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1.
EMBO J ; 29(14): 2301-14, 2010 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-20551903

RESUMEN

T-cell receptor (TCR) signalling is triggered and tuned at immunological synapses by the generation of signalling complexes that associate into dynamic microclusters. Microcluster movement is necessary to tune TCR signalling, but the molecular mechanism involved remains poorly known. We show here that the membrane-microfilament linker ezrin has an important function in microcluster dynamics and in TCR signalling through its ability to set the microtubule network organization at the immunological synapse. Importantly, ezrin and microtubules are important to down-regulate signalling events leading to Erk1/2 activation. In addition, ezrin is required for appropriate NF-AT activation through p38 MAP kinase. Our data strongly support the notion that ezrin regulates immune synapse architecture and T-cell activation through its interaction with the scaffold protein Dlg1. These results uncover a crucial function for ezrin, Dlg1 and microtubules in the organization of the immune synapse and TCR signal down-regulation. Moreover, they underscore the importance of ezrin and Dlg1 in the regulation of NF-AT activation through p38.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/inmunología , Sinapsis Inmunológicas , Activación de Linfocitos/inmunología , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Linfocitos T , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Homólogo 1 de la Proteína Discs Large , Activación Enzimática , Humanos , Sinapsis Inmunológicas/química , Sinapsis Inmunológicas/metabolismo , Sinapsis Inmunológicas/ultraestructura , Células Jurkat , Proteínas de la Membrana/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Linfocitos T/citología , Linfocitos T/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
2.
Proc Natl Acad Sci U S A ; 108(31): 12915-9, 2011 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-21753079

RESUMEN

The peripheral astrocyte process (PAP) preferentially associates with the synapse. The PAP, which is not found around every synapse, extends to or withdraws from it in an activity-dependent manner. Although the pre- and postsynaptic elements have been described in great molecular detail, relatively little is known about the PAP because of its difficult access for electrophysiology or light microscopy, as they are smaller than microscopic resolution. We investigated possible stimuli and mechanisms of PAP plasticity. Immunocytochemistry on rat brain sections demonstrates that the actin-binding protein ezrin and the metabotropic glutamate receptors (mGluRs) 3 and 5 are compartmentalized to the PAP but not to the GFAP-containing stem process. Further experiments applying ezrin siRNA or dominant-negative ezrin in primary astrocytes indicate that filopodia formation and motility require ezrin in the membrane/cytoskeleton bound (i.e., T567-phosphorylated) form. Glial processes around synapses in situ consistently display this ezrin form. Possible motility stimuli of perisynaptic glial processes were studied in culture, based on their similarity with filopodia. Glutamate and glutamate analogues reveal that rapid (5 min), glutamate-induced filopodia motility is mediated by mGluRs 3 and 5. Ultrastructurally, these mGluR subtypes were also localized in astrocytes in the rat hippocampus, preferentially in their fine PAPs. In vivo, changes in glutamatergic circadian activity in the hamster suprachiasmatic nucleus are accompanied by changes of ezrin immunoreactivity in the suprachiasmatic nucleus, in line with transmitter-induced perisynaptic glial motility. The data suggest that (i) ezrin is required for the structural plasticity of PAPs and (ii) mGluRs can stimulate PAP plasticity.


Asunto(s)
Astrocitos/metabolismo , Proteínas del Citoesqueleto/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapsis/fisiología , Animales , Astrocitos/citología , Astrocitos/ultraestructura , Células Cultivadas , Cricetinae , Proteínas del Citoesqueleto/genética , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Ácido Glutámico/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Mesocricetus , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Plasticidad Neuronal/fisiología , Embarazo , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Seudópodos/fisiología , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5 , Sinapsis/metabolismo
3.
J Biol Chem ; 287(41): 34583-95, 2012 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-22891241

RESUMEN

The amyloid precursor protein (APP) can be cleaved by α-secretases in neural cells to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic P2X7 receptor (P2X7R) triggers sAPPα shedding from neural cells. Here, we demonstrate that the activation of ezrin, radixin, and moesin (ERM) proteins is required for the P2X7R-dependent proteolytic processing of APP leading to sAPPα release. Indeed, the down-regulation of ERM by siRNA blocked the P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggered the phosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R. Using specific pharmacological inhibitors, we established the order in which several enzymes trigger the P2X7R-dependent release of sAPPα. Thus, a Rho kinase and the MAPK modules ERK1/2 and JNK act upstream of ERM, whereas a PI3K activity is triggered downstream. For the first time, this work identifies ERM as major partners in the regulated non-amyloidogenic processing of APP.


Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteolisis , Receptores Purinérgicos P2X7/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Línea Celular Tumoral , Proteínas del Citoesqueleto/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Ratones , Proteínas de Microfilamentos/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Purinérgicos P2X7/genética , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo
4.
EMBO J ; 27(1): 38-50, 2008 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-18046454

RESUMEN

The remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) results in the reorganization of actin cytoskeleton and cellular junctions. We previously showed that the membrane-cytoskeleton linker ezrin plays a major role in HGF-induced morphogenic effects. Here we identified a novel partner of phosphorylated ezrin, the Fes kinase, that acts downstream of ezrin in HGF-mediated cell scattering. We found that Fes interacts directly, through its SH2 domain, with ezrin phosphorylated at tyrosine 477. We show that in epithelial cells, activated Fes localizes either to focal adhesions or cell-cell contacts depending on cell confluency. The recruitment and the activation of Fes to the cell-cell contacts in confluent cells depend on its interaction with ezrin. When this interaction is impaired, Fes remains in focal adhesions and as a consequence the cells show defective spreading and scattering in response to HGF stimulation. Altogether, these results provide a novel mechanism whereby ezrin/Fes interaction at cell-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.


Asunto(s)
Movimiento Celular/fisiología , Proteínas del Citoesqueleto/fisiología , Factor de Crecimiento de Hepatocito/fisiología , Proteínas Proto-Oncogénicas c-fes/metabolismo , Animales , Adhesión Celular/fisiología , Comunicación Celular/fisiología , Uniones Célula-Matriz/fisiología , Activación Enzimática/fisiología , Células LLC-PK1 , Porcinos
5.
BMC Cancer ; 12: 82, 2012 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-22397367

RESUMEN

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.


Asunto(s)
Neoplasias de la Mama/patología , Carcinoma/patología , Proteínas del Citoesqueleto/fisiología , Animales , Western Blotting , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Movimiento Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Femenino , Ratones , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación , Células Tumorales Cultivadas , Tirosina/metabolismo
6.
Curr Opin Cell Biol ; 14(1): 104-9, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11792551

RESUMEN

The ERM (ezrin, radixin and moesin) family of proteins are linkers that tether actin microfilaments to the plasma membrane. Merlin, the NF2 tumor suppressor gene product, is highly homologous to ERM proteins. In ERM proteins and merlin, interdomain binding promotes auto-inhibition and homo-oligomerization or hetero-oligomerization. Recent studies have revealed that ERM proteins transduce growth signals, and have shed new light on how merlin links cell growth to the cytoskeleton.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Microfilamentos/fisiología , Neurofibromina 2/fisiología , Animales , Proteínas Sanguíneas/fisiología , División Celular , Proteínas del Citoesqueleto/fisiología , Proteínas de la Membrana/fisiología , Microvellosidades/ultraestructura , Modelos Biológicos , Neoplasias/etiología , Fosfoproteínas/fisiología , Transducción de Señal
7.
Mol Biol Cell ; 18(12): 4780-93, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17881735

RESUMEN

The mechanisms underlying functional interactions between ERM (ezrin, radixin, moesin) proteins and Rho GTPases are not well understood. Here we characterized the interaction between ezrin and a novel Rho guanine nucleotide exchange factor, PLEKHG6. We show that ezrin recruits PLEKHG6 to the apical pole of epithelial cells where PLEKHG6 induces the formation of microvilli and membrane ruffles. These morphological changes are inhibited by dominant negative forms of RhoG. Indeed, we found that PLEKHG6 activates RhoG and to a much lesser extent Rac1. In addition we show that ezrin forms a complex with PLEKHG6 and RhoG. Furthermore, we detected a ternary complex between ezrin, PLEKHG6, and the RhoG effector ELMO. We demonstrate that PLEKHG6 and ezrin are both required in macropinocytosis. After down-regulation of either PLEKHG6 or ezrin expression, we observed an inhibition of dextran uptake in EGF-stimulated A431 cells. Altogether, our data indicate that ezrin allows the local activation of RhoG at the apical pole of epithelial cells by recruiting upstream and downstream regulators of RhoG and that both PLEKHG6 and ezrin are required for efficient macropinocytosis.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Proteínas del Citoesqueleto/genética , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Proteínas de Unión al GTP rho/genética
8.
Mol Biol Cell ; 18(8): 2935-48, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17538024

