The tumour suppressor LATS2 interacts with both Signalosome and E3 ubiquitin ligases : implications in control of cell cycle progression

SUMMARY LATS2 is a member of the Lats tumour suppressor gene family. The human LATS2 gene is located at chromosome 13q11-12, which has been shown to be a hot spot (67%) for LOH in nonsmall cell lung cancer. Both lats mosaic flies and LATS1 deficient mice spontaneously develop tumours, an observation that is explained by the function of LATS1 in suppressing tumourigenesis by negatively regulating cell proliferation by modulating Cdc2/Cyclin A activity. LATS1 also plays a critical role in maintenance of ploidy through its action on the spindle assembly checkpoint. Initial insights into the function of LATS2 reveals that the protein is involved in the G2/M transition of the cell cycle, whereby it controls the phosphorylation status of Cdc25C. The aim of the present study was to identify LATS2 interacting partners that would provide a more thorough understanding of the molecular pathways in which the protein is involved. The yeast two-hybrid system identified a number of candidate genes that interact with LATS2. Most of the interactions were confirmed biochemically by GST-pull down assays that enabled us to demonstrate that LATS2 is an integral component of the Signalosome complex. The Signalosome is thought to be required for the establishment of functional Cullin-based E3 ubiquitin ligases, the substrate-recognition elements of the ubiquitin-mediated protein proteolytic pathway. The findings that LATS2 also interacts with all of the components of the E3 enzymes allows us to postulate that LATS2 is probably involved in the regulation of this Signalosome-E3 super-complex. In addition, the discovery that LATS2 associates with multiple protein kinases localised at the cellular membrane and in various signalling cascades supports the idea that LATS2 functions as an integrator of signals which allows it to monitor the activity of these pathways and translate these signals through its action on the Signalosome. Furthermore, the observation that a kinase-dead LATS2 mutant arrests at the G2/M phase of the cell cycle, demonstrates that the protein, through the action of its kinase domain, is crucial for progression through the cell cycle, an action in accordance to its proposed role as a regulator of E3 ubiquitin ligases. The findings presented herein provide evidence that LATS2 associates with the Signalosome-E3 ubiquitin ligases super-complex which governs protein stability. Any alteration of the protein would have a strong impact on pathways that modulate cell proliferation, as shown by its implication in tumourigenesis. RESUME LATS2 est un membre de la famille de gènes suppresseurs de tumeurs LATS. Le gène humain LATS2 est situé sur le chromosome 13q11-12, une région qui s'est avérée être un point sensible (67%) dans la perte d'hétérozigosité (LOH) notamment pour le cancer du poumon. Le fait que des tumeurs se développent spontanément chez les souris qui sont déficientes pour le gène LATS1 ainsi que dans des cellules mutantes pour LATS chez la Drosophile, est expliqué Par la fonction de LATS1, qui est de supprimer l'apparition de tumeurs en réprimant la prolifération cellulaire à travers sa capacité à réguler l'activité de Cdc2/Cyciine A. LATS1 joue également un rôle important au niveau du maintient de la ploïdie de la cellule, au travers de son action sur les points de contrôle de l'assemblage du fuseau mitotique. Les premières études du gène LATS2 indiquent que la protéine est, par son contrôle des réactions de phosphorylation de la Cdc25C, impliquée dans la transition 021M. Le but de cette étude était d'identifier les protéines qui interagissent avec LATS2, en vue d'obtenir une compréhension plus approfondie des mécanismes moléculaires dans lesquels LATS2 se trouve engagée. Le système de double-hybride chez la levure a permis l'identification d'un grand nombre de gènes qui interagissent avec LATS2. La plupart des interactions ont été confirmées par GST «pull clown», une technique in vitro qui a permis de démontrer que LATS2 est un composant intégral du Signalosome. Ce complexe est supposé réguler l'activité des E3 ubiquitine-rigases, les éléments responsables du recrutement des substrats qui doivent être recyclés par la voie de dégradation ubiquitine-dépendante. Les résultats obtenus indiquent également que LATS2 interagit avec tous les composants des enzymes E3, ce qui nous permet de soumettre l'idée selon laquelle la protéine LATS2 est en fait impliquée dans la régulation du complexe Signalosorne-E3. De plus, la découverte que LATS2 se trouve associée à plusieurs protéines kinases localisées au niveau de la membrane cellulaire, ainsi que dans diverses voies de transduction, confirment l'idée que LATS2 fonctionne en tant que molécule qui intègre les signaux en provenance de ces différentes voies cellulaires. De ce fait, il lui serait possible de coordonner la destruction des protéines au moyen du complexe Signalosome, permettant ainsi de réprimer l'activité des voies de signalisation. En outre, l'introduction d'une mutation dans le domaine kinase de LATS2 résulte en l'arrêt du cycle cellulaire en G2/M, ce qui montre que la protéine, au travers de son domaine kinase, est cruciale pour le bon fonctionnement du cycle cellulaire, ceci en accord avec son rôle proposé comme régulateur des E3 ubiquitine-ligases. Les résultats présentés dans ce manuscrit démontrent que la protéine LATS2 se trouve associée au complexe Signalosome-E3 qui régule la dégradation des protéines. La moindre modification de la protéine engendrerait des répercussions importantes au niveau des voies de transduction qui contrôlent fa prolifération ceilulaire, ce qui atteste du rôle déterminant que joue LAT32 dans la tumorigénèse.

