ERM proteins 和肿瘤转移

www.landesbioscience.com Cell Adhesion & Migration 199 Cell Adhesion & Migration 5:2, 199-206; March/April 2011; © 2011 Landes Bioscience SpeCiAL FoCuS: ACtin-Linked reguLAtory MoLeCuLeS SpeCiAL FoCuS: ACtin-Linked reguLAtory MoLeCuLeS; review Introduction Epithelial cells are characterized by a high degree of asymme- try critical for their function. Cell-cell and cell-matrix adhesion play a key role in the acquisition of structurally and function- ally distinct domains of the plasma membrane and cytoplasm. In particular, junctions between neighboring cells ensure the main- tenance of distinct apical and basolateral compartments, whereas adhesion to the extracellular matrix (ECM) defines the axis of polarity. Cell-cell adhesion is initiated by homotypic interactions between the membrane proteins, cadherins, which induce the reorganization of the actin cytoskeleton and membrane proteins on the cytoplasmic surface of the cells. Likewise, cell adhesion to the ECM via the heterodimeric transmembrane receptors, inte- grins, triggers the assembly of the actin cytoskeleton and regula- tory proteins that altogether form a large multiprotein complex named the adhesome.1 Through their association with the actin cytoskeleton and signaling molecules, adhesion complexes create *Correspondence to: Monique Arpin; Email: marpin@curie.fr Submitted: 10/02/10; Accepted: 02/07/11 DOI: 10.4161/cam.5.2.15081 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. Emerging role for ERM proteins in cell adhesion and migration Monique Arpin,* dafne Chirivino, Alexandra naba† and ingrid Zwaenepoel institut Curie-unité Mixte de recherche 144 (uMr144) Centre national de la recherche Scientifique (CnrS)/Morphogenèse et Signalisation Cellulaires; paris, France †Current address: koch institute for integrative Cancer research; Massachusetts institute of technology; Cambridge, MA uSA Key words: epithelial cells, membrane-cytoskeleton interface, morphogenesis, ERM proteins, cell adhesion elaborate networks that control a variety of cellular processes in normal and pathological conditions including cell morpho- genesis, migration, proliferation, survival and also invasion and metastasis. A family of closely related proteins, the ERM (Ezrin, Radixin, Moesin) proteins, participates in the regulation of these networks through their versatile interaction with membrane proteins, the actin cytoskeleton and signaling molecules. This review highlights the roles of ERM proteins in epithelial cell morphogenesis with particular emphasis on the signaling path- ways underlying their functions in cell adhesion and migration. ERM Proteins Are Conserved through Evolution The highly homologous ERM proteins2 belong to a superfamily whose founding member is band Four point one (4.1). Within this superfamily the members share a common domain, the FERM domain (Four point one, ERM), which in most cases mediates their association with the membrane. The genes encod- ing ERM proteins are found in all sequenced metazoans, but are absent from unicellular organisms such as yeast. Therefore their appearance coincides with the apparition of multicellularity and the generation of intercellular junctions. Whereas vertebrate genomes contain three genes encoding the three paralogs ezrin, radixin and moesin, only one ERM gene is found in the genome of non-vertebrate organisms that have been sequenced. ERM proteins from evolutionarily diverse species all share the same structural organization. They are composed of an amino-termi- nal FERM domain followed by a region rich in α-helices and a carboxy-terminal domain containing the F-actin binding site3 (Fig. 1A). Moreover, ERM proteins are highly conserved in their primary structure throughout evolution with the highest level of identity observed in the FERM domains and the F-actin bind- ing sites. Indeed these domains display ~76% identity between human ezrin and their homologs Dmoesin from Drosophila mela- nogaster or ERM-1 from Caenorhabditis elegans. Regulated Linkage of ERM Proteins to the Membrane and to the Actin Cytoskeleton Biological and biochemical studies have provided important insights into how ERM proteins interact with both membrane components and the actin cytoskeleton. Expressing either full- length ezrin or its N-/C-terminal domains in cells led to the 200 Cell Adhesion & Migration volume 5 issue 2 intramolecular interaction between the N-terminal domain and the C-terminal ~100 amino acids called N-/C-ERMAD (ERM Association Domain). In this closed or inactive conformation the actin and membrane binding sites are masked.5 In addition to biochemical studies, considerable insights into how the structure of ERM proteins contributes to their functions have been provided by structural studies. The X-ray structures of the FERM domains,6-8 the FERM domain of moesin com- plexed with the C-ERMAD (inactive FERM domain conforma- tion),9 and more recently the structure of the full-length moesin from Spodoptera frugiperda (Sfmoesin),10 have been solved. The FERM domain is composed of three subdomains, F1, F2 and F3, that adopt a clover leaf-like arrangement. The structure of the moesin FERM domain complexed to the C-ERMAD revealed that the C-ERMAD adopts an extended structure that covers a large region of domains F2 and F3 masking the F-actin and membrane binding sites.9 Interestingly the structure of the full- length Sfmoesin uncovered an extensive interaction of the cen- tral α-helical region with the FERM domain, indicating that this α-helical region contributes with the C-ERMAD to the masking of a large area of the FERM domain (Fig. 1B). Moreover, it sug- gests an unexpected role for this α-helical region in ERM protein activation. Indeed, any partners of ERM proteins binding to this α-helical region may contribute to conformational changes and therefore to the activation process. Activation through the release of the N-/C-ERMAD interac- tion is therefore required for the unmasking of the membrane and actin binding sites. Numerous studies have indicated that the dissociation of the N-/C-ERMAD interaction is triggered by the binding of the FERM domain to phosphatidyl-inositol-4,5-bi- phosphate (PIP2) and phosphorylation of a conserved threonine residue present in the F-actin binding site (Thr558, Thr564, Thr567 in human moesin, radixin and ezrin respectively). Initial observations showed that the association of ERM proteins with the cytoplasmic tail of the hyaluronan receptor CD44 requires PIP2 in vitro and in vivo suggesting that PIP2 binding induces a change in the conformation that exposes the membrane protein binding sites in the FERM domain.11,12 Comparison of the crys- tal structure of the free radixin FERM domain with that in com- plex with the head group of PIP2, inositol-(1,4,5)-triphosphate (IP3), indicates that structural changes in the FERM domain are associated with IP3 binding. It has been proposed that these changes may contribute to the activation mechanism of ERM proteins.7 The binding of ezrin to PIP2 has also been shown to be a critical step in the activation of ERM proteins in vivo.13 However, the structure of Sfmoesin suggests that the PIP2 bind- ing site is masked in the full-length proteins by the linker region since IP3 is located in a basic cleft between subdomains F1 and F3 in which three lysines and one arginine contact the phosphate groups of IP3,7,10 (Fig. 1B). Therefore how ERM proteins interact with PIP2 at the membrane remains an open question. As indicated above, phosphorylation of the conserved thre- onine residue present in the F-actin binding site has also been implicated in the activation of ERM proteins. Moesin was found phosphorylated at threonine 558 upon platelet activation14 as were ERM proteins following stimulation of Swiss3T3 cells with observation that ezrin interacts with membrane components via its N-terminal domain and with the actin cytoskeleton via its C-terminal domain.4 Moreover, whereas both full-length ezrin and its N-terminal domain concentrate at the plasma mem- brane, the C-terminal domain alone associates with the cortical actin cytoskeleton and actin stress fibers, suggesting that regu- latory mechanisms might exist that restrict the localization of the full-length protein to the cell cortex. Biochemical studies demonstrated that ERM proteins are negatively regulated by an Figure 1. (A) domain organization of human ezrin and Sfmoesin. All erM proteins display similar domain organization. they are composed of a globular n-terminal domain (FerM domain) followed by a domain rich in α-helices, a linker region and the C-terminal domain. the F- actin binding site has been mapped to the last 30 carboxy-terminal aminoacids.3 tyrosines 145 and 477 are phosphorylated by Src kinases downstream growth factor stimulation.28,63 these two tyrosines are not conserved in Sfmoesin. (B) Structure of full-length Spodoptera frugiperda moesin; protein data bank (pdB) identifier 2i1J. the three lobes that composed the FerM domain (F1, F2 and F3) are colored in dark blue, the α-helical domain in turquoise, the linker region in red and the C-terminal domain in yellow. in the closed state, the α-helical region and the C-terminal domain make extensive interactions with the FerM domain that mask the ligand binding sites.10 ip3 binds a basic cleft be- tween domains F1 and F3 of the free FerM domain.7 phosphorylation of threonine 556, highlighted in green, weakens the interaction between the FerM and the C-terminal domains. www.landesbioscience.com Cell Adhesion & Migration 201 without moesin show reduced cortical actin cytoskeleton, lack junctional markers, detach from and migrate out of the epithe- lium suggesting a role of Dmoesin in the stabilization of the apical-junctional domain.29 Similarly, ERM-1 in C. elegans func- tions in apical membrane morphogenesis of epithelial cells.30,31 In adult mammals, the expression of the three ERM proteins is rather tissue-specific with ezrin found primarily in epithelial cells, moesin in endothelial cells and radixin in hepatocytes and hair cells. Inactivation of either ezrin or radixin has provided insights into their function in the particular epithelial cell types where they are expressed. Ezrin knockout mice die soon after birth. Intestinal epithelial cells in these mice present a disorgani- zation of both the terminal web and microvilli and the adhesion complexes appear more elongated and twisted.32 Loss of ezrin leads also to reduction in the apical microvilli of Müller cells and in the microvilli and basal unfoldings of retinal pigment epithe- lium with, as a consequence, a retardation in the development of photoreceptors.33 In gastric epithelial cells, called parietal cells, ezrin is a key regulator of acid secretion that occurs following the fusion of H+, K+ATPase-rich tubulovesicles with the apical mem- brane.34 Ezrin knockdown mice suffer from severe achlorhydria due to a defect in the expansion of apical secretory canaliculi leav- ing the cytoplasm with densely packed tubulovesicles.35 Radixin inactivation causes hyperbilirubinemia likely due to a defect in the localization of Mrp2 (Multidrug resistance protein 2, a protein involved in the secretion of conjugated bilirubin) in the bile canalicular membranes36 and deafness due to progressive degeneration of cochlear stereocilia.37 In addition to these cellular defects in organisms lacking ERM proteins developmental defects are observed in the for- mation of epithelia that line tubular organs. Loss of the single ortholog erm-1 in Caenorhabditis elegans impairs lumen forma- tion of tubular organ epithelia.30,31 Luminal cysts are observed in the epithelia upon depletion of ERM-1 due to incomplete tubulogenesis. Moreover, intestine-specific phenotypes such as constriction or obstruction are observed.30 It was suggested that these phenotypes are not due to a defect in epithelial cell polar- ization, but rather arise from premature arrest in the reposition- ing of the apical junctions to the apico-lateral position during lumen formation resulting in the obstructions observed in the intestine.30 In this latter study, a genetic interaction was found between ERM-1 and the cadherin and catenin homologs HMR-1 and HMP-1, respectively. A possible role for ERM-1 would be to link the cadherin/catenin complex to actin cytoskeleton since the enrichment in F-actin at the apical pole is lost in the intestine of erm-1 (RNAi) embryos. Similarly, deletion of ezrin results in defects in mouse intes- tinal villus morphogenesis. Generation of individual villi occurs during embryonic development when the stratified intestinal epi- thelium is converted into a monolayer of epithelial cells covering the villi. In ezrin-/- mice, villus formation is abnormal. Instead of individual villi observed in wild-type mice, the intestine of ezrin-/- mice displays fused villi. Therefore, the transition from a strati- fied epithelium to a monolayer, which is initiated by a remodeling of the cell-cell contacts, is altered indicating that ezrin plays a regulatory role in the assembly of the junctions.32 lysophosphatidic acid.15 Several kinases were shown to phos- phorylate this conserved threonine in vertebrates ERM proteins including Protein kinase Cα (PKCα, θ), NIK (NFκB-inducing kinase), LOK (Lymphocyte-Oriented Kinase) and MST4 sug- gesting that the phosphorylation of ERM proteins may be induced through different pathways.16-20 This threonine is buried in the N-/C-ERMAD interface and its phosphorylation induces steric and electrostatic hindrance preventing the N-/C-ERMAD association.9,15,21 In vivo, the sequence of events consisting of binding to PIP2 followed by phosphorylation at threonine 567 is critical for the proper activation of ezrin, since impairing one of these steps alters its correct localization and functions in epi- thelial cells.13 Numerous membrane or membrane-associated proteins have been shown to bind the FERM domain when ERM proteins are in an active conformation. ERM proteins interact with the cytoplas- mic domain of several membrane proteins including the hyaluro- nan receptor CD44 which mediates cell adhesion and migration.22 Other membrane proteins such as ion channels, transporters and receptors can bind indirectly through the scaffolding factors NHERF1 (also named EBP50 for ERM-binding phosphopro- tein 50) or NHERF2 (Na+-H+ exchanger regulatory factor).23 These proteins contain two PDZ (Postsynaptic Density Protein) domains known to mediate protein interaction followed by an ERM protein binding site.24 Comparison of the crystal structure of the radixin FERM domain complexed with a CD44 cytoplas- mic peptide25 and of the moesin FERM domain in complex with a peptide from the C-terminal domain of EBP50 indicates that these two ligands bind to distinct sites of the FERM domain.26 So far few proteins have been shown to interact with the C-terminal domain of ERM proteins. The regulatory subunit RII of protein kinase A (PKA) associates with an α-helical region in ERM proteins (amino acids 417–430 in ezrin). Recently, two subunits of the HOPS complex (Homotypic fusion and Protein Sorting) have been shown to interact with the α-helical domains of ERM proteins.27 Both the regulatory subunit RII of protein kinase A (PKA) and the subunits of the HOPS complex interact with full-length ERM protein suggesting that their binding sites are not masked. The structure of Sfmoesin also indicates that the linker region in ERM proteins is likely to be accessible when ERM proteins are in a closed conformation. Specific to ezrin is the pres- ence in this region of tyrosine at position 477 which is phosphory- lated by Src kinase.28 This linker region likely represents a site for the docking of signaling proteins as discussed below. ERM Protein Functions in Epithelial Cell Morphogenesis: Insights from Genetic Analyses Genetic analyses of ERM proteins in different model systems have revealed that they have a crucial role in epithelial cell archi- tecture and in the formation of tubular organ epithelia during development. In all epithelial cell types analyzed a defect in the organization of the actin-rich apical plasma membrane has been observed. In D. melanogaster and C. elegans ERM proteins are essential for viability. In flies, Dmoesin has an important role in epithelial cell integrity and polarity. Imaginal epithelial cells 202 Cell Adhesion & Migration volume 5 issue 2 by antisense oligonucleotides affects cell-cell and cell-matrix adhesion of epithelial MTD-1A cells.44 Likewise, ERM proteins were shown to be required for the formation of focal adhesion complexes and stress fibers downstream of Rho GTPases activa- tion.45 Subsequently, ERM proteins were implicated in pathways regulating the dynamic of cell-cell junctions, the Rho pathways and growth factor signaling. GTPases of the Rho family play important and complex roles in the regulation of cell-cell and cell-matrix adhesion.46 ERM proteins have the potential to act either as effectors of Rho GTPases45 or as regulators of their activity in cell adhesion regu- lation. Thus, the loss of epithelial cell integrity and junctional markers in dmoesin mutants correlates with excess Rho1 activity, the fly RhoA ortholog29,47 suggesting that Dmoesin negatively regulates RhoA activity. In mammalian cells, the GTPase Rac has been shown to have both positive and negative roles in cell- cell junction dynamics. The production of an ezrin mutant mim- icking its constitutive phosphorylation on threonine 567 (ezrin T567D) leads to the formation of lamellipodia in non conflu- ent epithelial cells and to the disruption of cell-cell contacts due to the activation of the small GTPase Rac.48 Not only does the active form of ezrin disrupt the junctions, but it also prevents the proper assembly of cell-cell junctions. Epithelial cells producing this ezrin T567D were neither able to form hollow cysts when grown in a type 1 collagen matrix and remained aggregated nor were they able to organize into tubules, indicating that these cells were not able to establish functional cell-cell contacts.49 Rounds of activation and downregulation of Rac and Rho GTPases have been observed during formation of cell-cell adhesion.50 A consti- tutively active form of ezrin may prevent the downregulation of Rac therefore perturbing the assembly of the junctions. How do ERM proteins activate Rho GTPases? Rac activation may result from the association of ERM proteins with regulators of small GTPases including Rho GDP-dissociation inhibitor (GDI),51 Rho guanine nucleotide exchange factor (GEF),52,53 and Rho GTPase-activating prot