The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells

INTRODUCTION: The membrane cytoskeletal crosslinker ezrin participates in several functions including cell adhesion, motility and cell survival, and there is increasing evidence that it regulates tumour progression. However, the role played by ezrin in breast cancer metastasis has not been clearly delineated. METHODS: We examined the role of ezrin in metastasis using a highly metastatic murine mammary carcinoma cell line, namely AC2M2. Stable cell clones that overexpress wild-type ezrin or a dominant-negative amino-terminal domain of ezrin were selected. They were then tested for cell motility and invasion in vitro, and metastasis in a mouse in vivo tumour transplantation model. RESULTS: Parental AC2M2 cells and cells overexpressing wild-type ezrin were transplanted into the mammary fat pad of syngeneic recipient mice; these animals subsequently developed lung metastases. In contrast, expression of the dominant-negative amino-terminal ezrin domain markedly inhibited lung metastasis. Consistent with this effect, we observed that the expression of amino-terminal ezrin caused strong membrane localization of cadherin, with increased cell–cell contact and a decrease in cell motility and invasion, whereas cells expressing wild-type ezrin exhibited strong cytoplasmic expression of cadherins and pseudopodia extensions. In addition, inhibitors of phosphatidylinositol 3-kinase and c-Src significantly blocked cell motility and invasion of AC2M2 cells expressing wild-type ezrin. We further found that overexpression of amino-terminal ezrin reduced levels of Akt pS473 and cytoskeletal-associated c-Src pY418 in AC2M2 cells, which contrasts with the high levels of phosphorylation of these proteins in cells expressing wild-type ezrin. Phosphorylated Erk1/2 was also reduced in amino-terminal ezrin expressing cells, although a mitogen-activated protein kinase kinase (MEK) inhibitor had no detectable effect on cell motility or invasion in this system. CONCLUSION: Our findings indicate that ezrin is required for breast cancer metastasis, and that c-Src and phosphatidylinositol 3-kinase/Akt are effectors of ezrin in the cell motility and invasion stages of the metastatic process. Together, these results suggest that blocking ezrin function may represent a novel and effective strategy for preventing breast cancer metastasis

Ezrin phosphorylation on tyrosine 477 regulates invasion and metastasis of breast cancer cells

Background The membrane cytoskeletal crosslinker, ezrin, a member of the ERM family of proteins, is frequently over-expressed in human breast cancers, and is required for motility and invasion of epithelial cells. Our group previously showed that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in deregulation of cell-cell contacts and scattering of epithelial cells. In particular, ezrin phosphorylation on Y477 by Src is specific to ezrin within the ERM family, and is required for HGF-induced scattering of epithelial cells. We therefore sought to examine the role of Y477 phosphorylation in ezrin on tumor progression. Methods Using a highly metastatic mouse mammary carcinoma cell line (AC2M2), we tested the effect of over-expressing a non-phosphorylatable form of ezrin (Y477F) on invasive colony growth in 3-dimensional Matrigel cultures, and on local invasion and metastasis in an orthotopic engraftment model. Results AC2M2 cells over-expressing Y477F ezrin exhibited delayed migration in vitro, and cohesive round colonies in 3-dimensional Matrigel cultures, compared to control cells that formed invasive colonies with branching chains of cells and numerous actin-rich protrusions. Moreover, over-expression of Y477F ezrin inhibits local tumor invasion in vivo. Whereas orthotopically injected wild type AC2M2 tumor cells were found to infiltrate into the abdominal wall and visceral organs within three weeks, tumors expressing Y477F ezrin remained circumscribed, with little invasion into the surrounding stroma and abdominal wall. Additionally, Y477F ezrin reduces the number of lung metastatic lesions. Conclusions Our study implicates a role of Y477 ezrin, which is phosphorylated by Src, in regulating local invasion and metastasis of breast carcinoma cells, and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic/predictive marker or treatment target for invasive human breast cancer.Canadian Breast Cancer Research Alliance (BEE, 017374)Canadian Institutes of Health Research (BEE, 102644)Physicians Society Inc.Association pour le développement de la recherche sur le cancer (France

Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin

Ezrin, a membrane–actin cytoskeleton linker, which participates in epithelial cell morphogenesis, is held inactive in the cytoplasm through an intramolecular interaction. Phosphatidylinositol 4,5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (T567) are involved in the activation process that unmasks both membrane and actin binding sites. Here, we demonstrate that ezrin binding to PIP2, through its NH2-terminal domain, is required for T567 phosphorylation and thus for the conformational activation of ezrin in vivo. Furthermore, we found that the T567D mutation mimicking T567 phosphorylation bypasses the need for PIP2 binding for unmasking both membrane and actin binding sites. However, PIP2 binding and T567 phosphorylation are both necessary for the correct apical localization of ezrin and for its role in epithelial cell morphogenesis. These results establish that PIP2 binding and T567 phosphorylation act sequentially to allow ezrin to exert its cellular functions