RESUMEN

Establishment of polarized cell morphology is a critical factor for migration and requires precise spatial and temporal activation of the Rho GTPases. Here, we describe a novel role of the actin-binding ezrin/radixin/moesin (ERM)-protein ezrin to be involved in recruiting Cdc42, but not Rac1, to lipid raft microdomains, as well as the subsequent activation of this Rho GTPase and the downstream effector p21-activated kinase (PAK)1, as shown by fluorescence lifetime imaging microscopy. The establishment of a leading plasma membrane and the polarized morphology necessary for random migration are also dependent on ERM function and Cdc42 in motile breast carcinoma cells. Mechanistically, we show that the recruitment of the ERM-interacting Rho/Cdc42-specific guanine nucleotide exchange factor Dbl to the plasma membrane and to lipid raft microdomains requires the phosphorylated, active conformer of ezrin, which serves to tether the plasma membrane or its subdomains to the cytoskeleton. Together these data suggest a mechanism whereby precise spatial guanine nucleotide exchange of Cdc42 by Dbl is dependent on functional ERM proteins and is important for directional cell migration.


Asunto(s)
Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Microdominios de Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Línea Celular , Activación Enzimática , Ácido Glutámico/genética , Humanos , Mutación/genética , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteína de Unión al GTP rac1/metabolismo
9.
J Cell Biol ; 164(5): 653-9, 2004 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-14993232

RESUMEN

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.


Asunto(s)
Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositoles/metabolismo , Fosfoproteínas/metabolismo , Animales , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Polaridad Celular , Tamaño de la Célula , Proteínas del Citoesqueleto , Células Epiteliales/citología , Células Epiteliales/metabolismo , Morfogénesis/fisiología , Fosforilación , Mutación Puntual , Unión Proteica , Treonina/metabolismo
10.
Mol Biol Cell ; 17(6): 2661-73, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16540524

RESUMEN

Based on physiological studies, the epithelial brush-border (BB) Na+/H+ antiporter3 (NHE3) seems to associate with the actin cytoskeleton both by binding to and independently of the PDZ domain containing proteins NHERF1 and NHERF2. We now show that NHE3 directly binds ezrin at a site in its C terminus between aa 475-589, which is separate from the PSD95/dlg/zonular occludens-1 (PDZ) interacting domain. This is an area predicted to be alpha-helical, with a positive aa cluster on one side (K516, R520, and R527). Point mutations of these positively charged aa reduced (NHE3 double mutant [R520F, R527F]) or abolished (NHE3 triple mutant [K516Q, R520F, R 527F]) ezrin binding. Functional consequences of these NHE3 point mutants included the following. 1) A marked decrease in surface amount with a greater decrease in NHE3 activity. 2) Decreased surface expression due to decreased rates of exocytosis and plasma membrane delivery of newly synthesized NHE3, with normal total expression levels and slightly reduced endocytosis rates. 3) A longer plasma membrane half-life of mutant NHE3 with normal total half-life. 4) Decreased BB mobile fraction of NHE3 double mutant. These results show that NHE3 binds ezrin directly as well as indirectly and suggest that the former is related to 1) the exocytic trafficking of and plasma membrane delivery of newly synthesized NHE3, which determines the amount of plasma membrane NHE3 and partially determines NHE3 activity, and 2) BB mobility of NHE3, which may increase its delivery from microvilli to the intervillus clefts, perhaps for NHE3-regulated endocytosis.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Aparato Yuxtaglomerular/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Membrana Celular/metabolismo , Secuencia Conservada , Citoplasma/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Transporte de Proteínas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Intercambiadores de Sodio-Hidrógeno/química
11.
Biochim Biophys Acta ; 1773(5): 653-60, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-16904765

RESUMEN

ERM (Ezrin, Radixin, Moesin) proteins are membrane-cytoskeleton linkers that regulate the structure and the function of specific domains of the plasma membrane. ERM proteins are expressed in all metazoan analyzed so far. Genetic analysis of ERM protein functions has recently been performed simultaneously in three different organisms, mouse, Drosophila melanogaster and C. elegans. These studies have revealed a remarkable conservation of the protein functions through evolution. Moreover they have shed light on the crucial role these proteins play in various physiological processes that occur in epithelial cells.


Asunto(s)
Membrana Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/fisiología , Células Epiteliales/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Caenorhabditis elegans/fisiología , Proteínas del Citoesqueleto/genética , Drosophila melanogaster/fisiología , Proteínas de la Membrana/genética , Ratones , Proteínas de Microfilamentos/genética
12.
Mol Biol Cell ; 14(5): 2181-91, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12802084

RESUMEN

Ezrin, a membrane cytoskeleton linker, is involved in cellular functions, including epithelial cell morphogenesis and adhesion. A mutant form of ezrin, ezrin T567D, maintains the protein in an open conformation, which when expressed in Madin-Darby canine kidney cells causes extensive formation of lamellipodia and altered cell-cell contacts at low cell density. Furthermore, these cells do not form tubules when grown in a collagen type I matrix. While measuring the activity of Rho family GTPases, we found that Rac1, but not RhoA or Cdc 42, is activated in ezrin T567D-expressing cells, compared with cells expressing wild-type ezrin. Together with Rac1 activation, we observed an accumulation of E-cadherin in intracellular compartments and a concomitant decrease in the level of E-cadherin present at the plasma membrane. This effect could be reversed with a dominant negative form of Rac1, N17Rac1. We show that after a calcium switch, the delivery of E-cadherin from an internalized pool to the plasma membrane is greatly delayed in ezrin T567D-producing cells. In confluent cells, ezrin T567D production decreases the rate of E-cadherin internalization. Our results identify a new role for ezrin in cell adhesion through the activation of the GTPase Rac1 and the trafficking of E-cadherin to the plasma membrane.


Asunto(s)
Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Fosfoproteínas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Sustitución de Aminoácidos , Animales , Membrana Celular/metabolismo , Proteínas del Citoesqueleto , Perros , Fosfoproteínas/genética , Transporte de Proteínas
13.
Breast Cancer Res ; 7(3): R365-73, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15987432

RESUMEN

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.


Asunto(s)
Movimiento Celular , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Animales/patología , Metástasis de la Neoplasia/fisiopatología , Fosfoproteínas/fisiología , Animales , Proteína Tirosina Quinasa CSK , Proteínas del Citoesqueleto , Neoplasias Pulmonares/fisiopatología , Ratones , Invasividad Neoplásica , Neoplasias Experimentales , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Células Tumorales Cultivadas , Familia-src Quinasas
14.
Mol Biol Cell ; 23(6): 1080-94, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22262457

RESUMEN

The mechanisms that regulate actin filament polymerization resulting in the morphogenesis of the brush border microvilli in epithelial cells remain unknown. Eps8, the prototype of a family of proteins capable of capping and bundling actin filaments, has been shown to bundle the microvillar actin filaments. We report that Eps8L1a, a member of the Eps8 family and a novel ezrin-interacting partner, controls microvillus length through its capping activity. Depletion of Eps8L1a leads to the formation of long microvilli, whereas its overexpression has the opposite effect. We demonstrate that ezrin differentially modulates the actin-capping and -bundling activities of Eps8 and Eps8L1a during microvillus assembly. Coexpression of ezrin with Eps8 promotes the formation of membrane ruffles and tufts of microvilli, whereas expression of ezrin and Eps8L1a induces the clustering of actin-containing structures at the cell surface. These distinct morphological changes are neither observed when a mutant of ezrin defective in its binding to Eps8/Eps8L1a is coexpressed with Eps8 or Eps8L1a nor observed when ezrin is expressed with mutants of Eps8 or Eps8L1a defective in the actin-bundling or -capping activities, respectively. Our data show a synergistic effect of ezrin and Eps8 proteins in the assembly and organization of actin microvillar filaments.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Túbulos Renales Proximales/citología , Microvellosidades/metabolismo , Actinas/metabolismo , Animales , Células Epiteliales/metabolismo , Células LLC-PK1 , Dominios y Motivos de Interacción de Proteínas , Sus scrofa , Porcinos
15.
PLoS One ; 7(5): e37490, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22629406

RESUMEN

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 PPVY(477) present in ezrin's C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY(477) 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.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genética
16.
Cell Adh Migr ; 5(2): 199-206, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21343695

RESUMEN

The highly related ERM (Ezrin, Radixin, Moesin) proteins provide a regulated linkage between the membrane and the underlying actin cytoskeleton. They also provide a platform for the transmission of signals in responses to extracellular cues. Studies in different model organisms and in cultured cells have highlighted the importance of ERM proteins in the generation and maintenance of specific domains of the plasma membrane. A central question is how do ERM proteins coordinate actin filament organization and membrane protein transport/stability with signal transduction pathways to build up complex structures? Through their interaction with numerous partners including membrane proteins, actin cytoskeleton and signaling molecules, ERM proteins have the ability to organize multiprotein complexes in specific cellular compartments. Likewise, ERM proteins participate in diverse functions including cell morphogenesis, endocytosis/exocytosis, adhesion and migration. This review focuses on aspects still poorly understood related to the function of ERM proteins in epithelial cell adhesion and migration.


Asunto(s)
Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Actinas/metabolismo , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Caenorhabditis elegans , Adhesión Celular , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/genética , Drosophila melanogaster , Células Epiteliales/citología , Células Epiteliales/metabolismo , Expresión Génica , Humanos , Uniones Intercelulares/fisiología , Proteínas de la Membrana/genética , Ratones , Proteínas de Microfilamentos/genética , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal
17.
Mol Biol Cell ; 22(3): 375-85, 2011 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-21148287

RESUMEN

In the degradative pathway, the progression of cargos through endosomal compartments involves a series of fusion and maturation events. The HOPS (homotypic fusion and protein sorting) complex is part of the machinery that promotes the progression from early to late endosomes and lysosomes by regulating the exchange of small GTPases. We report that an interaction between subunits of the HOPS complex and the ERM (ezrin, radixin, moesin) proteins is required for the delivery of EGF receptor (EGFR) to lysosomes. Inhibiting either ERM proteins or the HOPS complex leads to the accumulation of the EGFR into early endosomes, delaying its degradation. This impairment in EGFR trafficking observed in cells depleted of ERM proteins is due to a delay in the recruitment of Rab7 on endosomes. As a consequence, the maturation of endosomes is perturbed as reflected by an accumulation of hybrid compartments positive for both early and late endosomal markers. Thus, ERM proteins represent novel regulators of the HOPS complex in the early to late endosomal maturation.


Asunto(s)
Proteínas del Citoesqueleto/fisiología , Endosomas/metabolismo , Receptores ErbB/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Microfilamentos/fisiología , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/genética , Receptores ErbB/análisis , Células HeLa , Humanos , Lisosomas/metabolismo , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/genética , Transporte de Proteínas , Proteínas de Transporte Vesicular/análisis , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7
18.
Blood ; 111(6): 3163-72, 2008 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-18182570

RESUMEN

The model of erythroleukemia caused by Spi-1/PU.1 transgenesis in mice is a multistage disease. A preleukemic step is characterized by an acute proliferation of proerythroblasts due to the arrest of differentiation provoked by Spi-1/PU.1. Later on, a blastic crisis occurs associated with somatic oncogenic mutations in the stem cell factor (SCF) receptor kit. To gain insights into the mechanisms of the leukemic progression, we performed proteomic profiling analyses of proerythroblasts isolated at the 2 stages of the disease. Our results indicate that the level of ezrin, a membrane cytoskeletal crosslinker, is increased in the leukemic cells. We show that Kit oncogenic forms are responsible for ezrin phosphorylation and that phosphorylation rather than overexpression is essential in the leukemic proerythroblasts. Using expression of dominant-negative forms of ezrin, we show that phosphorylation of ezrin on residue Y353 participates in apoptosis resistance, whereas phosphorylation on residue Y145 promotes proliferation of the leukemic cells in vitro and in vivo. Another recurrent oncogenic form of tyrosine kinases (Flt3) most frequently involved in human myeloid leukemia was also able to phosphorylate ezrin. These findings point to a new role for ezrin as signaling player in the development of leukemia, being a downstream effector of oncogenic tyrosine kinases in leukemic blasts.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Animales , Línea Celular , Proliferación Celular , Proteínas del Citoesqueleto/genética , Eritroblastos/citología , Eritroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/patología , Ratones , Ratones Transgénicos , Mutación/genética , Péptidos/genética , Péptidos/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética
20.
Genome Res ; 15(3): 376-84, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15710747

RESUMEN

The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.


Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Animales , Secuencia de Bases , ADN Complementario/genética , Proteínas de Drosophila/química , Biblioteca de Genes , Genes de Insecto , Genes ras , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Especificidad de la Especie , Técnicas del Sistema de Dos Híbridos
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