|
Report |
Address correspondence to Monique Arpin, Laboratoire de Morphogenèse et Signalisation Cellulaires, UMR144 CNRS, 26 Rue d'Ulm, Institut Curie, Paris, Cedex 05, 75248 France. Tel.: 33-1-4234-6372. Fax: 33-1-4234-6377. email: marpin{at}curie.fr
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: ERM proteins; PIP2; actin cytoskeleton; epithelial cell morphogenesis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
ERM proteins are recruited to the plasma membrane via their NH2-terminal domain (300 residues), which contains both protein and phosphatidylinositol 4,5-bisphosphate (PIP2) binding sites (Algrain et al., 1993; Niggli et al., 1995). They bind to F-actin through the last 34 aa of their COOH-terminal domain (Turunen et al., 1994). In the cytoplasm, ERM proteins are maintained in an inactive conformation through an intramolecular interaction between their NH2-terminal ERM association domain (N-ERMAD) and the last 100 residues of the COOH-terminal ERM association domain (C-ERMAD). This interaction masks membrane and F-actin binding sites (Gary and Bretscher, 1995; Magendantz et al., 1995).
The activation of ERM proteins, resulting in the unmasking of their functional binding sites, occurs through conformational changes triggered by events including the binding to PIP2 and the phosphorylation of a conserved threonine in the actin binding site of the C-ERMAD (T567 in ezrin). ERM proteins phosphorylated at this conserved threonine are localized in membrane extensions rich in actin (Nakamura et al., 1996; Hayashi et al., 1999). Expression of ERM mutants mimicking phosphorylated forms (threonine mutated to aspartic acid, T567D mutation in ezrin) induces the formation of actin-rich membrane projections such as lamellipodia or microvilli (Oshiro et al., 1998; Yonemura et al., 1999; Gautreau et al., 2000). In vitro, the C-ERMAD phosphorylation strongly weakens its interaction with the N-ERMAD (Matsui et al., 1998; Simons et al., 1998). By looking at the crystal structure of a complex between the N- and C-ERMADs of moesin, the threonine residue is seen located at the interface of these two domains (Pearson et al., 2000). Its phosphorylation is predicted to destabilize the N-ERMADC-ERMAD interaction through both steric and electrostatic effects. These observations imply that phosphorylation of this conserved threonine contributes to the conformational activation of ERM proteins. However, although this phosphorylation is required, it is not sufficient in vitro for the association of ERM proteins with F-actin indicating that this phosphorylation event is only one step of the activation process (Nakamura et al., 1999).
The binding to PIP2 has also been proposed to play an essential role in the conformational activation of ERM proteins (Nakamura et al., 1999; Yonemura et al., 2002). A PIP2 binding mutant of ezrin is not recruited to the plasma membrane, suggesting that PIP2 binding is essential for the membrane localization of ERM proteins (Barret et al., 2000). Moreover, in vitro, PIP2 regulates the binding of ERM proteins to the cytoplasmic tail of several transmembrane proteins (Hirao et al., 1996; Heiska et al., 1998) and together with the C-ERMAD threonine phosphorylation, the binding to F-actin (Nakamura et al., 1999). The crystal structure of the N-ERMAD of radixin complexed with the polar headgroup of PIP2 shows a slight change of conformation in contrast to N-ERMAD alone (Hamada et al., 2000). These observations indicate that the binding to PIP2 is an additional step required in the conformational activation of ERM proteins.
To analyze the synergy between these two events in the conformational activation of ezrin in vivo, we made use of the mutations abolishing PIP2 binding alone or in combination with the T567D mutation. We demonstrate that PIP2 binding is the primary requirement in the conformational activation of ezrin followed by the threonine phosphorylation. Moreover, we show that this sequence of events is necessary for the apical targeting of ezrin and for the morphogenesis of epithelial cells.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Unlike T/D ezrin, PIP2- T/D ezrin does not alter epithelial cell morphology
We have shown previously that production of T/D ezrin in epithelial cells induced the formation of lamellipodia, membrane ruffles, tufts of microvilli, and impaired cellcell contacts indicating a critical role for ezrin in epithelial cell morphogenesis (Gautreau et al., 2000; Pujuguet et al., 2003). To test whether ezrin binding to PIP2 is necessary for its role in epithelial cell morphogenesis we analyzed the surface of cells expressing PIP2- and PIP2- T/D ezrin by scanning electron microscopy (Fig. 5). In contrast to cells producing T/D ezrin, we observed that cells producing PIP2- T/D ezrin formed a regular monolayer with surface microvilli resembling those of cells overexpressing wt ezrin (Fig. 5). This suggests that despite the ability of PIP2- T/D ezrin to act as a linker between the membrane and the actin cytoskeleton, its expression does not affect epithelial cell morphology as seen with T/D ezrin. This suggests that the conformational activation unmasking functional binding sites alone is not sufficient to allow ezrin to exert its signaling functions. Therefore, correct regulation of ezrin activation is dependent on the binding of ezrin to PIP2.
|
Recently, genetic analysis of Drosophila moesin, the only ERM protein in this organism, has stressed the role of this protein in the control of epithelial cell integrity and polarity (Polesello et al., 2002; Speck et al., 2003). Our experiments have demonstrated that to exert its cellular functions at the apical pole of epithelial cells, ezrin undergoes a conformational activation, which requires primarily PIP2 binding and the subsequent phosphorylation at T567. Future works using these well-characterized mutants of ezrin should further the understanding of how the regulated conformational activation of ezrin controls its signaling functions in the development and maintenance of epithelial cell polarity.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies
The mouse monoclonal antivesicular stomatitis virus glycoprotein (VSV G) antibody (clone P5D4) was described previously (Kreis, 1986). T567 phospho-ezrin pAb was purchased from Cell Signaling Technology.
DNA constructs and stable transfection
The pCB6 vectors containing cDNA coding for VSV Gtagged ezrin carrying the K253N, K254N, K262N, and K263N mutations (PIP2- ezrin) or the T567D mutation (T/D ezrin) were described previously (Barret et al., 2000; Gautreau et al., 2000). The pCB6 vector containing cDNA coding for VSV Gtagged Nter ezrin (1-309) was described previously (Algrain et al., 1993). The pCB6 vectors containing cDNA coding for wtPH and PIP2- PH ezrin were obtained by an in-frame insertion of a fragment coding for the PH domain of rat phospholipase C-1 (10-139) at the 3' end of the VSV Gezrin cDNA. All constructs were obtained by standard techniques and verified by sequencing. Stable LLC-PK1 clones were obtained as described previously (Gautreau et al., 2000). All experiments were performed with three independent clones for each construct and gave similar results.
Immunofluorescence
105 cells were seeded on 1-cm2 polyester filters (Transwell; Costar Corp.) and grown for 4 d. Cells were fixed with 3% PFA and permeabilized with 0.5% Triton X-100. Cells were subsequently incubated with antiVSV G antibody and then with Alexa 488conjugated goat antimouse secondary antibody (Jackson ImmunoResearch Laboratories) and with TRITC-labeled phalloidin (Sigma Chemical Co.). The samples were analyzed with an SP2 confocal laser scanning microscope (Leica). When indicated, living cells were incubated for 30 min with 10 mM CaCl2 and 30 µM ionomycin (Sigma Chemical Co.) in PBS. Extraction was performed before fixation by treating the cells for 1 min with a Triton X-100 buffer (50 mM MES, 3 mM EGTA, 5 mM MgCl2, 0.5% Triton X-100, pH 6.4) at 20°C.
Scanning electron microscopy
5 x 105 cells were seeded on 4.7 cm2 polycarbonate filters and grown for 4 d. Samples were fixed with 2.5% glutaraldehyde, dehydrated in a graded series of ethanol incubation, dried by the critical point method using liquid CO2, coated with gold palladium, and observed with a microscope (model JSM 840A; JEOL).
Immunoprecipitation
Immunoprecipitations were performed as described previously (Gautreau et al., 2000). When indicated, immunoprecipitated proteins were treated with -phosphatase (New England Biolabs, Inc.) according to the manufacturer's instructions.
Analysis of detergent-soluble and -insoluble fractions
Cellular fractions were obtained from 6-well plates confluent cultures. Total cellular fractions were collected with Laemmli buffer at 100°C. Soluble fractions were prepared by a 1-min extraction with Triton X-100 buffer at 20°C and supplemented with 3x Laemmli buffer. The insoluble fractions were extracted with Laemmli buffer at 100°C. Samples were analyzed by SDS-PAGE and immunoblotting. Densitometric analysis were performed with the Scion image program (NIH image).
Cytosol/membrane fractionation
Confluent cultures from 10-cm dishes were mechanically disrupted using a cell cracker in 10 mM Hepes, 1 mM EDTA, 150 mM NaCl, pH 7.4, buffer containing a cocktail of protease inhibitors. The homogenates were clarified by centrifugation at 600 g. An aliquot of the supernatant was supplemented with 3x Laemmli buffer and corresponded to the total fraction. The supernatant was subjected to a 30-min centrifugation at 100,000 g. The resulting supernatant was supplemented with 3x Laemmli buffer (cytosolic fraction) and the membrane pellets were solubilized in Laemmli buffer (membrane fraction). Samples were analyzed by SDS-PAGE and immunoblotting.
![]() |
Acknowledgments |
---|
This work was supported by grants from Ligue Nationale contre le Cancer (équipe labellisée), Association pour la Recherche contre le Cancer (ARC 5599 and 4601). B.T. Fievet is a recipient of a fellowship from the Ministère de l'Education Nationale, de la Recherche et de la Technologie.
Submitted: 8 July 2003
Accepted: 15 January 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Algrain, M., O. Turunen, A. Vaheri, D. Louvard, and M. Arpin. 1993. Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membranecytoskeletal linker. J. Cell Biol. 120:129139.[Abstract]
Barret, C., C. Roy, P. Montcourrier, P. Mangeat, and V. Niggli. 2000. Mutagenesis of the phosphatidylinositol 4,5-biphosphate (PIP2) binding site in the NH2-terminal domain of ezrin correlates with its altered cellular distribution. J. Cell Biol. 151:10671079.
Bretscher, A., K. Edwards, and R.G. Fehon. 2002. ERM proteins and merlin: integrators at the cell cortex. Nat. Rev. Mol. Cell Biol. 3:586599.[CrossRef][Medline]
Gary, R., and A. Bretscher. 1995. Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol. Biol. Cell. 6:10611075.[Abstract]
Gautreau, A., D. Louvard, and M. Arpin. 2000. Morphogenic effects of ezrin require a phosphorylation-induced transition from oligomers to monomers at the plasma membrane. J. Cell Biol. 150:193203.
Gautreau, A., D. Louvard, and M. Arpin. 2002. ERM proteins and NF2 tumor suppressor: the Yin and Yang of cortical actin organization and cell growth signaling. Curr. Opin. Cell Biol. 14:104109.[CrossRef][Medline]
Hamada, K., T. Shimizu, T. Matsui, S. Tsukita, S. Tsukita, and T. Hakoshima. 2000. Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain. EMBO J. 19:44494462.
Hayashi, K., S. Yonemura, T. Matsui, and S. Tsukita. 1999. Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues. J. Cell Sci. 112:11491158.
Heiska, L., K. Alfthan, M. Grönholm, P. Vilja, A. Vaheri, and O. Carpén. 1998. Association of ezrin with intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2). J. Biol. Chem. 273:2189321900.
Hirao, M., N. Sato, T. Kondo, S. Yonemura, M. Monden, T. Sasaki, Y. Takai, S. Tsukita, and S. Tsukita. 1996. Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and rho-dependent signaling pathway. J. Cell Biol. 135:3751.[Abstract]
Kreis, T.E. 1986. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 5:931941.[Abstract]
Magendantz, M., M.D. Henry, A. Lander, and F. Solomon. 1995. Interdomain interactions of radixin in vitro. J. Biol. Chem. 270:2532425327.
Matsui, T., M. Maeda, Y. Doi, S. Yonemura, M. Amano, K. Kaibuchi, S. Tsukita, and S. Tsukita. 1998. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J. Cell Biol. 140:647657.
Nakamura, F., M.R. Amieva, C. Hirota, Y. Mizuno, and H. Furthmayr. 1996. Phosphorylation of T-558 of moesin detected by site-specific antibodies in RAW264.7 macrophages. Biochem. Biophys. Res. Commun. 226:650656.[CrossRef][Medline]
Nakamura, F., L. Huang, K. Pestonjamasp, E.J. Luna, and H. Furthmayr. 1999. Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and polyphosphatidylinositides. Mol. Biol. Cell. 10:26692685.
Niggli, V., C. Andréoli, C. Roy, and P. Mangeat. 1995. Identification of a phosphatidylinositol-4, 5-biphosphate-binding domain in the N-terminal region of ezrin. FEBS Lett. 376:172176.[CrossRef][Medline]
Oshiro, N., Y. Fukata, and K. Kaibuchi. 1998. Phosphorylation of moesin by Rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol. Chem. 273:3466334666.
Pearson, M.A., D. Reczek, A. Bretscher, and P.A. Karplus. 2000. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell. 101:259270.[Medline]
Polesello, C., I. Delon, P. Valenti, P. Ferrer, and F. Payre. 2002. Dmoesin controls actin-based cell shape and polarity during Drosophila melanogaster oogenesis. Nat. Cell Biol. 4:782789.[CrossRef][Medline]
Pujuguet, P., L. Del Maestro, A. Gautreau, D. Louvard, and M. Arpin. 2003. Ezrin regulates E-cadherin-dependent adherens junction assembly through Rac1 activation. Mol. Biol. Cell. 14:21812191.
Simons, P.C., S.F. Pietromonaco, D. Reczek, A. Bretscher, and L. Elias. 1998. C-terminal threonine phosphorylation activates ERM proteins to link the cell's cortical lipid bilayer to the cytoskeleton. Biochem. Biophys. Res. Commun. 253:561565.[CrossRef][Medline]
Speck, O., S.C. Hughes, N.K. Noren, R.M. Kulikauskas, and R.G. Fehon. 2003. Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity. Nature. 421:8387.[CrossRef][Medline]
Turunen, O., T. Wahlström, and A. Vaheri. 1994. Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J. Cell Biol. 126:14451453.[Abstract]
Várnai, P., and T. Balla. 1998. Visualization of phosphoinositides that bind pleckstrin homolgy domains: calcium- and agonist-induced dynamic changes and relationship to Myo-[3H]inositollabeled phosphoinositide pools. J. Cell Biol. 143:501510.
Yonemura, S., S. Tsukita, and S. Tsukita. 1999. Direct involvement of ezrin/radixin/moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated proteins. J. Cell Biol. 145:14971509.
Yonemura, S., T. Matsui, S. Tsukita, and S. Tsukita. 2002. Rho-dependent and -independent activation mechanisms of ezrin/radixin/moesin proteins: an essential role for polyphosphoinositides in vivo. J. Cell Sci. 115:25692580.