Ezrin Ubiquitylation by the E3 Ubiquitin Ligase, WWP1, and Consequent Regulation of Hepatocyte Growth Factor Receptor Activity

The membrane cytoskeleton linker ezrin participates in several functions downstream of the receptor Met in response to Hepatocyte Growth Factor (HGF) stimulation. Here we report a novel interaction of ezrin with a HECT E3 ubiquitin ligase, WWP1/Aip5/Tiul1, a potential oncogene that undergoes genomic amplification and overexpression in human breast and prostate cancers. We show that ezrin binds to the WW domains of WWP1 via the consensus motif PPVY477 present in ezrin’s C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY477 motif. Interestingly ezrin ubiquitylation does not target the protein for degradation by the proteasome. We find that ezrin ubiquitylation by WWP1 in epithelial cells leads to the upregulation of Met level in absence of HGF stimulation and increases the response of Met to HGF stimulation as measured by the ability of the cells to heal a wound. Interestingly this effect requires ubiquitylated ezrin since it can be rescued, after depletion of endogenous ezrin, by wild type ezrin but not by a mutant of ezrin that cannot be ubiquitylated. Taken together our data reveal a new role for ezrin in Met receptor stability and activity through its association with the E3 ubiquitin ligase WWP1. Given the role of Met in cell proliferation and tumorigenesis, our results may provide a mechanistic basis for understanding the role of ezrin in tumor progression

Etude des mécanismes moléculaires et cellulaires régulant l'activité des protéines ERM et de la schwannomine

L'EZRINE ET LA SCHWANNOMINE (SCH) RELIENT LE CYTOSQUELETTE D'ACTINE A LA MEMBRANE PLASMIQUE ET SONT IMPLIQUEES DANS LA MORPHOGENESE ET LA SIGNALISATION CELLULAIRES. NOUS AVONS ANALYSE LES MECANISMES QUI CONTROLENT L'ACTIVITE DE CES DEUX PROTEINES APPARENTEES. L'ACTIVITE DE L'EZRINE EST REGULEE PAR DES CHANGEMENTS DE CONFORMATION, ELLE EXISTE SOUS UNE FORME CYTOSOLIQUE INACTIVE DANS UNE CONFORMATION FERMEE. UN MECANISME D'ACTIVATION PERMET D'OUVRIR LA MOLECULE ET D'ATTEINDRE UNE CONFORMATION ACTIVE A LA MEMBRANE. DEUX FACTEURS SONT IMPLIQUES DANS CE MECANISME : LA PHOSPHORYLATION DE LA T567 ET LA LIAISON AU PIP2. NOS RESULTATS DEMONTRENT QUE LA LIAISON AU PIP2 ET LA PHOSPHORYLATION DE LA T567 AGISSENT SEQUENTIELLEMENT DANS LE MECANISME D'ACTIVATION DE L'EZRINE CETTE SEQUENCE D'EVENEMENTS EST NECESSAIRE POUR L'ADRESSAGE SPECIFIQUE AU POLE APICAL DE L'EZRINE ET POUR SON ROLE DANS LA MORPHOGENESE EPITHELIALE. LA SCH EST IMPLIQUEE DANS LA REGULATION DE LA CROISSANCE CELLULAIRE. ELLE EST LE PRODUIT D'UN GENE SUPPRESSEUR DE TUMEUR. DES MUTATIONS DANS CE GENE SONT RESPONSABLES DE LA NEUROFIBROMATOSE DE TYPE 2 (NF2) QUI SE CARACTERISE PAR LA FORMATION DE SCHWANNOMES. COMME L'EZRINE, L'ACTIVITE DE LA SCH EST REGULEE PAR DES CHANGEMENTS DE CONFORMATIONS, NOUS AVONS ANALYSE L'EFFET DE MUTATIONS TROUVEES CHEZ DES PATIENTS ATTEINTS DE NF2 SUR LA CONFORMATION DE LA SCH. NOS RESULTATS MONTRENT QUE CES MUTATIONS, EN AFFECTANT SEVEREMENT LA CONFORMATION DE LA PROTEINE, PROVOQUENT SA RAPIDE DEGRADATION PAR LA VOIE UBIQUITINE/PROTEASOME OU LA FORMATION D'AGGRESOMES. NOS RESULTATS DEMONTRENT QUE CES DEUX VOIES SONT INDEPENDANTES POUR L'ELIMINATION DES PROTEINES MAL REPLIEES.WE ANALYSED THE MECHANISMS THAT CONTROL THE ACTIVITY OF EZRIN AND SCHWANNOMIN. THESE TWO RELATED PROTEINS ACT AS LINKERS BETWEEN THE PLASMA MEMBRANE AND THE ACTIN CYTOSKELETON AND ARE INVOLVED IN EPITHELIAL CELL MORPHOGENESIS AND SIGNAL TRANSDUCTION PATHWAYS. EZRIN ACTIVATION REQUIRES CONFORMATIONAL REARRANGEMENTS THAT ALLOW THESE PROTEINS TO SWITCH FROM A DORMANT FORM IN THE CYTOPLASM TO AN ACTIVE FORM AT THE APICAL MEMBRANE. IN VITRO, TWO FACTORS ARE INVOLVED IN THIS MECHANISM OF ACTIVATION, THE PHOSPHORYLATION OF THE T567 AND THE BINDING TO PHOSPHATIDYLINOSITOL 4-5 BISPHOSPHATE (PIP2). WE DEMONSTRATED THAT PIP2 BINDING IS THE PRIMARY REQUIREMENT IN THE CONFORMATIONAL ACTIVATION OF EZRIN FOLLOWED BY THE THREONINE PHOSPHORYLATION AT THE MEMBRANE. MOREOVER, WE SHOWED THAT THIS SEQUENCE OF EVENTS IS NECESSARY FOR THE APICAL TARGETING OF EZRIN AND FOR THE MORPHOGENESIS OF EPITHELIAL CELLS. SCHWANNOMIN IS INVOLVED IN CELL GROWTH REGULATION. IT IS THE PRODUCT OF A TUMOUR SUPPRESSOR GENE MUTATIONS IN THIS GENE IS RESPONSIBLE FOR THE NEUROFIBOMATOSIS TYPE 2 (NF2) THAT IS CHARACTERISED BY THE PREDISPOSITION TO DEVELOP SCHWANNOMAS. LIKE EZRIN, THE ACTIVITY OF SCHWANNOMIN IS REGULATED BY CONFORMATIONAL CHANGES. WE HAVE ANALYSED THE EFFECTS OF PATHOGENIC MUTATIONS FOUND IN NF2 PATIENTS ON THE CONFORMATION OF SCHWANNOMIN. WE SHOWED THAT MUTATIONS DRASTICALLY AFFECTING THE FOLDING OF SCHWANNOMIN INDUCED ITS DEGRADATION BY THE UBIQUITIN-PROTEASOME PATHWAY. WE ALSO DEMONSTRATED THAT UNFOLDED SCHWANNOMIN EXPOSES AN AGGRESOME DETERMINANT RESPONSIBLE FOR AGGRESOME FORMATION THAT IS INDEPENDENT OF THE UBIQUITIN-PROTEASOME DEGRADATION PATHWAY.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Ezrin role in tyrosine kinase receptors endocytosis and stability