On ubiquitin ligases and cancer.

Protein kinase genes account for almost 10% of all currently known cancer genes, highlighting the role of signal transduction in oncogenesis. A reexamination of the literature and available databases shows that E3 ubiquitin ligases are also key mediators of tumorigenesis. Altogether kinase and E3 genes represent more than 15% of the known cancer genes, underlining the importance of phosphorylation and ubiquitylation signaling pathways in cancer formation. Considering the recent literature reporting correlations between alterations in ubiquitylation processes and oncogenesis, this percentage is likely to increase even further in the future. Finally, E3 genes could serve as baits for the identification of additional cancer genes (e.g. their interacting partners). In contrast, deubiquitinases, like phosphatases, are not overrepresented among cancer genes. The same holds for E1 and E2 genes. Thus, kinase and E3 genes represent primary targets as cancer susceptibility genes for mutation screening and for the design of novel therapies.

Ubiquitin Ligases of the N-End Rule Pathway: Assessment of Mutations in UBR1 That Cause the Johanson-Blizzard Syndrome.

Background: Johanson-Blizzard syndrome (JBS; OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons) recognized by UBR1 are destabilizing N-terminal residues of protein substrates.Methodology/Principal Findings: Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R) was inactive in yeast-based activity assays, the other one (Q1224E) had a detectable but weak activity, and the third one (V146L) exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients.Conclusions/Significance: These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS.

Regulation of the voltage-gated sodium channel Nav1.7 by ubiquitin ligase Nedd4-2

Background and aim: Neuropathic pain (NP) is a frequent and disabling disorder occurring as a consequence of a direct lesion of the nervous system and recurrently associated with a positive shift toward nervous system excitability. Peripheral nerve activity is mainly carried by voltage-gated sodium channels (VGSC), with Nav1.7 isoform being an important candidate since loss of function mutations of its gene is associated with congenital inability to experience pain. Interestingly, ubiquitin ligases from the Nedd4 family are well known proteins that regulate the turnover of many membrane proteins such as VGSC and we showed Nedd2-2 is downregualted in experimental models of chronic pain. The aim of this study was to investigate the importance of Nedd4-2 in the modulation of Nav1.7 at the membrane. Methods: In vitro: whole cell patch clamp on HEK293 cell line stably expressing Nav1.7 was used to record sodium currents (INa), where the peak current of INa reflects the quantity of functional Nav1.7 expressed at the membrane. The possibility that Nedd4-2 modulates the currents was assessed by investigating the effect of its cotransfection on INa. Biotinylation of cell surface was used to isolate membrane-targeted Nav1.7. Furthermore, as the interaction between Nedd4-2 and Nav isoforms was previously reported to rely on an xPPxYx sequence (PY-motif), we mutated this latter to study its impact in the specific interaction between Nav1.7 and Nedd4-2. GST-fusion proteins composed of the Nav1.7 c terminal 66 amino acids (wild-type or PY mutated) and GST were used to pull-down Nedd4-2 from lysates. Results: Co-transfection of Nav1.7 with Nedd4-2 reduced the Nav1.7 current amplitude by ~80% (n = 36, p <0.001), without modifying the biophysical properties of INa. In addition, we show that the quantity of Nav1.7 at the membrane was decreased when Nedd4-2 was present. This effect was dependent on the PY-motif since mutations in this sequence abolished the down-regulatory effect of Nedd4-2. The importance of this motif was further confirmed by pull down experiments since the PY mutant completely eliminate the interaction with Nedd4-2. Perspectives: Altogether, these results point to the importance of Nedd4-2 as a Nav1.7 regulator through cell surface modulation of this sodium channel. Further experiments in freshly dissociated neurons from wild type and Scn1bflox/Nedd4-2Cre mice are needed to confirm in vivo these preliminary data.

Ubiquitin- and ubiquitin-like proteins-conjugating enzymes (E2s) in breast cancer.

Breast cancer is the most common malignancy in women and a significant cause of morbidity and mortality. Sub-types of breast cancer defined by the expression of steroid hormones and Her2/Neu oncogene have distinct prognosis and undergo different therapies. Besides differing in their phenotype, sub-types of breast cancer display various molecular lesions that participate in their pathogenesis. BRCA1 is one of the common hereditary cancer predisposition genes and encodes for an ubiquitin ligase. Ubiquitin ligases or E3 enzymes participate together with ubiquitin activating enzyme and ubiquitin conjugating enzymes in the attachment of ubiquitin (ubiquitination) in target proteins. Ubiquitination is a post-translational modification regulating multiple cell functions. It also plays important roles in carcinogenesis in general and in breast carcinogenesis in particular. Ubiquitin conjugating enzymes are a central component of the ubiquitination machinery and are often perturbed in breast cancer. This paper will discuss ubiquitin and ubiquitin-like proteins conjugating enzymes participating in breast cancer pathogenesis, their relationships with other proteins of the ubiquitination machinery and their role in phenotype of breast cancer sub-types.

The E3 ubiquitin ligase Itch regulates sorting nexin 9 through an unconventional substrate recognition domain.

The level of intracellular proteins is mainly regulated through modifications by ubiquitin ligases that target them for degradation. Members of the NEDD4 family of E3 ubiquitin ligases, such as Itch (atrophin-1 interacting protein 4), possess up to four WW domains for specific association with PY motif-containing substrates. We have identified sorting nexin 9 (SNX9), a protein involved in endocytic processes, as a new substrate of Itch. Itch ubiquitylates SNX9 and regulates intracellular SNX9 levels. Using truncated proteins, we found that the interaction with SNX9 is mediated by the proline-rich domain (PRD) of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. Furthermore, this effect is specific for Itch, as NEDD4, a related PRD-containing E3 ligase, does not bind SNX9. SNX18, a second member of the SNX family containing an SH3 domain, was also found to bind to Itch. Our results indicate that the pool of substrates of NEDD4 family E3 ubiquitin ligases extends beyond proteins containing PY motifs.

Role of the ubiquitin system in regulating ion transport.

Ion channels and transporters play a critical role in ion and fluid homeostasis and thus in normal animal physiology and pathology. Tight regulation of these transmembrane proteins is therefore essential. In recent years, many studies have focused their attention on the role of the ubiquitin system in regulating ion channels and transporters, initialed by the discoveries of the role of this system in processing of Cystic Fibrosis Transmembrane Regulator (CFTR), and in regulating endocytosis of the epithelial Na(+) channel (ENaC) by the Nedd4 family of ubiquitin ligases (mainly Nedd4-2). In this review, we discuss the role of the ubiquitin system in ER Associated Degradation (ERAD) of ion channels, and in the regulation of endocytosis and lysosomal sorting of ion channels and transporters, focusing primarily in mammalian cells. We also briefly discuss the role of ubiquitin like molecules (such as SUMO) in such regulation, for which much less is known so far.

The Cul3-KLHL21 E3 ubiquitin ligase targets aurora B to midzone microtubules in anaphase and is required for cytokinesis.

Cul3 (Cullin3)-based E3 ubiquitin ligases recently emerged as critical regulators of mitosis. In this study, we identify two mammalian BTB (Bric-a-brac-Tramtrack-Broad complex)-Kelch proteins, KLHL21 and KLHL22, that interact with Cul3 and are required for efficient chromosome alignment. Interestingly, KLHL21 but not KLHL22 is necessary for cytokinesis and regulates translocation of the chromosomal passenger complex (CPC) from chromosomes to the spindle midzone in anaphase, similar to the previously described BTB-Kelch proteins KLHL9 and KLHL13. KLHL21 directly binds to aurora B and mediates ubiquitination of aurora B in vitro. In contrast to KLHL9 and KLHL13, KLHL21 localizes to midzone microtubules in anaphase and recruits aurora B and Cul3 to this region. Together, our results suggest that different Cul3 adaptors nonredundantly regulate aurora B during mitosis, possibly by ubiquitinating different pools of aurora B at distinct subcellular localizations.

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain.

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.

Keeping the leaves green above us.

The plant immune system relies to a great extent on the highly regulated expression of hundreds of defense genes encoding antimicrobial proteins, such as defensins, and antiherbivore proteins, such as lectins. The expression of many of these genes is controlled by a family of mediators known as jasmonates; these cyclic oxygenated fatty acid derivatives are reminiscent of prostaglandins. The roles of jasmonates also extend to the control of reproductive development. How are these complex events regulated? Nearly 20 members of the jasmonate family have been characterized. Some, like jasmonic acid, exist in unmodified forms, whereas others are conjugated to other lipids or to hydrophobic amino acids. Why do so many chemically different forms of these mediators exist, and do individual jasmonates have unique signaling properties or are they made to facilitate transport within and between cells? Key features of the jasmonate signal pathway have been identified and include the specific activation of E3-type ubiquitin ligases thought to target as-yet-undescribed transcriptional repressors for modification or destruction. Several classes of transcription factor are known to function in the jasmonate pathway, and, in some cases, these proteins provide nodes that integrate this network with other important defensive and developmental pathways. Progress in jasmonate research is now rapid, but large gaps in our knowledge exist. Aimed to keep pace with progress, the ensemble of jasmonate Connections Maps at the Signal Transduction Knowledge Environment describe (i) the canonical signaling pathway, (ii) the Arabidopsis signaling pathway, and (iii) the biogenesis and structures of the jasmonates themselves.

Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.

Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.

Characterization of the expression, regulation and function of Sorting Nexin 9 and Sorting Nexin 18

Summary : Sorting nexin (SNX) family members play important roles in intracellular protein and membrane trafficking, The membrane-tubulating SNX9 protein has been shown to interact with multiple components of the endocytic machinery and to participate in clathrin-mediated endocytosis of cell surface receptors. It has not been investigated if SNX9 may also participate in other protein sorting pathways that involve vesicular transport, specifically the biogenesis of lysosome-related organelles (LROs). Closely related to SNX9 is SNXl8, whose function is largely unknown. In this work, we have characterized the expression of SNX9 and SNXl8 in LRO-containing cells and investigated their role in protein trafficking during the formation of LROs. Our results indicate that SNX9 and SNXl8 are not essential for the formation of LROs, nor for the sorting of melanosomal proteins. We investigated how the level of intracellular SNX9 protein is regulated and found that it is a substrate of the ubiquitin ligase Itch, a member of the NEDD4 family of E3 ubiquitin ligases. Itch ubiquitylates SNX9 and regulates SNX9 levels by enhancing its degradation. Using ? truncated proteins we found that the interaction with SNX9 is mediated by the proline-rich domain of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. We further showed that Itch binding is not affected by tyrosine phosphorylation of SNX9. Using lentivector-mediated siRNA techniques, we found that Itch regulates the level of melanosomal proteins, while knock-down of SNX9 does not alter their level. Interestingly, we revealed that silencing of SNXIS affects the amount of the melanosomal protein Melan-A, but also of SNX9, and that SNXl8 can interact with SNX9. Taken together, our results highlight that the pool of substrates of NEDD4 family E3 ligases extends to proteins containing SH3 domains and provide insight into the potential functions of SNXI8. Résumé : Les membres de la famille des Sorting Nexins (SNX) jouent des rôles importants dans le trafic intracellulaire de protéines et membranes. Il a été démontré que la protéine SNX9, qui génère les tubules membranaires, interagit avec plusieurs composants de la machinerie d'endocytose et participe à l'endocytose des récepteurs de surface mediée par la clathrine. Aucune étude n'a investigué si SNX9 pourrait aussi participer à d'autres voies de trafic de protéines tel que le transport vésiculaire, et plus particulièrement la biogenèse des organites lysosomaux ("lysosome-related organelles", LR©s). SNXl8 est similaire à SNX9, mais sa fonction est largement inconnue. Dans ce travail, nous avons caractérisé l'expression de SNX9 et SNX18 dans des cellules contenants des LROs et investigué leur rôle dans le trafic de protéines pendant la formation des LROS. Nos résultats indiquent que SNX9 et SNXI8 ne sont essentiels ni pour la formation des LR©s, ni pour le trafic de protéines mélanosomales. Nous avons examiné la régulation du niveau intracellulaire de la protéine SNX9 et avons trouvé qu'elle est un substrat de l'ubiquitine ligase Itch, un membre de la famille NEDD4 des ubiquitine ligases E3. Itch ubiquitine SNX9 et régule les niveaux de SNX9 en augmentant sa dégradation. En utilisant des protéines mutées nous avons découvert que l'interaction avec SNX9 est médiée par le domaine riche en proline de Itch, qui est différent du domaine conventionnel de reconnaissance WW, et par le domaine SH3 de SNX9. L'interaction avec le domaine riche en proline de Itch est essentielle pour l'ubiquitination et la dégradation de SNX9. De plus, nous avons montré que cette liaison n'est pas affectée par la phosphorylation des résidus tyrosine de SNX9. En utilisant des vecteurs lentiviraux exprimant des siARN, nous avons trouvé que Itch régule les niveaux de protéines mélanosomales, alors que l'extinction de l'expression de SNX9 ne change pas leurs niveaux. En autre, nous avons révélé que la diminution de SNXl8 affecte le niveau de la protéine mélanosomale Melan-A et de SNX9, et aussi que SNXl8 peut interagir avec SNX9. En résumé, nos résultats démontrent que l'ensemble des substrats de la famille NEDD4 des ubiquitine ligases E3 s'élargit aux protéines contenant des domaines SH3 et ouvrent des perspectives sur les fonctions potentielles de SNXl8.

Regulation of the cardiac voltage-gated sodium channel Nav1.5 : regulation of Nav1.5 by ubiquitin-protein ligases of the Nedd4/Nedd4-like family and characterisation of two novel mutations in the gene encoding Nav1.5 (SCN5A) implicated in the Brugada syndrome triggered by fever

Abstract: The genesis of the cardiac action potential, which accounts for the cardiac contraction, is due to the sodium current INa mediated by the voltage-gated sodium channel Nav1.5. Several cardiac arrhythmias such as the Brugada syndrome are known te be caused by mutations in SCN5A, the gene encoding Nav1.5. Studies of these mutations allowed a better understanding of biophysical and functional properties of Nav1.5. However, only few investigations have been performed in order to understand the regulation of Nav1.5. During my thesis, I investigated different mechanisms of regulation of Nav1.5 using a heterologous expression system, HEK293 cells, coupled with a technique of sodium current recording: the patch clamp in whole cell configuration. In previous studies it has been shown that an enzyme of the Nedd4 family (Nedd4-2) regulates an epithelial sodium channel via the interaction with PY-motifs present in the latter. Interestingly, Nav1.5 contains a similar PY-motif, which motivated us to study the role of Nedd4-2 expressed in heart for the regulation of Nav1.5. In a second study, we investigated the implication of two Nav1.5 mutants, which were either less functional or net functional (Nav1.5 R535X and Nav1.5 L325R respectively) implied in the genesis of the Brugada syndrome by fever. Our results established two mechanisms implied in Nav1.5 regulation. The first one implies that following the interaction between the PY-motif of Nav1.5 and Nedd4- 2 Nav1.5 is ubiquitinated by Nedd4-2. This ubiquitination leads to the internalization of Nav1 .5. The second mechanism is a phenomenon called the "dominant negative" effect of Nav1.5 L325R on Nay1.5 where the decrease of 'Na is potentially due to the retention of Nav1.5 by Nav1.5 L325R in an undefined intracellular compartment. These studies defined two mechanisms of Nav1.5 regulation, which could play an important role for the genesis of cardiac arrhythmias where molecular processes are still poorly understood. Résumé La genèse du potentiel d'action cardiaque, permettant la contraction cardiaque, est due au courant sodique INa issu des canaux sodiques cardiaques dépendants du voltage Nav1.5. Nombreuses arythmies cardiaques telles que le syndrome de Brugada sont connues pour être liées à des mutations du gène SCN5A, codant pour Nav1.5. L'étude de ces mutations a permis une meilleure compréhension des propriétés structurelles et fonctionnelles de Nav1.5 et leurs implications dans la genèse de ces pathologies. Néanmoins peu d'études ont été menées afin de comprendre les mécanismes de régulation de Nav1.5. Mon travail de thèse a consisté à étudier des mécanismes de régulation de Nav1.5 en utilisant un système d'expression hétérologue, les cellules HEK293, couplé à une technique d'enregistrement des courants sodiques, le "patch clamp" en configuration cellule entière. La présence sur Nav1.5 d'un motif-PY similaire à ceux nécessaires pour la régulation d'un canal épithélial sodique par une enzyme de la famille de Nedd4, nous a amenée à étudier le rôle de ces ubiquitine-ligases, en particulier Nedd4-2, dans la régulation de Nav1.5. La seconde étude s'est intéressée aux conséquences de deux mutations de SCN5A codant pour deux mutants peu ou pas fonctionnels (Nav1.5 L325R et Nav1.5 R535X respectivement) retrouvées chez des patients présentant un syndrome de Brugada exacerbé par un état fébrile. Nos résultats ont permis d'établir deux mécanismes de régulation de Nav1.5 L'un par Nedd4-2 qui implique rubiquitination de Nav1.5 par cette ligase suite à l'interaction entre le motif-PY de Nav1.5 et Nedd4-2. Cette modification déclenche l'internalisation du canal impliquée dans la diminution d'INa. Le second mécanisme quant à lui est un effet "dominant négatif" de Nav1.5 L325R sur Nav1.5 aboutissant à une diminution d'INa suite à la séquestration intracellulaire potentielle de Nav1.5 par Nav1.5 L325R. Ces études ont mis en évidence deux mécanismes de régulation de Nav1.5 pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des arythmies cardiaques dont les processus moléculaires au sein des cardiomyocytes, impliquant des modifications du courant sodiques, sont encore mal compris. Résumé destiné à un large public La dépolarisation électrique de la membrane des cellules cardiaques permet la contraction du coeur. La génèse de cette activité électrique est due au courant sodique issu d'un type de canal à sodium situé dans la membrane des cellules cardiaques. De nombreuses pathologies provoquant des troubles du rythme cardiaque sont issues de mutations du gène qui code pour ce canal à sodium. Ces canaux mutants, entrainant diverses pathologies cardiaques telles que le syndrome de Brugada, ont été largement étudiées. Néanmoins, peu de travaux ont été réalisés sur les mécanismes de régulation de ce canal à sodium non muté. Mon travail de thèse a consisté à étudier certains des mécanismes de régulation de ce canal à sodium en utilisant une technique permettant l'enregistrement des courants sodiques issus de l'expression de ces canaux à sodium à la membrane de cellules mammifères. La présence sur ce canal à sodium d'une structure spécifique, similaire à celle nécessaire pour la régulation d'un canal épithélial à sodium par une enzyme appelée Nedd4-2, nous a amenée à étudier le rôle de cette enzyme dans la régulation de ce canal à sodium. La seconde étude s'est intéressée aux rôles de deux mutations du gène codant pour ce canal à sodium retrouvées chez des patients présentant un syndrome de Brugada exacerbé par la fièvre. Nos résultats nous ont permis d'établir deux mécanismes de régulation de ce canal à sodium diminuant le courant sodique l'un par l'action de l'enzyme Nedd4-2, suite à son interaction avec ce canal, qui modifie ce canal à sodium (ubiquitination) diminuant de ce fait la densité membranaire du canal. L'autre par un mécanisme suggérant un effet négatif de l'un des canaux mutants sur l'expression à la membrane du canal à sodium non muté. Ces études ont mis en évidence deux mécanismes de régulation de ce canal à sodium pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des troubles du rythme cardiaques dont les mécanismes cellulaires sont encore incompris.

Mineralocorticoid receptor degradation is promoted by Hsp90 inhibition and the ubiquitin-protein ligase CHIP.

The mineralocorticoid receptor (MR) plays a crucial role in the regulation of Na(+) balance and blood pressure, as evidenced by gain of function mutations in the MR of hypertensive families. In the kidney, aldosterone binds to the MR, induces its nuclear translocation, and promotes a transcriptional program leading to increased transepithelial Na(+) transport via the epithelial Na(+) channel. In the unliganded state, MR is localized in the cytosol and part of a multiprotein complex, including heat shock protein 90 (Hsp90), which keeps it ligand-binding competent. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin antibiotic that binds to Hsp90 and alters its function. We investigated whether 17-AAG affects the stability and transcriptional activity of MR and consequently Na(+) reabsorption by renal cells. 17-AAG treatment lead to reduction of MR protein level in epithelial cells in vitro and in vivo, thereby interfering with aldosterone-dependent transcription. Moreover, 17-AAG inhibited aldosterone-induced Na(+) transport, possibly by interfering with MR availability for the ligand. Finally, we identified the ubiquitin-protein ligase, COOH terminus of Hsp70-interacting protein, as a novel partner of the cytosolic MR, which is responsible for its polyubiquitylation and proteasomal degradation in presence of 17-AAG. In conclusion, 17-AAG may represent a novel pharmacological tool to interfere with Na(+) reabsorption and hypertension.

Lats2, a novel regulator of Elongin BC ubiquitin ligase

Résumé : Le Large tumor suppressor, Lats2, est une protéine humaine homologue au suppresseur de tumeur Warts (Lats) de Drosophila melanogaster, qui réprime la prolifération des cellules en altérant leur cycle au niveau des transitions Gl/S et G2/M, et en induisant l'apoptose. Pourtant, la voie moléculaire par laquelle Lats2, une sériase-thréonine kinase, déclenche l'arrêt du cycle cellulaire, est toujours inconnue. Notre équipe a d'abord déterminé que Lats2 était un gène de réponse à la protéine p53 (Kostic et al., 2000). Par la suite, nous avons identifié des protéines interagissant avec Lats2, notamment les modules de reconnaissance du substrat des ligases Colline E3 (des protéines contenant Socs box ou F box) ainsi que deux Bous-unités du Signalosome CSN: CSN4 et CSNS. En outre, Lats2 est connue pour s'associer au Super-complexe composé de CSN et des ligases Colline E3 (Rongere, thesis, 2004; Rongere, unpublished results, 2005). Le travail présenté ici sur Lats2 a confirmé que cette protéine est une kinase associée à CSN. Nous avons caractérisé les interactions spécifiques de domaines de Lats2 avec hSocs3, hWsb 1 (des protéines Socs box) et hFBX-7 (une protéine F box), ainsi que les conséquences physiologiques des interactions avec hSocs3, hWsb1 et hSocs1. Des expériences de GST pull-down ont montré que les deux domaines, N-terminal et kinase, de Lats2 interagissent avec hSocs3, hWsb1 et hFBX-7, ce qui suggère aussi que l'ensemble de la protéine Lats2 est impliqué dans ces interactions. Une étude approfondie des interactions entre Lats2 et hSocs3 indique que le domaine kinase de Lats2 interagit avec la région de hSocs3 contenant un domaine SH2, situé en amont du domaine Socs box de hSocs3. Par ailleurs, Lats2 phosphoryle des régions spécifiques entre les domaines N-terminal et SH2 (Sl), et, entre les domaines SH2 et Socs box (S3) de la protéine hSocs3. Ces résultats révèlent que hSocs3 est un.nouveau substrat de Lats2. Des modifications de l'activité kinase ont aussi révélé que la protéine sauvage Lats2 (wt Lats2) était capable de phosphoryler hSocs3, alors qu'un mutant dead du domaine kinase Lats (poche ATP délétée, Lats2OATP) non. L'analyse des mutations a permis d'identifier deux résidus sériase situés aux positions 1441145 (S3), spécifiquement phosphorylés par wt Lats2. La phosphorylation des protéines représentant un signal de dégradation protéolytique, nous avons envisagé que Lats2 pouvait cibler hSocs3 pour une dégradation protéasomale. Lorsque wt Lats2 est surexprimée dans des cellules HEK293T et COS7, la demi-vie de hSocs3, un élément de la ligase Elongine BC-Colline É3 (ligase EBC), diminue significativement, effet que n'a pas la surexpression de Lats2OATP. De plus, la stabilité de hSocs3 dépend de la phosphorylation des résidus sériase aux positions 144/145 par wt Lats2. Bien que les sites de phosphorylation ne soient pas définis pour les deux autres modules de reconnaissance du substrat de la ligase EBC: hWsb 1 et hSocsl, leurs demi-vies diminuent également quand wt Lats2 est surexprimée. Pour les tests in vivo, nous avons synthétisé des esiRNA pour diminuer l'expression du gène endogène lats2, ce qui a entraîné une augmentation d'un facteur 2 de la demi-vie de hSocs3 et de hWsbl dans les cellules HEK293T. En conclusion, nos résultats suggérent que Lats2, une kinase associée au CSN, est un nouveau régulateur de la fonction des ligases EBC, agissant sur le renouvellement des protéines hSocs3, hSocs1 et hWsb1. Ainsi, Lats2 altère la spécificité et la capacité des ligases EBC, régulant par là même la stabilité de nombreuses protéines, ciblées par les ligases EBC pour une dégradation protéasomale. D'autres études devraient révéler si la modification observée de la fonction de la ligase EBC par Lats2, associée au Super-complexe, est également responsable du renouvellement des régulateurs du cycle cellulaire et des changements dans ce même cycle observés lors de la surexpression de Lats2. Summary : The Large tumor suppressor 2 (Lats2) is a human homologue of the Drosophila melanogaster tumor suppressor Warts (Cats) who negatively regulates cell proliferation by altering cell cycle Gl/S and G2/M transition and inducing apoptosis. However, the molecular pathway by which Lats2, a serine-threonine kinase, mediates cell cycle arrest is still unknown. Lats2 was initially identified to be a p53 response gene by our group (Kostic et al., 2000). Subsequently, our group identified interacting candidates of Lats2, including substrate recognition modules of Cullin-based E3 ligases (Socs box or F-box containing proteins) as well as two subunits of the Signalosome (CSN), CSN4 and CSNS. Additionally, Lats2 was shown to associate with a Super-complex, composed of CSN and Cullin-based E3 ligases (Rongere, thesis, 2004; Rongere, unpublished results, 2005) We hypothesized that Lats2 may perform its physiological function through interaction with CSN and Cullin-based E3 ligases. The present work on Lats2 has confirmed that Lats2 is a CSN associated kinase. We defined the domain specific interactions of Lats2 with hSocs3, hWsb1 (Sots box proteins) and hFBX-7 (F box protein), as well as the physiological consequences of interaction with hSocs3, hWsb1 and hSocs1. Both the N-terminal and the kinase domains of Lats2 interact with full-length hSocs3, hWsb1 and hFBX-7, determined in GST pull-down assays suggesting that full-length Lats2 protein is involved in interactions. Refinement of the Lats2 interaction with hSocs3 indicated that the kinase domain of Lats2 interacts with a region of hSocs3 containing a SH2 domain located upstream of the Socs box domain of the hSocs3. Moreover, Lats2 phosphorylated specific regions between the N-terminal and SH2 domain (S l) as well as between the SH2 domain and Socs box domain of hSocs3 (S3).These results indicate that hSocs3 is a novel Lats2 substrate. The kinase assay has also demonstrated that wt Lats2 was able to phosphorylate hSocs3, but not Lats2 kinase dead mutant (deleted ATP pocket, Lats20ATP). Mutational analysis identified two serine residues located at positions 144/145 (S3) to be specifically phosphorylated by wt Lats2. Phosphorylation of proteins has been shown to be a signal for proteolytic degradation of many characterized proteins. Thus we hypothesized that Lats2 could target hSocs3 for proteasomal degradation. When wt Lats2 was over-expressed in HEK293T cells and COST cells, the half-life of hSocs3, as a component of Elongin BC Cullin-based E3 ubiquitin ligase (EBC ligase), decreased significantly. In contrast, aver-expression of the Lats2OATP did not alter the half-life of hSocs3. Furthermore, the stability of hSocs3 depended on phosphorylation of serine residues at positions 144/145 by wt Lats2. Although the sites of phosphorylation were not defined for two other substrate recognition modules of EBC ligasehWsbl and hSocsl, their half-lives also decreased when wt Lats2 was over-expressed. To test in vivo, we synthesized esiRNA to knock-down endogenous Lats2 and subsequently we measured the half-lives of hSocs3 and hVVsb l . Here we demonstrated that the half-lives of hSocs3 and hWsbl were increased by the factor of two in Lats2-depleted HEK293T cells. In conclusion, our findings suggest that Lats2, a CSN associated kinase, is a novel regulator of EBC ligase function by regulating the turn-over of hSocs3, hSocs1 and hWsb1. Thus, Lats2 alters the specificity and capacity of EBC ligases regulating thereby the stability of numerous proteins which are targeted by EBC ligases for proteasomal degradation. Further studies should reveal whether the observed modulation of EBC ligase function by Lats2 associated with a Super-complex is also responsible for the turn-over of cell cycle regulators and the observed alteration in cell cycle by Lats2 over-expression.