La protéine suppresseur de tumeurs PTEN (Phosphatase and tensin deleted on chromosome 10) est une phosphatase lipidique. En déphosphorylant le phosphatidylinositol (3,4,5) trisphosphate (PIP3) en PI(4,5) P2, PTEN contre-régule la voie PI3K/Akt et inhibe la prolifération. D’autres fonctions de PTEN peuvent être indépendantes de son activité phosphatase lipidique, notamment l’inhibition de la migration. Bien que PTEN soit, après p53, le suppresseur de tumeurs le plus muté dans un large panel de cancers (gliomes, prostate, sein, endomètre…), les mécanismes par lesquels ses fonctions sont régulées ne sont pas entièrement élucidés. Par une approche de double-hybride, notre équipe a identifié que les β-arrestines (β-arrs), des protéines d’échafaudage, interagissent avec PTEN. Nos travaux mettent en évidence que l’interaction entre PTEN et les β-arrs permet de moduler ses deux activités dépendantes ou non de son activité phosphatase lipidique. D’une part, les β-arrs augmentent l’activité phosphatase lipidique de PTEN in vitro. La GTPase RhoA et sa kinase d’aval ROCK activent PTEN, et ceci se fait par l’intermédiaire des β-arrs. La stimulation du récepteur à l’acide lysophosphatidique (LPA), qui active la voie RhoA/ROCK, augmente la formation du complexe PTEN/β-arrs et permet le recrutement du complexe à la membrane. Par l’effet positif sur l’activité phosphatase lipidique de PTEN, les β-arrs participent à l’inhibition d’Akt et de la prolifération dans les fibroblastes embryonnaires de souris (MEF). A l’inverse dans les gliomes U373, les β-arrs lèvent l’inhibition de la migration exercée par le domaine C2 de PTEN, indépendamment de son activité phosphatase lipidique. En aval de l’activation de RhoA induite par blessure du tapis cellulaire, les β-arrs interagissent davantage avec PTEN et rétablissent la migration des gliomes. De ce fait, les β-arrs régulent différentiellement les fonctions de PTEN importantes pour le contrôle de la prolifération cellulaire et la migration. Enfin, l’activité et la localisation de PTEN sont modulées par des interactions intramoléculaires entre ses domaines catalytiques, C2 et sa queue C-terminale régulatrice. Ces interactions régulent le passage d’une conformation fermée vers une conformation ouverte et active de PTEN. Grâce au développement d’un biosenseur de PTEN basé sur le transfert d’énergie par résonnance (RET), nous pouvons suivre pour la première fois les changements conformationnels de PTEN dans les cellules vivantes. En utilisant ce biosenseur nous montrons que la mutation des résidus impliqués dans les interactions intramoléculaires entraine des changements de conformation détectés par des variations de RET. De plus, l’activation de voies de signalisation connues pour activer PTEN, entrainent des changements conformationnels qui corrèlent avec l’augmentation de l’activité phosphatase lipidique de PTEN. Nos données montrent que le biosenseur peut être utilisé comme outil pour détecter les changements d’activité de PTEN dans les cellules vivantes. L’axe suppresseur de tumeurs/oncogène PTEN/PI3K/Akt joue un rôle essentiel dans la progression tumorale et constitue une cible thérapeutique pour le cancer. L’ensemble de nos travaux permet d’ajouter un degré de compréhension dans la régulation de PTEN, tant par les β-arrs que par l’interaction intramoléculaire et les changements conformationnels. / The Tumour Suppressor protein PTEN (Phosphatase and tensin deleted on chromosome 10) is a lipid phosphatase. By converting phosphatidylinositol (3,4,5) trisphosphate (PIP3) to PI(4,5)P2, PTEN inhibits the PI3K/Akt signalling pathway and cell proliferation. Other functions attributed to PTEN, including the inhibition of cell migration, can occur independently of its lipid phosphatase activity. Although PTEN function is dysregulated in a broad range of cancers (gliomas, prostate, breast, endometrium…), the mechanisms by which it is regulated are far from being completely elucidated. Using a two-hybrid approach, our team identified that the molecular scaffolds, β-arrestins (β-arrs), interact with PTEN.Our studies demonstrate that β-arrs modulate distinct functional outputs of PTEN that in turn are dependent or independent on its lipid phosphatase activity. β-arrs increase the lipid phosphatase activity of PTEN in vitro. The small GTPase RhoA and its downstream effector ROCK activate PTEN and this effect requires β-arrs. The stimulation of the lysophosphatidic acid receptor 1 (LPA1-R) receptor, that activates the RhoA/ROCK pathway, was found to increase the association of β-arrs with PTEN and induced plasma membrane translocation of the complex. Through their stimulatory effect on the lipid phosphatase activity of PTEN, β-arrs inhibit the PI3K/Akt pathway and proliferation of mouse embryonic fibroblasts. In contrast, in U373 glioma cells, βarrs release the brake on cell migration, which is mediated by the C2 domain of PTEN independently of its lipid phosphatase activity. Following wounding of a cell monolayer, and RhoA activation, β-arrs show increased association with PTEN, and rescue glioma cell migration. β-arrs therefore differentially regulate functions of PTEN important in the control of cell proliferation and migration.The activity and localization of PTEN are under tight control of intramolecular interactions between its regulatory C-terminal tail, and catalytic and C2 domains. These intramolecular interactions regulate a switch between a closed form of PTEN, and an open and active form that is targeted to the membrane. We have developed a resonance energy transfer (RET)-based biosensor that permits the monitoring of PTEN conformational change in live cells. Using the biosensor we demonstrate that mutation of residues implicated in the intramolecular switch produce conformational rearrangement of PTEN, detected by changes in RET. Furthermore, activation of signalling pathways known to activate PTEN, elicit conformational changes that parallel increased PTEN lipid phosphatase activity in living cells. Combined, these data demonstrate that the biosensor can be used as a tool to detect changes in PTEN tumour suppressor activity in live cells.The tumour suppressor/oncogene PTEN/PI3K/Akt axis plays a key role in tumour progression and represents a major therapeutic target in the treatment of cancer. Our studies help to further our understanding of how tumour suppressor PTEN is controlled by inter- and intramolecular interactions and provide a biosensor that can report changes in PTEN activity.

Níveis elevados de hormônios tireoidianos (HTs) são comumente associados à ativação do sistema renina angiotensina local e ao desenvolvimento da hipertrofia cardíaca. O envolvimento do receptor de angiotensina II tipo 1 (AT1R) nos efeitos hipertróficos dos HTs fora descrito previamente. No entanto, os mecanismos subjacentes a essa interação ainda são desconhecidos. O AT1R pertence à família dos receptores acoplados à proteína G e, portanto, promove a transdução de sinal por mecanismos dependentes e independentes de proteína G. Recentemente, a sinalização dependente de beta-arrestinas (independente de proteína G) tem sido descrita por contribuir com a resposta hipertrófica em diferentes modelos experimentais. Assim, no presente estudo investigou-se o envolvimento da sinalização dependente de beta-arrestinas nos efeitos hipertróficos dos HTs, mediados pelo AT1R, bem como a participação de ERK½ nesse processo. Culturas primárias de cardiomiócitos foram estimuladas com T3 (triiodotironina; 15nM) para indução da hipertrofia. O tratamento dos cardiomiócitos com T3 por tempos rápidos (5-30 min) resultou na ativação transiente de ERK½, a qual foi parcialmente atenuada quando da administração de Losartan (1µM), antagonista do AT1R. A contribuição de ERK½ na hipertrofia dos cardiomiócitos foi verificada através do uso de PD98059 (20µM), inibidor de MEK½, o qual preveniu a transcrição de marcadores hipertróficos. Ensaios de imunoprecipitação revelaram o aumento da interação entre AT1R e beta-arrestina 2 sob estímulo do T3, sugerindo o recrutamento de beta-arrestina 2 e, possível, internalização do AT1R. Através de ensaios de imunofluorescência e fracionamento subcelular, foi demonstrado que o T3 estimula a translocação do AT1R, amentando sua expressão no núcleo dos cardiomiócitos. Além disso, tanto a ativação de ERK½ quanto a hipertrofia cardiomiocítica mostraram-se sensíveis à inibição da endocitose, a qual foi avaliada através de Concanavalina A (0,5µg/ml). Ensaios de silenciamento gênico por RNA de interferência foram eficientes em demonstrar o envolvimento de beta-arrestina 2 na ativação de ERK½ e na hipertrofia cardiomiocítica induzida por T3. Desta forma, os resultados evidenciam o envolvimento da sinalização dependente de beta-arrestina 2 na ativação de ERK½, através do AT1R, a qual contribui com a hipertrofia cardiomiocítica promovida pelo T3. / Elevated levels of thyroid hormones (THs) are commonly associated with activation of the local renin angiotensin system and the development of cardiac hypertrophy. The involvement of the angiotensin II receptor type 1 (AT1R) in the hypertrophic effects of the THs was previously described. However, the mechanisms underlying this interaction are still unknown. AT1R belongs to the G-protein coupled receptor family and promotes its signal transduction by G-protein dependent and independent mechanisms. Recently, beta-arrestin signaling (G-protein independent) has been described as contributing to the hypertrophic response in different experimental models. Thus, the present study investigated the involvement of beta-arrestin signaling in the hypertrophic effects of THs mediated by AT1R, as well as the participation of ERK½ in this process. Primary cardiomyocytes cultures were stimulated with T3 (triiodothyronine; 15nM) for the induction of hypertrophy. Cardiomyocytes acutely treated with T3 (5-30 min) resulted in transient activation of ERK½, which was partially attenuated upon Losartan (1µM) administration, an AT1R antagonist. The contribution of ERK½ to cardiomyocyte hypertrophy was verified by using PD98059 (20µM), a MEK½ inhibitor, which prevented the transcription of hypertrophic markers. Immunoprecipitation assays revealed increased interaction between AT1R and beta-arrestin 2 under T3 stimulation, suggesting the recruitment of beta-arrestin 2 and, possibly, the internalization of AT1R. Through immunofluorescence and subcellular fractionation assays, T3 has been shown to stimulate AT1R translocation, enhancing its expression in the cardiomyocyte nucleus. In addition, both ERK½ activation and cardiomyocyte hypertrophy were sensitive to the inhibition of endocytosis, which was assessed by Concanavalin A (0.5µg/ml). Interfering RNA assays were efficient in demonstrating the involvement of beta-arrestin 2 in ERK½ activation and in T3-induced cardiomyocyte hypertrophy. Therefore, the results evidenced the involvement of beta-arrestin-2-dependent signaling in the activation of ERK½, through the AT1R, which contributes to the cardiomyocyte hypertrophy promoted by T3.