L ezrine appartient à la famille des protéines ERM. Ces protéines assurent la liaison entre la membrane plasmique et le cytosquelette d actine. L ezrine est impliquée dans le trafic de protéines membranaires, cependant sa fonction dans ce processus n est pas connue à ce jour. Des cribles doubles hybrides utilisant l ezrine comme appât ont permis l identification de protéines impliquées dans le transport de protéines membranaires. Nous avons étudié la fonction de l interaction de l ezrine avec la sous unité Vps11 du complex HOPS et WWP1, une ubiquitine ligase de type E3.Le complexe HOPS orchestre la fusion de compartiments intracellulaires car il coordonne les activités des protéines Rab et SNARE. Nous avons étudié comment l interaction de l ezrine avec Vps11 régule le transport du récepteur de l EGF vers la voie de dégradation par les lysosomes. Nous avons montré que cette interaction promeut la maturation des endosomes et qu elle est nécessaire au transport du récepteur à l EGF des endosomes précoces aux tardifs ainsi participant à la régulation de la dégradation du récepteur à l EGF. WWP1 a été impliquée dans la régulation et la dégradation des récepteurs aux facteurs de croissance ainsi que des facteurs de transcription. Nous avons montré que l interaction entre l ezrine et WWP1 induit l ubiquitylation de l ezrine sans toutefois entraîner sa dégradation. Nous avons montré que l interaction de l ezrine avec WWP1 stabilise le récepteur c-Met. Nos résultats indiquent que l ezrine participe au contrôle de la dégradation des récepteurs de type tyrosine kinase en régulant l assemblage de complexes multiprotéiques impliqués dans le transport et l ubiquitylation des protéines.Ezrin is a member of the ERM protein family, which provides a regulated linkage between the plasma membrane and the actin cytoskeleton. Ezrin has been involved in the trafficking of membrane proteins however its function in this process is as of yet unknown. Two-hybrid screens performed with ezrin as baits led to the identification of proteins involved in membrane protein transport. We analyzed the function of ezrin association with two new interactors: the HOPS complex subunit Vps11 and the E3 ubiquitin ligase WWP1.Vps11 is a member of the tethering HOPS complex that coordinates Rab and SNARE functions during vesicular fusion along the endocytic pathway. We have investigated the role of ezrin/Vps11 interaction in the endocytosis of the EGF receptor. We have shown that the interaction between ezrin and the HOPS complex promotes endosome maturation and is necessary for EGF receptor transport from early to late endosomes, therefore participating in the regulation of EGFR endocytosis and degradation.WWP1 is an E3 ubiquitin ligase of the Nedd4 family. A role for WWP1 was identified in the regulation and degradation of growth factors receptors and transcription factors. We have shown that Ezrin/WWP1 interaction results in ezrin ubiquitylation, but is not involved in regulating ezrin degradation. Rather, we were able to show that this ubiquitylation likely mediates protein interaction and that the binding between ezrin and WWP1 promotes c-Met receptor stabilization. Altogether our results indicate that ezrin participates in the control of tyrosine kinase receptor degradation by regulating the assembly of multimeric complexes involved in protein trafficking and ubiquitylation.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Rôle de l'ezrine dans le contrôle de la morphogenèse et de l'adhérence des cellules épithéliales

