and localization of this pathway by additional GTPases. We conclude that, at basal levels, ELMO/DOCK180 is complexed, with ELMO in an autoinhibited state in the cytosol. Through cell stimulation, certain GTPases will be activated that now bind the ELMO RBD and alleviate the intramolecular contacts. In this way, the GTPase anchors the activated ELMO/DOCK180 module in place for proper spatio-temporal regulation of Rac activation and signaling

The effect of a VAChT-saporin immunotoxin on retinal cholinergic amacrine cells during post-natal development in rats

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Identification of synthetic lethality of PRKDC in MYC-dependent human cancers by pooled shRNA screening

BACKGROUND: MYC family members are among the most frequently deregulated oncogenes in human cancers, yet direct therapeutic targeting of MYC in cancer has been challenging thus far. Synthetic lethality provides an opportunity for therapeutic intervention of MYC-driven cancers. METHODS: A pooled kinase shRNA library screen was performed and next-generation deep sequencing efforts identified that PRKDC was synthetically lethal in cells overexpressing MYC. Genes and proteins of interest were knocked down or inhibited using RNAi technology and small molecule inhibitors, respectively. Quantitative RT-PCR using TaqMan probes examined mRNA expression levels and cell viability was assessed using CellTiter-Glo (Promega). Western blotting was performed to monitor different protein levels in the presence or absence of RNAi or compound treatment. Statistical significance of differences among data sets were determined using unpaired t test (Mann-Whitney test) or ANOVA. RESULTS: Inhibition of PRKDC using RNAi (RNA interference) or small molecular inhibitors preferentially killed MYC-overexpressing human lung fibroblasts. Moreover, inducible PRKDC knockdown decreased cell viability selectively in high MYC-expressing human small cell lung cancer cell lines. At the molecular level, we found that inhibition of PRKDC downregulated MYC mRNA and protein expression in multiple cancer cell lines. In addition, we confirmed that overexpression of MYC family proteins induced DNA double-strand breaks; our results also revealed that PRKDC inhibition in these cells led to an increase in DNA damage levels. CONCLUSIONS: Our data suggest that the synthetic lethality between PRKDC and MYC may in part be due to PRKDC dependent modulation of MYC expression, as well as MYC-induced DNA damage where PRKDC plays a key role in DNA damage repair

The Arf Family Gtpase Arl4a Complexes with Elmo Proteins to Promote Actin Cytoskeleton Remodeling and Reveals a Versatile Ras-Binding Domain in the Elmo Proteins Family

The prototypical DOCK protein, DOCK180, is an evolutionarily conserved Rac regulator and is indispensable during processes such as cell migration and myoblast fusion. The biological activity of DOCK180 is tightly linked to its binding partner ELMO. We previously reported that autoinhibited ELMO proteins regulate signaling from this pathway. One mechanism to activate the ELMO-DOCK180 complex appears to be the recruitment of this complex to the membrane via the Ras-binding domain (RBD) of ELMO. In the present study, we aimed to identify novel ELMO-interacting proteins to further define the molecular events capable of controlling ELMO recruitment to the membrane. To do so, we performed two independent interaction screens: one specifically interrogated an active GTPase library while the other probed a brain cDNA library. Both methods converged on Arl4A, an Arf-related GTPase, as a specific ELMO interactor. Biochemically, Arl4A is constitutively GTP- loaded, and our binding assays confirm that both wild-type and constitutively active forms of the GTPase associate with ELMO. Mechanistically, we report that Arl4A binds the ELMO RBD and acts as a membrane localization signal for ELMO. In addition, we report that membrane targeting of ELMO via Arl4 A promotes cytoskeletal reorganization including membrane ruffling and stress fiber disassembly via an ELMO-DOCK1800- Rac signaling pathway. We conclude that ELMO is capable of interacting with GTPases from Rho and Arf families, leading to the conclusion that ELMO contains a versatile RBD. Furthermore, via binding of an Arf family GTPase, the ELMO- DOCK180 is uniquely positioned at the membrane to activate Rac signaling and remodel the actin cytoskeleton

Opening up on ELMO regulation: New insights into the control of Rac signaling by the DOCK180/ELMO complex

GTPases are central hubs for directing cytoskeletal reorganization and cell migration. The DOCK family enforces positive regulation of certain GTPases, leading to their activation in discrete areas of the cell. ELMO, a well-known DOCK180 binding partner, has been cast with the role of potentiating DOCK180-mediated Rac activation. Exactly how ELMO contributes to Rac signaling is only beginning to be understood. Here, we discuss our most recent research investigating ELMO regulation of the DOCK180/Rac pathway. Interestingly, we found that ELMO is autoinhibited via intramolecular contacts at basal levels and we explore the novel domains that we identified at the heart of the auto-regulatory switch. We propose that the closed ELMO molecule masks protein-protein interfaces or domains with novel uncharacterized function; cell stimulation and GTPase binding to ELMO is proposed to activate (open) the protein and/or target the ELMO/DOCK180 complex to the cell membrane. In this manner, promiscuous signaling/activity downstream of ELMO/DOCK180 can be controlled for both spatially and temporally. Additionally, we report new data highlighting that DOCK proteins can form heterodimers, and we discuss possible mechanisms that could be implicated in controlling the ELMO activation state

The invadopodia scaffold protein Tks5 is required for the growth of human breast cancer cells in vitro and in vivo.

The ability of cancer cells to invade underlies metastatic progression. One mechanism by which cancer cells can become invasive is through the formation of structures called invadopodia, which are dynamic, actin-rich membrane protrusions that are sites of focal extracellular matrix degradation. While there is a growing consensus that invadopodia are instrumental in tumor metastasis, less is known about whether they are involved in tumor growth, particularly in vivo. The adaptor protein Tks5 is an obligate component of invadopodia, and is linked molecularly to both actin-remodeling proteins and pericellular proteases. Tks5 appears to localize exclusively to invadopodia in cancer cells, and in vitro studies have demonstrated its critical requirement for the invasive nature of these cells, making it an ideal surrogate to investigate the role of invadopodia in vivo. In this study, we examined how Tks5 contributes to human breast cancer progression. We used immunohistochemistry and RNA sequencing data to evaluate Tks5 expression in clinical samples, and we characterized the role of Tks5 in breast cancer progression using RNA interference and orthotopic implantation in SCID-Beige mice. We found that Tks5 is expressed to high levels in approximately 50% of primary invasive breast cancers. Furthermore, high expression was correlated with poor outcome, particularly in those patients with late relapse of stage I/II disease. Knockdown of Tks5 expression in breast cancer cells resulted in decreased growth, both in 3D in vitro cultures and in vivo. Moreover, our data also suggest that Tks5 is important for the integrity and permeability of the tumor vasculature. Together, this work establishes an important role for Tks5 in tumor growth in vivo, and suggests that invadopodia may play broad roles in tumor progression

Tks5 is required for tumor progression.

<p>MDA-MB-231-Luc cells were infected to stably express an inducible TetOn lentivirus where the levels of the Tks5 shRNA are under the control of the tetracycline promoter (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121003#sec002" target="_blank">Materials and methods</a> for the experimental procedure). A) Immunoblot demonstrating Tks5 expression reduction in the MDA-MB-231-Luc cell line in dose- and time-dependent fashion in response to doxycycline exposure. B) TetOn/D6 were injected orthotopically (blue, red, and green lines) compared to SCR and no doxycycline controls (grey and purple lines, respectively) under three conditions: when Tks5 was already reduced by <i>in vitro</i> exposure of the cells to doxycycline for 10 days (DOX A) (blue line), when unexposed cells were injected into the animal and the animal received doxycycline starting at the day of injection (DOX B) (red line), as well as when the animals received doxycycline in the drinking water for the first time after the tumor has been growing for 7 days (DOX C) (green line). C) Animals were given doxycycline 15 days after tumor cell injection and after randomization of the mice. TetOn/D6 mice were divided up in 3 groups where 2 groups received doxycycline in the drinking water at different time points and 1 group received doxycycline-free drinking water. Tumor volumes were measured at different time points as described in Materials and methods, and tumors were allowed to grow to a final volume of approximately 2cm³. N = 4 mice per tumor type. Experiments were performed in duplicate. D-I) Tumors from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121003#pone.0121003.g004" target="_blank">Fig 4C</a> were analyzed for vascularization by CD31 (panel D-E), for apoptosis by TUNEL (panel F-G) and for proliferation using Ki-67 (panel H-I) immunofluorescence staining. Scale bar: 100μm. Images are representative for all experiments performed. J-K) Quantification of positive immunohistochemical and immunofluorescence staining. Graphs show immunopositive cells for apoptosis (TUNEL) (panel J) and proliferative (Ki67) (panel K) markers at the day of dissection (endpoint of experiment). Data were expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate <i>p</i> values.</p

Reduction of Tks5 expression in tumor cells is associated with decreased angiogenesis.

<p>Tumors from Scr KD and Tks5 KD MDA-MB-231-Luc-orthotopic mouse models in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121003#pone.0121003.g002" target="_blank">Fig 2</a> were analyzed via immunohistochemistry and immunofluorescence in size-matched tumors. A-B) Vessel morphology and density was examined by staining tumor samples with CD31 (quantification panels K and L). C-D) Hematoxylin and eosin staining revealed altered vessel morphology and hemorrhaging in Tks5 KD tumors as compared to Scr KD tumors. Tumors were also analyzed for FITC-Dextran leakage (panels E-F, red = CD31; Green = FITC-dextran; quantification panel M), VEGF expression (red; panels G-H), and hypoxic areas (pimonidazole staining in panel I-J, quantification panel N). Red dashed lines delineate borders for areas of hypoxia. Scale bar: 100 μm, except panels C and D where scale bar: 50μm. Images are representative for all experiments performed. Data were expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate <i>p</i> values.</p

Tumor proliferation and apoptosis is affected by Tks5 knockdown.

<p>Tumors from Scr KD and Tks5 KD MDA-MB-231-Luc-orthotopic mouse models in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121003#pone.0121003.g002" target="_blank">Fig 2</a> were analyzed via immunohistochemistry and immunofluorescence, using size-matched tumors. A-D) TUNEL staining was used to visualize cell death in small (panels A-B) and large (panels C-D) tumors from Scr (panels A, C) and Tks5 (panels B, D) KD mice. Scale bar: 100μm. E-H) Ki-67 staining (nuclear protein marker) was used to visualize cell proliferation in small (panels E-F) and large (panels G-H) tumors from Scr (panels E, G) and Tks5 (panels F, H) KD mice. Scale bar: 50μm. Images are representative for all experiments performed. I-J) Quantification of positive immunohistochemical and immunofluorescence staining. Graphs show immune-positive cells for apoptosis (TUNEL) (panel I) and proliferative cells (Ki67) (panel J) at the day of dissection (endpoint of experiment). Data are expressed as mean ± SD. One-way ANOVA or a Student’s t test was used to calculate <i>p</i> values.</p