L ezrine est une protéine de liaison du cytosquelette d'actine à la membrane plasmique. Sa phosphorylation, en réponse à des signaux provenant des facteurs de croissance ou de la matrice extracellulaire, contrôle diverses étapes de la morphogenèse des cellules épithéliales. Ces signaux extracellulaires sont relayés par les kinases cytoplasmiques de la famille Src et nous avons montré que l ezrine est une cible de ces kinases. Au cours de ma thèse, j ai étudié deux nouveaux partenaires de l'ezrine phosphorylée par les kinases de la famille Src : la kinase Fes et les protéines de la famille Eps8. Nous avons montré que la kinase Fes interagit via son domaine SH2 avec la tyrosine 477 phosphorylée de l ezrine. Selon la confluence des cellules épithéliales, la forme activée de Fes se localise soit au niveau des adhérences focales soit au niveau des jonctions adhérentes. La localisation de Fes au niveau des jonctions adhérentes dépend de son interaction avec l ezrine. Ce recrutement est nécessaire, d une part pour l activation de la kinase Fes et d autre part pour la dissociation des cellules en réponse à une stimulation par l HGF (Hepatocyte Growth Factor). Cette étude nous a permis d impliquer l ezrine et la kinase Fes dans la régulation des jonctions cellulaires en aval de la voie de signalisation induite par l HGF. Les protéines de la famille Eps8 régulent le remodelage du cytosquelette d'actine en aval des voies de signalisation induites par les facteurs de croissance. Nos observations suggèrent un rôle pour l interaction ezrine/protéines de la famille Eps8 dans l organisation des filaments d actine du cortex cellulaire. Nos résultats indiquent que l interaction de l ezrine avec différents partenaires permet l organisation structurale et fonctionnelle de domaines spécifiques de la membrane plasmique des cellules épithéliales.Ezrin is a membrane-actin cytoskeleton linker that plays an essential role in epithelial cell morphogenesis. It participates to the transmission of signals that derive from either outside or inside the cells. In response to growth factors or to extracellular matrix signals, ezrin is phosphorylated by various kinases including Src kinases. We performed a twohybrid screen with ezrin phosphorylated by the Src family kinases to identify the downstream pathways activated through this phosphorylation. We analyzed two interacting partners of ezrin phosphorylated by Src: the Fes kinase and the proteins of the Eps8 family. We showed that the Fes kinase interacts, via its SH2 domain, with ezrin phosphorylated at tyrosine 477. In confluent epithelial cells, this interaction is necessary for the recruitment and the activation of the Fes kinase to the adherens junctions. If this interaction is impaired, the Fes kinase localizes to focal adhesions. As a consequence, the cells show delay in their spreading and they do no longer scatter in response to HGF treatment. Altogether, this study provides a novel mechanism whereby ezrin/Fes interaction at cell contacts plays an essential role in HGF-induced cell scattering and implicates Fes in the cross-talk between cell-cell and cell-matrix adhesion. The proteins that belong to the Eps8 family regulate the actin cytoskeleton remodeling in response to growth factor stimulations. Our observations suggest a possible role for the ezrin/Eps8 interaction in the organization of the cortical actin cytoskeleton. Altogether, our results indicate that the interaction of phosphorylated ezrin with its partners is required for the structural and functional organization of specific domains of the plasma membrane in epithelial cells.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF