Morphogenic Effects of Ezrin Require a Phosphorylation-induced Transit
http://www.100md.com
《细胞学杂志》
a Laboratoire de Morphogénèse et Signalisation Cellulaires, UMR 144 CNRS/Institut Curie, 75248 Paris Cedex 05, France
Correspondence to: Monique Arpin, Laboratoire de Morphogénèse et Signalisation Cellulaires, UMR 144 CNRS/Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France. Tel:33 1 42 34 63 72 Fax:33 1 42 34 63 77 E-mail:marpin@curie.fr.
Abstract
ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH2- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.
Key Words: ERM, head-to-tail interaction, conformation, actin, cytoskeleton
Introduction
ERM (ezrin, radixin, moesin)1 proteins act as linkers between the plasma membrane and the actin cytoskeleton. Inactivation studies indicated that these proteins play a role in the formation of microvilli, cell–cell junctions, and membrane ruffles, and also regulate substrate adhesion and motility (Takeuchi et al. 1994 ; Crepaldi et al. 1997 ; Lamb et al. 1997 ). Regulation of ERM linker function is thought to occur through conformational changes (Bretscher 1999 ).
ERM proteins possess two conserved domains. The NH2-terminal domain is responsible for membrane targeting, whereas the COOH-terminal domain contains an F-actin binding site (Algrain et al. 1993 ; Turunen et al. 1994 ). These two domains interact strongly with each other, and have been termed N- and C-ERMADs, standing for ERM association domain (Gary and Bretscher 1995 ; Magendantz et al. 1995 ). In ezrin, N-ERMAD has been mapped to the first 296 amino acids and C-ERMAD to the last 107 amino acids. Because of the intramolecular N/C-ERMAD interaction, most ERM proteins are in a cytosolic dormant form, in which binding sites for membrane components and F-actin are masked. In the NH2-terminal domain, cryptic binding sites have been identified for Rho-GDI and EBP-50 proteins (Reczek et al. 1997 ; Takahashi et al. 1997 ; Reczek and Bretscher 1998 ). Recently, the crystal structure of the moesin N-ERMAD bound to the C-ERMAD revealed a globular conformation for the N-ERMAD domain and an extended conformation for the C-ERMAD, which mutually mask binding sites (Pearson et al. 2000 ).
Intermolecular N/C-ERMAD interactions also form ERM oligomers. In purified placental microvilli, ezrin dimers, trimers, tetramers, and higher order oligomers were identified, suggesting a head-to-tail assembly (Berryman et al. 1995 ). These oligomers are proposed to be associated with the cytoskeleton and to be involved in microvillar morphogenesis. However, soluble ezrin dimers were also detected (Bretscher et al. 1995 ). Oligomerization is not specific to ezrin, since mixed oligomers containing different ERM members were observed (Gary and Bretscher 1993 ; Andreoli et al. 1994 ). To engage ERMADs in intermolecular interactions, cytosolic dormant monomers are thought to be subjected to a gross conformational change. This phenomenon does not occur spontaneously in vitro with purified ezrin, and probably requires an activation step (Bretscher et al. 1995 ).
Phosphorylation has been proposed to regulate ERM activation, since phosphorylation of ERM proteins correlates with their cytoskeletal association (Chen et al. 1994 ; Kondo et al. 1998 ; Simons et al. 1998 ). Ezrin is phosphorylated on tyrosine residues upon growth factor stimulation (Gould et al. 1989 ; Fazioli et al. 1993 ; Crepaldi et al. 1997 ). In response to EGF, ezrin phosphorylation on tyrosines 145 and 353 is concomitant with an increase in dimer formation, suggesting a causal relationship between phosphorylation and oligomerization (Krieg and Hunter 1992 ; Berryman et al. 1995 ). However, mutations of these tyrosines into phenylalanines does not alter ezrin localization in microvilli, and production of this mutated ezrin does not affect cell morphology (Crepaldi et al. 1997 ). Rather than controlling its cytoskeletal association, tyrosine phosphorylation of ezrin appears to transduce signals. For example, phosphorylation of tyrosine 353 was found to signal cell survival during epithelial differentiation (Gautreau et al. 1999 ).
Another phosphorylation site is a better candidate to activate ERM cytoskeletal linkage. A phosphothreonine residue, originally identified in moesin (Nakamura et al. 1995 ), is localized in a conserved COOH-terminal region of ERM proteins (T567 in ezrin, T564 in radixin, and T558 in moesin). Using phosphospecific antibodies, this phosphorylated residue was detected in ezrin, radixin, and moesin from a variety of cells and tissues, and phosphorylated ERM proteins were shown to be present in actin-rich membrane structures (Nakamura et al. 1996 ; Matsui et al. 1998 ; Oshiro et al. 1998 ; Hayashi et al. 1999 ). Two kinases, protein kinase C-theta (PKC-) and Rho-kinase, and two phosphatases, myosin phosphatase and type 2C protein phosphatase (PP2C), were found in different systems to regulate the phosphorylation status of this conserved threonine in ERM proteins (Fukata et al. 1998 ; Matsui et al. 1998 ; Pietromonaco et al. 1998 ; Hishiya et al. 1999 ).
The primary consequence of this phosphorylation event is to impair N/C-ERMAD interaction. In an overlay assay, phosphorylation of T564 in radixin COOH-terminal domain impairs its association with the NH2-terminal domain (Matsui et al. 1998 ). Similarly, the T558D mutation of moesin, which mimics the phosphorylated state, was shown to affect the N/C-ERMAD interaction (Huang et al. 1999 ). From the crystal structure, it appears that the phosphorylation of moesin T558 weakens the N/C-ERMAD interaction due to both electrostatic and steric effects (Pearson et al. 2000 ). The phosphorylation of an isolated COOH-terminal fragment of ERM proteins does not affect its association with F-actin (Matsui et al. 1998 ; Huang et al. 1999 ). However, in full-length ERM proteins, phosphorylation of this conserved threonine is required to bind to F-actin (Simons et al. 1998 ; Hishiya et al. 1999 ; Nakamura et al. 1999 ). These results suggest that phosphorylation of this residue activates ERM cytoskeletal association by unmasking the cryptic F-actin binding site. Furthermore, expression of T into D mutant forms of ezrin or moesin potentiates the formation of microvilli-like dorsal projections by growth factors (Oshiro et al. 1998 ; Yonemura et al. 1999 ), whereas transfection of the nonphosphorylatable T558A moesin inhibits RhoA-induced formation of these structures (Oshiro et al. 1998 ; Shaw et al. 1998 ).
Although phosphorylation of this conserved threonine residue regulates the activation of ERM cytoskeletal linkers, the mechanism of this regulation is still poorly understood. By disrupting N/C-ERMAD interaction, this phosphorylation event could trigger the opening of dormant monomers, could impair oligomerization, or both. To clarify the mechanism of ERM conformational regulation, we analyzed the role of ezrin T567 phosphorylation in LLC-PK1 epithelial cells. We found that T567D ezrin exhibited a drastic reduction in the amount of oligomers at the plasma membrane. Monomeric T567D ezrin was associated with the actin cytoskeleton and induced actin-rich membrane projections. Production of T567D ezrin strongly affected epithelial morphology and differentiation. In contrast, T567A ezrin exhibited a level of membrane oligomers similar to wild-type (wt), but was poorly associated with the actin cytoskeleton. These results suggest that phosphorylation of this conserved threonine regulates a membrane-specific transition from oligomers to monomers, which are active plasma membrane–actin cytoskeleton linkers.
Materials and Methods
Cells and Recombinant Proteins
LLC-PK1 cells (CCL 101; American Type Culture Collection) were cultured in DME containing 10% FCS and maintained at 37°C in 10% CO2. Recombinant NH2-terminal fragment 1–309 of ezrin was produced and purified as a GST fusion as previously described (Gautreau et al. 1999 ). GST moiety was cleaved off by thrombin digestion. Recombinant NH2-terminal fragment was biotinylated with NHS-LC-biotin (Pierce Chemical Co.) according to the manufacturer's instructions.
cDNA Constructs and Transfection
To substitute T567 with A567 or D567, PCR reactions were performed with oligonucleotides in which the codon ACG was replaced by GCG or GAC, respectively. The amplified fragments were subcloned into the pCB6 vector containing VSV G-tagged ezrin cDNA (Algrain et al. 1993 ). Myc-tagged ezrin was cloned in pCDNA 3.1 vector (Invitrogen). All PCR fragments were verified by sequencing.
For transfection, trypsinized cells were resuspended at a concentration of 2.5 x 107 cells/ml in 15 mM Hepes, pH 7.4, buffered medium. 200 μl of cell suspension was added to 50 μl of a solution containing 210 mM NaCl, 5 μg of plasmid DNA, and 30 μg of salmon sperm DNA carrier (Sigma-Aldrich). LLC-PK1 cells were electroporated with a BioRad Gene Pulser at 950 μF and 240 V using 4-mm width cuvettes. Transiently transfected cells were analyzed after 48 h of cDNA expression. Clones producing T567A and T567D ezrin were established as previously described, and were compared with the previously obtained clones transfected with the empty plasmid or producing wt ezrin (Crepaldi et al. 1997 ).
Cytosol/Membrane Fractionation and Gel Filtration Analysis
Cells from a confluent 10-cm dish (for standard immunoprecipitation), or from ten confluent 10-cm dishes (for the Coomassie blue-stained immunoprecipitation experiment or gel filtration analysis), were rinsed once with cold PBS, once with cold cyt buffer (10 mM Hepes, 1 mM EDTA, 150 mM NaCl, pH 7.4), and scraped off with a rubber policeman in 1 ml of cold cyt buffer supplemented with protease inhibitors (200 μg/ml pefabloc, 15 μg/ml benzamidine, 1 μg/ml pepstatin, 1 μg/ml antipain). Cells in suspension were mechanically disrupted by 10 strokes of a cell cracker. Debris and nuclei were pelleted by a 10-min centrifugation at 600 g at 4°C. The supernatant was then subjected to a 20-min centrifugation at 100,000 g using a TLA-120.2 rotor in an optima TLX ultracentrifuge (Beckman Coulter). This ultracentrifugation pellets crude membranes, whereas the supernatant is the cytosolic fraction.
For gel filtration analysis, membrane pellets were further extracted by a 15-min incubation in 200 μl of mbn buffer (10 mM Hepes, 1 mM EDTA, 600 mM KCl, 1% Triton X-100, pH 7.4) supplemented with protease inhibitors, and then ultracentrifuged again. 200 μl of cytosol or membrane extracts were loaded onto a superose-6 HR10/30 gel filtration column (Amersham Pharmacia Biotech), preequilibrated with cyt or mbn buffer respectively, and run at a flow rate of 300 μl/min. 250 μl fractions were collected. The column was calibrated with thyroglobulin, ferritin, aldolase, and BSA (Sigma-Aldrich) as standards. Thyroglobulin (Stokes radius of 85 ?), ferritin (61 ?), aldolase (48 ?), and BSA (35.5 ?) peaked at fractions 24, 32, 41, and 43, respectively. Void volume of the column emerged in fraction 3.
When endogenous ERM phosphorylation was examined, cyt and mbn buffers were also supplemented with 2 mM of sodium pyrophosphate and 1 μM of calyculinA (Upstate Biotechnology). When indicated, LLC-PK1 cells were pretreated for 10 min at 37°C with 300 nM of calyculinA. For oligomer and monomer samples of Fig 2, 150 μl of fractions 24–27 and 150 μl of fractions 35–38 were pooled and precipitated by 2 min at 100°C, followed by 10 min at 4°C. For efficient precipitation of the oligomeric fraction, 15 μg of BSA was added before boiling, because this fraction contained a low amount of proteins. The precipitates were pelleted by a 10-min centrifugation at 20,000 g at 4°C, and resuspended in SDS-loading buffer.
Figure 1. T567D ezrin has a reduced N-ERMAD binding activity in vitro, and does not oligomerize with moesin at the membrane in vivo. Throughout this study, clones producing VSV G-tagged wt ezrin, T567A ezrin, T567D ezrin (E, A, and D cells, respectively) or clones obtained after transfection of the empty plasmid (P cells) were compared. (A) Denatured extracts from P, E, A, and D clones were used to immunoprecipitate ezrin or VSV G-tagged wt, T567A, and T567D ezrin. The immunoprecipitates were probed with ezrin antibodies or with biotinylated ezrin N-ERMAD (1–309). (B) Ezrin from P, E, A, or D cell membrane extracts was immunoprecipitated with either anti-ezrin antibodies or anti-VSV G antibodies as indicated. After SDS-PAGE, the immunoprecipitates were stained by Coomassie blue to reveal ezrin (top), or immunoblotted with moesin-specific antibodies (bottom). Moesin associated with ezrin can be seen in the Coomassie blue–stained gel as a faint band just below the strong ezrin band.
Figure 2. T567D ezrin and endogenous phosphorylated ERM proteins are preferentially monomeric at the plasma membrane. A, Membrane or cytosolic extracts from P, E, A, and D cells were resolved by gel filtration chromatography on a superose-6 column. Fractions 15–41 were analyzed by SDS-PAGE and immunoblotted with antiezrin antibodies (P) or anti-VSV G antibodies (E, A, and D). T567D ezrin exhibited a strongly reduced amount of oligomers at the membrane, but not in the cytosol. B, Total, cytosolic, and membrane extracts were immunoblotted with 297S mAb, recognizing all three ERM proteins when phosphorylated on this conserved threonine (pERM), or with a mixture of antibodies specific for ERM. Ezrin and radixin comigrated at 80 kD, and moesin migrated at 75 kD. Phosphorylated ERM proteins were strongly enriched in the membrane fraction. C, LLC-PK1 cells were pretreated with calyculinA, a protein phosphatase inhibitor, and the membrane extract was resolved by gel filtration chromatography. Oligomeric and monomeric fractions were pooled, and immunoblotted as in B. Monomers were preferentially phosphorylated over oligomers.
Immunoprecipitations
Cytosol or total extracts were adjusted to 1 ml of RIPA buffer (50 mM Hepes, 10 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4). Membrane pellets were resuspended in RIPA buffer. RIPA extracts were supplemented with protease inhibitors. For the experiment in which denatured extracts are used, cells were lysed in 1 ml of 50 mM Hepes, 10 mM EDTA, 150 mM NaCl, 1% NP-40, pH 7.4, buffer. The extracts were adjusted to 1% SDS and boiled for 2 min. Denatured extracts were then put on ice for 10 min and diluted to 10 ml with a cold buffer reconstituting RIPA composition.
Extracts were clarified by 10 min centrifugation at 20,000 g at 4°C, and incubated with 10 μl of protein A–Sepharose fast-flow beads (Amersham Pharmacia Biotech) and 5 μg of affinity-purified ezrin or VSV G rabbit polyclonal antibodies for 2 h (1 ml vol) or overnight (10 ml vol). Beads were washed five times with 1 ml of RIPA buffer, and boiled 2 min in SDS loading buffer.
Overlay and Western Blotting
All blots were performed on nitrocellulose membranes (Protran Hybond). P5D4 anti-VSV G mAb, 9E10 anti-myc mAb, phosphospecific 297S mAb (a kind gift of Dr. S. Tsukita, Kyoto University, Japan), anti-moesin affinity-purified mouse polyclonal antibodies, anti-radixin affinity-purified guinea pig polyclonal antibodies (both kind gifts of Dr. P. Mangeat, University of Montpellier, France), anti-ezrin affinity-purified rabbit polyclonal antibodies (Algrain et al. 1993 ), or biotinylated NH2-terminal domain of ezrin were used as primary reagents, and alkaline phosphatase-coupled immunoglobulins or streptavidin, as secondary reagents (Promega). Blots were developed with nitroblue tetrazolium/5-bromo, 4-chloro, 3-indolyl phosphate as substrates (Promega). The intensities of bands were quantitated by scanning densitometry on a Bio-Profil station (Vilbert-Lourmat).
Immunofluorescence and Microscopy
Cell morphology was examined by phase-contrast optics or by Nomarski optics on a Leica microscope. For scanning EM, cells grown at confluence on 10-mm 0.2 μm anopore membrane filters (Nunc) were dehydrated in graded ethanol baths, dried by the critical point method using liquid CO2, coated with gold palladium, and observed with a JEOL microscope (JSM 840A).
Immunolocalization of exogenous ezrin was achieved with VSV G affinity-purified polyclonal antibodies at 10 μg/ml and Cy-2–conjugated goat anti–rabbit secondary antibodies (Jackson ImmunoResearch Laboratories) on fixed and permeabilized cells using the paraformaldehyde/Triton X-100 protocol previously described (Crepaldi et al. 1997 ). Samples were examined with a Leica confocal laser-scanning microscope, using the same settings for all acquisitions.
Analysis of Cytoskeletal Fraction
Confluent cultures from 6 well plates were rinsed quickly with PBS at room temperature. The soluble fraction was extracted by a 1-min incubation with 500 μl of a Triton X-100 buffer that preserves cytoskeleton-associated material (csk buffer: 50 mM MES, 3 mM EGTA, 5 mM MgCl2, 0.5% Triton X-100, pH 6.4) at room temperature. For immunofluorescence analysis, the insoluble material was immediately fixed in 3% paraformaldehyde. We verified that actin and microtubule cytoskeletons were not affected by this treatment. For immunoblot analysis, the insoluble material was quickly rinsed with 500 μl of csk buffer, and further extracted by a 1-min incubation with 500 μl of ice-cold RIPA buffer. When endogenous ERM phosphorylation was examined, csk and RIPA buffers were also supplemented with 2 mM of sodium pyrophosphate and 1 μM of calyculinA.
Development of Multicellular Epithelial Structures
For the suspension cyst assay, 25-ml siliconized erlenmeyers containing 5 ml of a cell suspension at 106 cells/ml in 15 mM Hepes, pH 7.4, buffered medium were rocked in a shaking incubator at 75 rpm (ROSI 1000; Thermolyne). Cultures were analyzed after 48 h. For the tubulogenesis assay, cells were seeded in a collagen type I gel, as previously described (Gautreau et al. 1999 ). 1-wk cultures, in DMEM containing 10% FCS and 100 U/ml of HGF, were examined. Importantly, in these two assays, all clones isolated in each category behaved similarly.
Results
T567D Ezrin Exhibits a Low Amount of Oligomers at the Membrane
We chose to study ezrin function in LLC-PK1 epithelial cells, which are derived from kidney proximal tubule. This cell line retains many features of proximal tubule cells, since it harbors numerous microvilli at its apical surface, and coexpresses ERM proteins (Berryman et al. 1993 ). To analyze the role of ezrin T567 phosphorylation, cDNAs encoding a nonphosphorylatable variant, T567A ezrin, and a pseudophosphorylated variant, T567D ezrin, were expressed in LLC-PK1 cells. We derived stably transfected clonal cell lines producing wt, T567A, or T567D ezrin, called E, A, or D cells, respectively, and we compared them to control clones transfected with the empty plasmid, called P cells. The exogenous proteins were tagged at the COOH terminus with a VSV G epitope so they could be readily distinguished from the endogenous ezrin. The amount of exogenous ezrin relative to endogenous ezrin was estimated to be about tenfold higher in the isolated clones, by immunoblotting serially diluted extracts with the antiezrin antibody (data not shown).
To assess if the T567D mutation of ezrin impairs the N/C-ERMAD interaction, we analyzed wt, T567A, and T567D ezrin for their binding to biotinylated N-ERMAD in a blot overlay assay. We immunoprecipitated endogenous ezrin or VSV G-tagged ezrin variants from denatured lysates derived from P, E, A, and D cells. This allowed the isolation of ezrin without associated proteins. An equivalent amount of ezrin and VSV G-tagged wt, T567A, and T567D ezrin was immunoprecipitated as revealed by ezrin immunoblotting (Fig 1 A). The endogenous ezrin and VSV G-tagged wt ezrin bound strongly to the N-ERMAD, indicating that the VSV G tag has little influence, if any, on the N/C-ERMAD interaction. In contrast to wt and T567A ezrin, which bind efficiently to the ezrin N-ERMAD, the ability of T567D ezrin to interact with N-ERMAD was substantially diminished. This result suggests that the T567D mutation of ezrin mimics the phosphorylation of the C-ERMAD, at least in its disruptive effect on the N/C-ERMAD interaction (Matsui et al. 1998 ; Huang et al. 1999 ; Pearson et al. 2000 ).
In our current view of ERM activation, a discrepancy remains unresolved. On one hand, the active ERM protein, which is bound to the plasma membrane and the actin cytoskeleton, is expected to be oligomeric (Bretscher 1999 ). On the other hand, an ERM protein requires the phosphorylation of this conserved threonine of the COOH-terminal domain to bind to F-actin (Nakamura et al. 1999 ). However, phosphorylation might disrupt the oligomeric form, through its impairment of intermolecular N/C-ERMAD interaction. To examine ERM oligomerization, we looked at whether the ezrin variants were associated with endogenous moesin. We immunoprecipitated ezrin from the membrane pool to enrich for the active form. The immunoprecipitates were either stained with Coomassie blue or immunoblotted with moesin-specific antibodies (Fig 1 B). A high amount of ezrin was precipitated with anti-ezrin or anti-VSV G antibodies, as seen by Coomassie blue staining. Moesin coprecipitated with endogenous ezrin, exogenous wt, and T567A ezrin, but not with T567D ezrin. This lack of hetero-oligomerization of T567D ezrin with moesin in the membrane fraction suggests that the T567D mutation of ezrin impairs intermolecular N/C-ERMAD interactions. An association with radixin could not be determined, because the immunoprecipitated ezrin gave a high background at the position of radixin, which migrates only slightly faster than ezrin in SDS-PAGE.
To examine directly the oligomeric status of T567 mutant forms of ezrin, we used a procedure that resolves ezrin oligomers from monomers (Berryman et al. 1995 ). First, we examined the distribution of endogenous ezrin in P cells. Ninefold less ezrin is found in the membrane fraction compared with the cytosolic fraction (Table 1). Membrane and cytosolic extracts from the P cells were applied to a superose-6 gel filtration column. Oligomers were detected in the cytosol, as well as in the membrane fraction (Fig 2 A). Even if most oligomers were cytosolic (Table 1), the relative level of oligomers over the total ezrin at the membrane was about twofold that in the cytosol (28% of oligomers at the membrane vs. 15% of oligomers in the cytosol). Then, we examined the oligomer profile for the ezrin variants (Fig 2 A). In the membrane extracts, ezrin oligomers were observed with wt and T567A ezrin. However, consistent with its lack of association with moesin, T567D ezrin from the membrane fraction was eluted essentially as monomers, with only a trace amount of oligomers. Interestingly, in the cytosol, no differences between ezrin variants were noted. Thus, T567D ezrin exhibited a strong reduction in the amount of oligomers exclusively at the membrane.
Table 1. Quantification of the Ezrin Content in the Different Pools
Phosphorylated ERM Proteins Are Preferentially Monomeric at the Membrane
The analysis of T567D ezrin indicated that the effect of phosphorylation occurs at the membrane. To get insight into the distribution of phosphorylated endogenous ERM proteins from LLC-PK1 cells, we used 297S, an mAb recognizing this conserved phosphorylated threonine of ERM COOH-terminal domain (Matsui et al. 1998 ). We examined the distribution of phosphorylated ERM proteins between the membrane and the cytosol. When a similar amount of ERM proteins from total, cytosol, and membrane fractions was blotted with 297S, phosphorylated ERM proteins appeared highly enriched in the membrane fraction (Fig 2 B). This result is consistent with the membrane-dependent effect of the ezrin T567D mutation.
Because the T567D mutation reduced the amount of ezrin oligomers at the membrane, we hypothesized that the phosphorylation of this threonine dissociates ezrin oligomers into monomers. If this hypothesis is correct, monomers should be more phosphorylated than oligomers. Our initial attempts to determine which of the monomers or the oligomers were preferentially phosphorylated failed because of dephosphorylation during the gel filtration procedure. To overcome this problem, we pretreated LLC-PK1 cells with calyculinA, a serine/threonine protein phosphatase inhibitor known to affect moesin phosphorylation (Nakamura et al. 1995 ). This treatment enhanced the phosphorylation signal and preserved it during gel filtration. Consistent with the preferential distribution of T567D ezrin in the monomeric fraction, ERM monomers were found to be preferentially phosphorylated over oligomers (Fig 2 C).
Membrane/Cytosol Distribution of T567D Ezrin Is Regulated through its Functional C-ERMAD Domain
These results suggest that phosphorylation of ezrin occurs at the membrane, and dissociates oligomers, through an impairment of intermolecular N/C-ERMAD interaction at the membrane. However, we were intrigued by the fact that T567D ezrin was not defective in cytosolic oligomerization. This result is compatible with the hypothesis that the C-ERMAD of T567D ezrin is functional for oligomerization in the cytosol, but not at the membrane. To confirm in vivo that T567D ezrin has a functional C-ERMAD in the cytosol, we compared T567D ezrin to 29 ezrin, a form in which the 29 COOH-terminal amino acids were eliminated. Such a deletion completely abrogates C-ERMAD activity, i.e., N-ERMAD binding (Gary and Bretscher 1995 ; data not shown). We devised a sensitive assay to study specifically ezrin homo-oligomerization. We transiently cotransfected LLC-PK1 cells with two ezrin cDNAs, one of them being tagged by the VSV G epitope, the other by the myc epitope, and examined the amount of ezrin–myc coprecipitating with ezrin–VSV G.
We analyzed the distribution of produced proteins between the membrane and cytosol pools (Fig 3). Half of the transfected cell sample was analyzed directly (total), and the other half was separated in cytosolic and membrane fraction before analysis, so that the three pools were comparable. In total lysates, similar amounts of wt, T567A, T567D, and 29 ezrin were detected. In all transfections, a similar amount of ezrin–myc was produced. Analysis of cytosolic and membrane fractions revealed that wt, T567A, and T567D ezrin were similarly distributed between cytosol and membrane, most of ezrin being in the cytosol. In contrast, 29 ezrin was highly enriched in the membrane fraction. VSV G-tagged proteins were immunoprecipitated, and the immunoprecipitates were analyzed by immunoblotting with either anti-VSV G or antimyc antibodies. In total lysates, wt, T567A, and T567D ezrin oligomerized to roughly the same extent, whereas 29 ezrin was completely unable to form oligomers. T567A ezrin exhibited no difference with wt ezrin in its oligomerization ability in the cytosol and at the membrane. Consistent with gel filtration analysis, oligomers of T567D ezrin were present in the cytosol, but only as a trace amount at the membrane.
Figure 3. In the cytosol, T567D ezrin has a functional C-ERMAD. Various VSV G-tagged cDNAs, wt, T567A, and T567D ezrin, or an ezrin construct lacking a C-ERMAD due to the deletion of the 29 COOH-terminal amino acids (29), were cotransfected with myc-tagged wt ezrin cDNA into LLC-PK1 cells to detect oligomerization. Total, cytosol, or membrane lysates (top) or VSV G immunoprecipitates of these lysates (bottom) were analyzed by immunoblotting with anti-VSV G or anti-Myc antibodies as indicated on the right of each panel. 29 ezrin was strongly enriched in the membrane fraction, and completely defective in oligomer formation. In contrast, T567D ezrin was correctly distributed between the cytosolic and the membrane fraction, and exhibited a strongly reduced amount of oligomers specifically at the membrane.
These data suggest that a functional C-ERMAD is needed both to mask membrane binding sites in the NH2-terminal domain, and to form oligomers. T567D ezrin has a functional C-ERMAD in the cytosol, since T567D ezrin forms cytosolic oligomers and is correctly distributed between the cytosol and the membrane. However, upon membrane recruitment, the N/C-ERMAD interaction is abolished by the T567D mutation, and membrane oligomers of T567D ezrin are dissociated.
Dramatic Morphological Changes of LLC-PK1 Cells Producing T567D Ezrin
We asked whether there was a consequence of producing monomeric T567D ezrin on actin-rich membrane structures. We examined the morphology of LLC-PK1 clones producing wt, T567A, or T567D ezrin and the control clones transfected with the empty plasmid. E clones formed typical epithelial islets in sparse cell culture (Fig 4 A), as did A and P clones (data not shown). These islets were composed of cells adhering to each other. The islet periphery was regular. In sharp contrast, all the D clones had an altered morphology. Some space between cells could be distinguished. The edges of D colonies were not smooth, but interrupted by wide lamellipodia. Those lamellipodia were sometimes at the tip of long extensions. By phase contrast, a refractile relief was also prominent at the position of the nucleus in most D cells. By scanning EM, this relief was shown to be due to extensive membrane ruffling (Fig 4 B).
Figure 4. Morphology of LLC-PK1 cells producing T567D ezrin. (A) Clones were examined by phase-contrast optics. P, E, and A cells grew in typical LLC-PK1 islets (only the E control is presented). D colonies exhibited a number of morphological changes. In D colonies, cells were not always adherent to each other. The periphery of D colonies was irregular with wide lamellipodia (arrowheads). Those lamellipodia were occasionally formed at the extremity of long extensions (arrow). In most D cells, the membrane area around the nuclei was highly refractile. Bar, 25 μm. (B) Scanning EM examination of E and D cell morphology. Extensive membrane ruffling was observed in the cell central area, probably corresponding to the refractility observed by phase-contrast optics. Bars: (A) 25 μm; (B) 1 μm.
We observed confluent cultures of P, E, A, and D cells for optimal epithelial polarization and development of microvilli. Microvilli containing apical surface of these cells were analyzed by scanning EM. Production of wt or T567A ezrin did not alter microvillar density, length or organization, which were similar to those of control P cells (Fig 5). In these confluent cultures, D cells formed a less organized layer of cells. The layer of flat cells was often interrupted by holes that exposed the filter surface, and some round cells attached above this flat cell layer were frequently observed. We verified that these round structures were indeed cells by fluorescent staining of nuclei (data not shown). Flat cells were covered with microvilli similar to controls, whereas round cells were covered with denser and longer microvilli. Occasionally, on the flat cell layer, microvilli developed aberrantly into tufts (Fig 5). Such a tree-like organization of microvilli was never observed in P, E, or A cultures. In conclusion, production of monomeric T567D ezrin in LLC-PK1 cells induces numerous actin-rich membrane structures, such as lamellipodia, ruffles, and projections that are covered with microvilli.
Figure 5. Scanning EM analysis of microvilli in clones producing T567A and T567D ezrin. Monolayers of P, E, and A cells exhibited comparable microvilli in density and length. In D cultures, above a layer of flat cells, which contain comparable microvilli to P, E, and A cells, some round cells containing numerous and long microvilli were frequently observed. In addition, tufts of microvilli occasionally emerged from flat D cells (right). Bars, 1 μm.
Ezrin Association with the Actin Cytoskeleton Requires Phosphorylation of T567
Because these ezrin variants displayed differential capacity to affect membrane morphogenesis, we compared their ability to associate with the actin cytoskeleton. Localization of exogenous ezrin by immunofluorescence was performed with anti-VSV G antibodies on confluent cultures of E, A, and D cells. Wt and T567A ezrin were detected in microvilli (Fig 6 A), in a pattern similar to the one of endogenous ezrin in LLC-PK1 cells (data not shown). In the D cultures, T567D ezrin was found in microvilli of both flat and round cells and in ruffles. To evaluate whether these ezrin variants were associated with the actin cytoskeleton, we used an extraction procedure with a Triton X-100 buffer, which preserves the cytoskeleton and cytoskeleton-associated proteins. After extraction, wt ezrin and T567D ezrin were still detected in microvilli. In sharp contrast, T567A ezrin was almost completely extracted. Consistently, when Triton X-100 buffer–extracted material and cytoskeleton-associated material were compared by immunoblotting, T567A ezrin was highly extracted (Fig 6 B). After densitometry of the signals, insoluble to soluble ratios were calculated. For exogenous ezrin, as well as endogenous ezrin, the insoluble pool was always less than the soluble pool. However, T567A ezrin was significantly less insoluble than endogenous ezrin, exogenous wt ezrin, or T567D ezrin (P < 10-3, ANOVA followed by a Bonferroni t test). Therefore, T567A ezrin binds inefficiently to the actin cytoskeleton. Then we examined the distribution of endogenous phosphorylated ERM proteins. Consistently, phosphorylated ERM proteins are enriched in the Triton X-100–insoluble fraction. Taken together, these results indicate that the phosphorylation of the C-ERMAD is required for the association of ERM proteins with the actin cytoskeleton.
Figure 6. Ezrin association with the actin cytoskeleton requires phosphorylation of T567. (A) Localization of ezrin variants with anti-VSV G antibodies by immunofluorescence and confocal microscopy in E, A, or D cells. Cells were also stained after extraction with a Triton X-100 buffer that preserves cytoskeleton-associated material (csk). A single apical section is shown. Wt, T567A, and T567D ezrin were observed in microvilli. T567D ezrin was also present in the membrane ruffles it induced. After extraction of ezrin-soluble pool, wt and T567D ezrin staining were preserved, whereas T567A ezrin staining was strongly reduced. Bar, 5 μm. (B) Western blot analysis of ezrin cytoskeletal association. Similar fractions of soluble material (Sol), extracted with the Triton X-100 buffer, and insoluble material (Ins), were immunoblotted with anti-ezrin antibodies for P cell extract or with anti-VSV G antibodies for E, A, and D cell extracts. A densitometric analysis was performed and the Ins/Sol ratio was calculated from data obtained from two to four independent experiments with three different A and D clones (mean ± SEM). (C) Soluble and insoluble fractions from LLC-PK1 cells were equalized for their ERM content and immunoblotted with either 297S mAb (pERM) or ERM antibodies. Phosphorylated ERM proteins are enriched in the insoluble fraction.
Effect of T567A and T567D Ezrin on the Development of Multicellular Epithelial Structures
Organization of the actin cytoskeleton is a crucial point for the establishment and the maintenance of epithelial polarity. Ezrin has been implicated in the development of multicellular epithelial structures (Crepaldi et al. 1997 ; Gautreau et al. 1999 ). Therefore, we investigated whether production of T567A ezrin or T567D ezrin affects the morphogenesis of LLC-PK1 cells into suspension cysts and into tubules (Fig 7).
Figure 7. Production of T567A and T567D ezrin affects the development of multicellular epithelial structures. (A) Morphogenesis of suspension cysts examined by phase-contrast optics. Aggregates of LLC-PK1 cells in suspension are able to form hollow epithelial cysts. P, E, and A cells were not affected in this process, whereas D cells formed loose aggregates in which individual cells could still be distinguished at the periphery. (B) Tubulogenesis assay examined by Nomarski optics. In three-dimensional collagen type I, in the presence of HGF, P cells are able to differentiate into multicellular tubules. Production of wt ezrin potentiated growth and branching morphogenesis of tubules. Production of both T567A and T567D ezrin impaired tubulogenesis. A cells exhibited a growth defect, whereas D cells grew in disorganized colonies. Bars, 50 μm.
When isolated LLC-PK1 cells are put in suspension, they aggregate. These aggregates compact in an epithelial cyst with a smooth outline (Wohlwend et al. 1985 ). By two days, one or several cavities form in these cysts. Cavitation is a hallmark of the development of epithelial polarity. In the hollow cyst, the apical pole is in contact with the medium, whereas the basal pole is in contact with the cavity (Wohlwend et al. 1985 ). Consistent with the physiology of kidney proximal tubule, formation of this cavity is thought to reflect the vectorial transport of solutes and water from the medium. P, E, and A cells were similarly efficient in the development of hollow suspension cysts. In contrast to these cells, D cells were able to aggregate, but remained in clumps, in which individual round cells could still be distinguished at the periphery. Neither compaction nor cavitation occurred in D cells. This suggests that T567D ezrin impairs the development of epithelial polarity, probably by inducing constant membrane projections.
Consistent with the morphology of kidney proximal tubule, LLC-PK1 cells are able to develop into elongated polarized epithelial structures. Tubulogenesis occurs in a one-week culture in presence of HGF after seeding isolated cells in a three-dimensional collagen type I gel. P cells were able to differentiate into elongated tubules. Overproduction of wt ezrin in E cells potentiated this process and led to long branched tubules, as we previously reported (Crepaldi et al. 1997 ). In this sensitive assay for ezrin function, production of T567A ezrin impaired tubule formation by affecting clonal growth. By counting cell number, we observed that colonies formed by A cells were always composed of less than five cells, instead of tens of cells for P and E tubules (data not shown). In this assay also, production of T567D ezrin severely impairs epithelial organization of colonies. Colonies of D cells developed into loose aggregates with peripheral cells extending processes in the collagen matrix. D colonies never developed along a well-defined axis. In conclusion, we found that production of T567A ezrin affects tubule development, but not suspension cyst morphogenesis, whereas production of T567D ezrin impairs the establishment of epithelial polarity in both assays.
Discussion
In this report, we have examined in vivo the role of ezrin T567 phosphorylation by deriving stable clones producing wt, T567A, and T567D ezrin from the kidney epithelial cell line, LLC-PK1. Since ezrin is present in microvilli both as oligomers and as monomers, it was not known whether the active form of this plasma membrane–actin cytoskeleton linker is monomeric or oligomeric (Berryman et al. 1995 ; Bretscher 1999 ). Our study of T567A and T567D ezrin suggests that the active ezrin linker is a monomer. Given the high conservation of this threonine and of the amino acids forming the interface between the N- and C-ERMAD in ERM from vertebrates, and in homologues from invertebrates, the mechanism of conformational activation by phosphorylation of this conserved threonine from the C-ERMAD probably applies to all members of the ERM family (Pearson et al. 2000 ).
The Active Plasma Membrane–Actin Cytoskeleton Linker Is a Phosphorylated Monomer
T567A ezrin is poorly associated with microvillar cytoskeleton, as evidenced by the fact that this variant is almost completely extracted by a buffer preserving cytoskeleton-associated material, whereas a significant fraction of wt ezrin is not. This observation confirms in vivo the recent finding that phosphorylation of the homologous threonine 558 in moesin is required for F-actin binding in vitro (Hishiya et al. 1999 ; Nakamura et al. 1999 ). Although T567A ezrin is inactive as a cytoskeletal linker, its level of oligomers at the membrane is similar to that of wt ezrin. Thus, the oligomeric species are not sufficient to form active linkers.
The T567D mutation mimics the phosphorylation of this conserved threonine, since both disrupt the N/C-ERMAD interaction in vitro (our results; Matsui et al. 1998 ). In contrast to T567A ezrin, T567D ezrin is associated with the actin cytoskeleton. Moreover, T567D ezrin is a strongly morphogenic variant. It triggers the formation of wide lamellipodia, extensive membrane ruffles, and microvilli-rich projections, in which T567D ezrin is present. This finding is consistent with T567D ezrin being an active cytoskeletal linker. By three independent experiments, gel filtration analysis, assay of ezrin homo-oligomers, and ezrin-moesin hetero-oligomers, the level of T567D ezrin oligomers at the membrane was found to be strongly reduced. Thus, the oligomeric species are not necessary to form active linkers. Our study of these two mutant forms of ezrin, which uncouple oligomerization and cytoskeleton binding, provides strong evidence that the active form of this cytoskeletal linker is a phosphorylated monomer. However, it should be pointed out that phosphorylated active ezrin linkers might represent only a minor fraction of membrane monomers, since T567A ezrin exhibits as high a level of monomers at the membrane as wt ezrin.
To confirm this finding made with these ezrin variants, we have analyzed the endogenous phosphorylated ERM proteins. Consistently, we have found that the phosphorylated ERM proteins consist mainly of monomers at the membrane. Moreover, these phosphorylated ERM proteins are Triton X-100–insoluble, suggesting an association with the actin cytoskeleton. Therefore, it is likely that the T567D mutation mimics the phosphorylated state of ezrin in vivo, as well as in vitro, and thus, represents a useful tool to study ezrin function.
Phosphorylation Impairs N/C-ERMAD Interaction of Membrane-bound ERM Proteins
Since T567D ezrin shows a strongly reduced level of membrane oligomers and that phosphorylated ERM proteins are membrane monomers, it is likely that phosphorylation of this conserved threonine in ERM proteins dissociates membrane-bound oligomers into active monomers. It is striking that the phosphorylation-dependent impairment of N/C-ERMAD interaction occurs only on membrane-bound molecules, since the T567D mutation affects neither formation of cytosolic oligomers, nor the correct distribution between cytosol and membrane. Our in vivo analysis suggests the following model of ERM activation. The phosphorylation of a membrane-bound ERM molecule might disrupt intermolecular N/C-ERMAD interaction, thereby dissociating oligomers, and might prevent intramolecular N/C-ERMAD interaction from reforming, thereby exposing its F-actin binding site.
In vitro, phosphorylated moesin binding to F-actin was also found to be strongly dependent on the addition of phosphatidyl-inositol (4,5) bisphosphate or a charged detergent molecule (Nakamura et al. 1999 ). This result and our in vivo observations suggest that, for the maintenance of the active state, N/C-ERMAD interaction should be abrogated by both phosphorylation of the C-ERMAD and lipid binding to the N-ERMAD. This double regulation of ERM proteins is highly significant for these plasma membrane–actin cytoskeleton linkers. This double regulation might also explain that, despite the strong morphogenic effects of T567D ezrin, no more T567D ezrin than wt ezrin is Triton X-100–insoluble. This suggests that an activation factor other than phosphorylation is limiting the amount of cytoskeleton-bound ezrin. Since inactivation of ERM linkers occurs presumably through dephosphorylation of this conserved threonine (Fukata et al. 1998 ; Hishiya et al. 1999 ), T567D ezrin might be morphogenic, because, in contrast to wt ezrin, this variant cannot be dephosphorylated. The T567D mutation presumably locks the membrane pool of T567D ezrin in its active conformation.
What Is the Role of ERM Oligomers?
Since the dormant cytosolic form of ERM proteins is monomeric and the active plasma membrane–cytoskeleton linker is also monomeric, the role of oligomers in the activation pathway is rather intriguing. Formation of oligomers requires the conformational opening of monomers and the condensation of two, or more, opened monomers. The molecular components of the machinery required for oligomer formation are unknown. Membrane binding sites are cryptic in the dormant monomer, since a deletion of the C-ERMAD is sufficient to allow membrane recruitment of 29 ezrin. This truncated molecule is monomeric, indicating that oligomerization is not required, per se, for membrane recruitment. Also intriguing is the observation that 29 ezrin, despite having an exposed N-ERMAD, does not form oligomers, or at least dimers. One possible explanation is that the machinery for oligomer formation is only present in the cytosol, and is not accessible to the membrane-bound 29 ezrin. Since ezrin oligomers exist in cytosolic and membrane pools, asymmetric oligomers, having an exposed N-ERMAD (Bretscher 1999 ), might be in equilibrium between the cytosol and the membrane.
Phosphorylation of Ezrin on T567 Transduces Morphogenic and Growth Signals
ERM proteins are known to be essential for actin-rich membrane projections (Takeuchi et al. 1994 ; Crepaldi et al. 1997 ; Lamb et al. 1997 ; Bretscher 1999 ). Various stimuli that trigger the formation of these membrane structures induce ERM phosphorylation on this threonine residue (Nakamura et al. 1995 ; Matsui et al. 1999 ; Yonemura et al. 1999 ). Moreover, we showed that the production of T567D ezrin in LLC-PK1 cells induces a variety of actin-rich membrane projections, wide lamellipodia, membrane ruffling, and projections covered with microvilli, appearing as tufts of microvilli. Therefore, phosphorylation of this threonine residue on membrane ERM proteins appears to be critical for the generation of actin-rich membrane projections.
However, it is surprising that the mere production of an active linker tethering actin cytoskeleton to the plasma membrane induces such structures. Indeed, these membrane projections require several other coordinated processes, such as actin polymerization and cross-linking of actin filaments. Thus, T567D ezrin, in addition to being an active linker, induces a complete program for the formation of lamellipodia and ruffles. This is evidence of signaling events from active ezrin to the machineries controlling actin polymerization and cross-linking. These constitutive signaling events from T567D ezrin to actin dynamics and the consequent formation of membrane projections might explain why LLC-PK1 cells producing this ezrin variant fail to achieve epithelial polarity, as seen in suspension cyst and tubulogenesis assays.
In contrast, production of the inactive linker T567A ezrin did not affect epithelial polarity, as evidenced by the development of suspension cysts. In LLC-PK1 cells, production of the inactive T567A ezrin does not impair the formation of epithelial microvilli, whereas in transiently transfected COS7 cells, T558A moesin was reported to inhibit the RhoA-dependent formation of microvilli-like structures (Oshiro et al. 1998 ). However, T567A ezrin was found to be dominant negative in a tubulogenesis assay.
Differentiation of tubules in three-dimensional collagen matrix in the presence of HGF is a cellular assay critically dependent on ezrin function. Some phenotypes associated with expression of wt or mutant ezrin cDNAs are detected uniquely in these differentiation conditions. As reported previously, ezrin overproduction potentiates elongation and branching of tubules (Crepaldi et al. 1997 ). Production of Y353F ezrin specifically triggered apoptosis in this tubulogenesis assay (Gautreau et al. 1999 ). Here, production of T567A ezrin affected growth of LLC-PK1 cells in these conditions. One possible mechanism for this dominant negative effect is that overproduced inactive T567A ezrin is membrane-recruited instead of endogenous ezrin, and thereby impairs its function. In this tubulogenesis assay, phosphorylation of ezrin T567, and activation of its linker function, are needed for proliferation signaling. In 3T3 cells, T567A ezrin was also found to inhibit Ras- and Rho-dependent cellular transformation (Tran Quang, C., A. Gautreau, M. Arpin, and R. Treisman, manuscript submitted for publication). How ezrin signals proliferation is presently not known, but this signaling ability of ezrin relates to the activation of its linker function by phosphorylation of T567.
In the present study, we described the role of ezrin phosphorylation on T567 in vivo. This phosphorylation event regulates a membrane-specific transition of this actin cytoskeleton linker from inactive oligomers to active monomers. This unanticipated step of ezrin activation is critical for cell shape and growth during epithelial differentiation.
References
Algrain, M., Turunen, O., Vaheri, A., Louvard, D., and Arpin, M. 1993. Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane–cytoskeletal linker. J. Cell Biol. 120:129-139.
Andréoli, C., Martin, M., Le Borgne, R., Reggio, H., and Mangeat, P. 1994. Ezrin has properties to self-associate at the plasma membrane. J. Cell Sci. 107:2509-2521.
Berryman, M., Franck, Z., and Bretscher, A. 1993. Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J. Cell Sci. 105:1025-1043.
Berryman, M., Gary, R., and Bretscher, A. 1995. Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis. J. Cell Biol. 131:1231-1242.
Bretscher, A. 1999. Regulation of cortical structure by the ezrin-radixin-moesin protein family. Curr. Opin. Cell Biol. 11:109-116.
Bretscher, A., Gary, R., and Berryman, M. 1995. Soluble ezrin purified from placenta exists as stable monomers and elongated dimers with masked C-terminal ezrin-radixin-moesin association domains. Biochemistry. 34:16830-16837.
Chen, J., Doctor, R.B., and Mandel, L.J. 1994. Cytoskeletal dissociation of ezrin during renal anoxia: role in microvillar injury. Am. J. Physiol. 267:C784-C795.
Crepaldi, T., Gautreau, A., Comoglio, P.M., Louvard, D., and Arpin, M. 1997. Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells. J. Cell Biol. 138:423-434.
Fazioli, F., Wong, W.T., Ullrich, S.J., Sakaguchi, K., Appella, E., and Di Fiore, P.P. 1993. The ezrin-like family of tyrosine kinase substrates: receptor-specific pattern of tyrosine phosphorylation and relationship to malignant transformation. Oncogene. 8:1335-1345.
Fukata, Y., Kimura, K., Oshiro, N., Saya, H., Matsuura, Y., and Kaibuchi, K. 1998. Association of the myosin binding subunit of myosin phosphatase and moesin: dual regulation of moesin phosphorylation by rho-associated kinase and myosin phosphatase. J. Cell Biol. 141:409-418.
Gary, R., and Bretscher, A. 1993. Heterotypic and homotypic associations between ezrin and moesin, two putative membrane–cytoskeletal linking proteins. Proc. Natl. Acad. Sci. USA. 90:10846-10850.
Gary, R., and Bretscher, A. 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. 6:1061-1075.
Gautreau, A., Poullet, P., Louvard, D., and Arpin, M. 1999. Ezrin, a plasma membrane–microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway. Proc. Natl. Acad. Sci. USA. 96:7300-7305.
Gould, K.L., Bretscher, A., Esch, F.S., and Hunter, T. 1989. cDNA cloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1. EMBO (Eur. Mol. Biol. Organ.) J. 8:4133-4142.
Hayashi, K., Yonemura, S., Matsui, T., and Tsukita, S., Tsukita, S. 1999. Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues: application of a novel fixation protocol using trichloroacetic acid (TCA) as a fixative. J. Cell Sci. 112:1149-1158.
Hishiya, A., Ohnishi, M., Tamura, S., and Nakamura, F. 1999. Protein phosphatase 2C inactivates F-actin binding of human platelet moesin. J. Biol. Chem. 274:26705-26712.
Huang, L.Q., Wong, T.Y.W., Lin, R.C.C., and Furthmayr, H. 1999. Replacement of threonine 558, a critical site of phosphorylation of moesin in vivo, with aspartate activates F-actin binding of moesin: regulation by conformational change. J. Biol. Chem. 274:12803-12810.
Kondo, T., Takeuchi, K., Doi, Y., Yonemura, S., Nagata, S., and Tsukita, S. 1998. ERM (ezrin/radixin/moesin)-based molecular mechanism of microvillar breakdown at an early stage of apoptosis. J. Cell Biol. 139:749-758.
Krieg, J., and Hunter, T. 1992. Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin. J. Biol. Chem. 267:19258-19265.
Lamb, R.F., Ozanne, B.W., Roy, C., McGarry, L., Stipp, C., Mangeat, P., and Jay, D.G. 1997. Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts. Curr. Biol. 7:682-688.
Magendantz, M., Henry, M.D., Lander, A., and Solomon, F. 1995. Interdomain interactions of radixin in vitro. J. Biol. Chem. 270:25324-25327.
Matsui, T., Maeda, M., Doi, Y., Yonemura, S., Amano, M., Kaibuchi, K., and Tsukita, S. 1998. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J. Cell Biol. 140:647-657.
Matsui, T., Yonemura, S., and Tsukita, S., Tsukita, S. 1999. Activation of ERM proteins in vivo involves phosphatidylinositol 4-phosphate 5-kinase and not ROCK kinases. Curr. Biol. 9:1259-1262.
Nakamura, F., Amieva, M.R., and Furthmayr, H. 1995. Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets. J. Biol. Chem. 270:31377-31385.
Nakamura, F., Amieva, M.R., Hirota, C., Mizuno, Y., and Furthmayr, H. 1996. Phosphorylation of 558T of moesin detected by site-specific antibodies in RAW264.7 macrophages. Biochem. Biophys. Res. Comm. 226:650-656.
Nakamura, F., Huang, L., Pestonjamasp, K., Luna, E.J., and Furthmayr, H. 1999. Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and polyphosphatidylinositides. Mol. Biol. Cell 10:2669-2685.
Oshiro, N., Fukata, Y., and Kaibuchi, K. 1998. Phosphorylation of moesin by rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol. Chem. 273:34663-34666.
Pearson, M.A., Reczek, D., Bretscher, A., and Karplus, P.A. 2000. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell. 101:259-270.
Pietromonaco, S.F., Simons, P.C., Alman, A., and Elias, L. 1998. Protein kinase C-theta phosphorylation of moesin in the actin-binding sequence. J. Biol. Chem. 273:7594-7603.
Reczek, D., and Bretscher, A. 1998. The carboxy-terminal region of EBP50 binds to a site in the amino-terminal domain of ezrin that is masked in the dormant molecule. J. Biol. Chem. 273:18452-18458.
Reczek, D., Berryman, M., and Bretscher, A. 1997. Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family. J. Cell Biol. 139:169-179.
Shaw, R.J., Henry, M., Solomon, F., and Jacks, T. 1998. RhoA-dependent phosphorylation and relocalization of ERM proteins into apical membrane/actin protrusions in fibroblasts. Mol. Biol. Cell. 9:403-419.
Simons, P.C., Pietromonaco, S.F., Reczek, D., Bretscher, A., and Elias, L. 1998. C-terminal threonine phosphorylation activates ERM proteins to link the cell's cortical lipid bilayer to the cytoskeleton. Biochem. Biophys. Res. Comm. 253:561-565.
Takahashi, K., Sasaki, T., Mammoto, A., Takaishi, K., Kameyama, T., Tsukita, S., and Takai, Y. 1997. Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J. Biol. Chem. 272:23371-23375.
Takeuchi, K., Sato, N., Kasahara, H., Funayama, N., Nagafuchi, A., Yonemura, S., and Tsukita, S. 1994. Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J. Cell Biol. 125:1371-1384.
Turunen, O., Wahlstrom, T., and Vaheri, A. 1994. Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J. Cell Biol. 126:1445-1453.
Wohlwend, A., Montesano, R., Vassalli, J.D., and Orci, L. 1985. LLC-PK1 cysts: a model for the study of epithelial polarity. J. Cell. Physiol. 125:533-539.
Yonemura, S., and Tsukita, S., Tsukita, S. 1999. Direct involvement of ezrin/radixin/moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins. J. Cell Biol. 145:1497-1509.(Alexis Gautreaua, Daniel Louvarda, and M)
濠电姷鏁告慨鐑藉极閸涘﹥鍙忛柣鎴f閺嬩線鏌熼梻瀵割槮缁炬儳娼¢弻鐔衡偓鐢登瑰瓭缂備浇缈伴崐婵嬪蓟閿曗偓铻e〒姘煎灡閿涘棗鈹戦悙鍙夆枙濞存粍绻堝畷鎴﹀箛閻楀牆浠梺鎼炲労娴滄粓鎯冮悜妯镐簻闁挎棁顕ч獮鏍煃鐟欏嫬鐏撮柟顔规櫇缁辨帒螣婵犳碍鏆樺┑鐘垫暩閸嬫盯鎯囨导鏉戠9婵犻潧顑冮埀顑跨椤繈鎳滈悽闈涘Ц闁诲骸绠嶉崕鍗炍涘☉銏犲偍闁告鍋愰弨浠嬫煟閹邦厽缍戦柣蹇嬪劤閳ь剝顫夊ú锕傚礈閻斿鍤曢柟闂寸鍥撮梺绯曞墲閸旀帞鑺辨繝姘拺闁告劕寮堕幆鍫ユ煕閻曚礁浜扮€规洏鍨介獮姗€顢欓悾灞藉箰闂佽绻掗崑娑欐櫠娴犲鐓″鑸靛姈閻撳啴鏌﹀Ο渚▓婵☆垪鍋撻柣搴ゎ潐濞测晝鎹㈠┑瀣畺婵炲棗娴氶崯鍛亜閺傚墽鐭欓柣娑橀叄濮婂宕掑▎鎴М闂佸湱鈷堥崑濠呮濡炪倖鍔х粻鎴︽偪閻愵剛绡€濠电姴鍊搁幃鈺傜箾閹存瑥鐏╃紒鈧崘鈹夸簻闁哄啫娲らˉ宥夋煙閼恒儲绀€闁宠鍨块幃娆撳级閹寸姳妗撻梺鑹帮骏閸婃繈寮婚敍鍕ㄥ亾閿濆簼閭柛娆忓閺岋綁骞欓崘銊ゅ枈閻庤娲栭悥鍏间繆閹间焦鏅濋柍褜鍓熼幆鍐箣閿旇В鎷虹紓浣割儐椤戞瑩宕曢幇鐗堢厵婵繂鑻崥鍦磼椤旂⒈鍎旀鐐村浮瀵剟宕崟顏勵棜闂備焦瀵х换鍌滆姳閼测晞濮冲ù鐘差儐閻撴洟鏌¢崘鈺傚暗闁伙綀椴搁妵鍕敂閸曨偅娈绘繝纰樷偓宕囧煟鐎规洖宕灃闁逞屽墮宀e潡骞嬮敂瑙f嫼缂備礁顑嗛娆撳磿閹扮増鐓欓柣鐔哄閸犳ḿ鈧鍠涢褔鍩ユ径鎰潊闁炽儱鍘栧Ч妤呮煟鎼淬値娼愭繛鍙夌墪鐓ら柕蹇嬪€曢悞鍨亜閹哄秶顦︾紒妤佽壘鑿愰柛銉戝秷鍚悗瑙勬穿缁叉儳顕ラ崟顒傜瘈闁稿被鍊栫紞瀣⒒閸屾瑧顦﹂柣銈呮喘閿濈偞寰勯幇顒€鐎梺鍓茬厛閸犳岸寮抽敃鍌涚厱妞ゆ劧绲剧粈鈧梺缁樻尰閿曘垽寮婚敐澶婄疀闂傚牊绋戦~顐g箾鐎涙ḿ鐭嬮柣鐔叉櫅椤繘鎼圭憴鍕彴闂佸搫琚崕娲焵椤掍緡娈樼紒杈ㄥ浮閹晠鎳犻顐庡應鍋撶憴鍕婵炶尙鍠庨悾鐑芥晸閻樺啿鈧鈧箍鍎卞Λ娑㈠煡婢舵劖鐓冮柦妯侯樈濡偓婵犳鍠掗崑鎾绘⒑缂佹﹫鑰挎繛浣冲嫭鍙忛柨鏃€鍨濈换鍡涙煟閹板吀绨婚柍褜鍓氶悧婊堝极椤曗偓楠炴帒顪冨┑鍡樺枠妞ゃ垺鐩幃娆撳级閹存柨浜鹃柛顐f礃閻撱儵鏌¢崘銊モ偓濠氬箺閸屾稓绠鹃柛顐g矌閻瑩鏌″畝瀣М妤犵偛娲、妤佹媴閸欏浜為梻鍌欒兌鏋悗娑掓櫅椤繗銇愰幒瀣у亾閸愵喖唯闁冲搫鍊搁埀顒傚厴閺屾稑鈻庤箛锝喰﹂梺鍝勵儑婵挳鈥旈崘顔嘉ч柛鎰╁妿娴犳儳鈹戦埥鍡椾簻闁哥噥鍋婇幃楣冩倻閽樺)鈺呮煃閸濆嫸鏀婚柡鍜冪秮濮婅櫣绱掑Ο鍝勵潚濠电偘鍖犻崶褏顔愮紓浣割儏缁ㄩ亶寮ㄦ禒瀣厽婵☆垵娅f禒娑㈡煛閸″繑娅婇柡灞剧〒閳ь剨缍嗛崜娆撳煝閸噥娈介柣鎰摠瀹曞本銇勯姀鈩冾棃鐎规洖宕灃闁告劦浜為幗宀勬⒒閸屾瑦绁版い鏇熺墵瀹曡绺介崨濠傗偓鍫曟煙闁箑鍔掓繛宸憾閺佸倿鏌涢弴銊ュ季闁告艾鎳樺缁樻媴閾忕懓绗¢梺鎸庡哺閺屾稑顫濋敐鍛缂傚倸鍊烽悞锕傘€冮崨姝ゅ洭妫冨☉杈ㄧ稁濠电偛妯婃禍婊呯不娴兼潙绠归弶鍫濆⒔閹ジ鏌i敐鍥ㄦ毈婵﹥妞介幃鐑藉级閹稿巩鈺侇渻閵堝棗鐏ユい锔诲灦閹箖鎮滈懞銉ヤ缓缂備礁顑呭ḿ锟犲船鐠鸿 鏀介柣姗嗗枛閻掑搫霉濠婂嫮绠撻摶鐐烘煕閹伴潧鏋涢柡瀣╃窔閺屾稓浠﹂崜褏鐓傛俊鐐额潐婵炲﹪骞冨Δ鍛櫜閹煎瓨绻勯惄搴㈢節绾版ǚ鍋撻悙钘変划濠殿喖锕︾划顖炲箯閸涙潙宸濆┑鐘插暙閺嬫垿姊绘担鍛靛綊顢栭崶顒€纾婚柕鍫濐槺瀹撲線鏌熼悧鍫熺凡缂佺媭鍣i弻锕€螣娓氼垱歇闂佺濮ゅú鐔奉潖濞差亜浼犻柛鏇ㄥ墮閸嬪秹姊洪幖鐐插缂傚秴锕幃浼搭敋閳ь剚鎱ㄩ埀顒勬煏閸繃顥犻柛妯绘倐濮婃椽骞栭悙鎻掑闂佸搫鎳忕粙鎴︽偤椤撶偐鏀介柣妯活問閺嗩垶鏌嶈閸撴繄浜稿▎鎾村剹婵炲棙鎸婚悡娑氣偓鍏夊亾閻庯綆鍓涜ⅵ濠电姵顔栭崰鎺楀磻閹剧粯鈷戦悗鍦У閵嗗啴鏌ょ€圭姴鐓愮紒鍌涘浮閸╋繝宕橀鍡闯闂備胶枪閺堫剙顫濋妸銉ф懃闂傚倷鐒︾€笛兠洪敂鐣岊洸妞ゅ繐鐗婇崑妯汇亜閺冨倹娅曠紒鈾€鍋撻梻浣规偠閸庮垶宕濆鍛瀺闁靛繈鍊栭埛鎴犵磽娴e箍鈧帡宕烽婵堝墾濠电偛妫欓幐濠氬磻椤忓牊鐓冪憸婊堝礈濮橆厾鈹嶅┑鐘叉处閸婇攱銇勮箛鎾愁仱闁稿鎹囧浠嬵敇閻愭鍞跺┑掳鍊х徊浠嬪疮椤愩倕顥氬┑鐘崇閻撶喖鏌¢崒娑橆嚋闁规彃鎲¢妵鍕煛閸愩劌骞嬮梺鍝勭灱閸犳牠鐛澶樻晩缁炬媽椴稿В澶愭⒒娴g懓鈻曢柡鈧柆宥呭瀭闁秆勵殔閽冪喐绻涢幋娆忕仾闁哄懏绮撻幃褰掑炊閵娿儳绁风紓浣诡殔閻倸顫忓ú顏呭殥闁靛牆鎳忛悗楣冩⒑闂堚晝绋绘繛鍏肩懅閸欏懎顪冮妶鍛閻庢埃鍋撻悷婊勬瀵鈽夐姀鐘栥劍銇勯弮鈧崕宕囨暜閵夈儮鏀介柣鎰硾娴滃綊鏌涢悩宕囧ⅹ妞ゎ偄绻愮叅妞ゅ繐瀚ˇ銊╂⒑閸涘﹦鎳冩い锔垮嵆瀹曟洟骞樼紒妯锋嫼闂佽崵鍠愬姗€寮虫潏銊﹀弿婵鐗嗛々顒勬煙楠炲灝鐏╂い顐g箞閹虫粎鍠婂Ο璇差伜婵犵數鍋犻幓顏嗗緤娴犲绠犳繛鍡樺竾娴滃綊鏌熼悜妯虹仴闁绘繃锚椤啴濡堕崱妤冪懆闂佺ǹ锕ら…鐑藉箚鐏炶В鏋庨柟鎯ь嚟閸樼敻姊虹紒妯虹仸闁挎洍鏅涢埢鎾诲即閵忥紕鍘辨繝鐢靛Т閸婄粯绂掑☉銏$厪闁搞儜鍐句純閻庢鍣崜鐔风暦瑜版帩鏁婇悷娆忓閻﹁京绱撻崒娆掑厡闁稿鎹囧畷鏇㈠箮閽樺)褔鏌熼梻瀵割槮闁藉啰鍠栭弻銊╂偄閸濆嫅銏㈢磼閳ь剟宕橀鐣屽帾婵犵數鍋涢悘婵嬪礉濡ゅ懏鐓曢悗锝庡亝鐏忕數绱掗鐣屾噰妤犵偞鍔栭幆鏃堝閵忊槅娼犳繝鐢靛Х閺佸憡鎱ㄩ悜钘夋瀬闁告稑锕ラ崣蹇斻亜閹惧崬鐏╃紒鐙€鍨堕弻娑㈠箛闂堟稒鐏堢紓浣插亾閻庯綆鍠楅悡鏇熴亜椤撶喎鐏ュù婊呭仱閺屸剝寰勬繝鍕ㄩ梺鍝勭焿缁查箖骞嗛弮鍫濐潊闁宠桨鐒﹀▍娑㈡⒒娴e憡鎲稿┑顔炬暬閹囨偐鐠囪尙鐣洪梺闈涱槴閺呮粓宕戦崒鐐茬閺夊牆澧界粙缁樼箾閸喎鍔ら柍瑙勫灴椤㈡瑦鎱ㄩ幇顏嗘崟闂備胶枪椤戝棝骞愰幖浣哥厴闁硅揪绠戦悞娲煕閹板吀绨奸柟鍙夌懃椤啴濡惰箛娑欘€嶆繝鐢靛仜閿曨亜顕g拠娴嬫闁靛繒濮烽鎺楁⒑閸濆嫷妲归柛銊潐缁傛帡宕妷褏锛濇繛杈剧秬濞咃絿鏁☉姘辩<閻庯綆鍋呭畷宀勬煕閳规儳浜炬俊鐐€栫敮濠囨嚄閸洖鐓€闁哄洨鍋熼弳鍡涙煥濠靛棙鍣洪柛瀣ㄥ劦閺岀喖顢涘顓熸嫳缂備胶绮换鍫濈暦閹达附鍤嶉柕澶涘瘜濡啴姊虹拠鈥虫灀闁哄懐濮撮悾鐑芥晲閸℃绐為柣搴秵娴滄繈鎮楁ィ鍐┾拻闁稿本鐟︾粊鐗堜繆濡炵厧濡挎繛鍡愬灲閺佹捇鎮╅弻銉︼紬闂備焦瀵х换鍌炈囬銏犵劦妞ゆ垼娉曠粣鏃傗偓娈垮枟閹歌櫕鎱ㄩ埀顒勬煃閳轰礁鏆為柣婵囨礋濮婄粯鎷呴挊澹捇鏌ㄥ顓滀簻闁挎棁娉曢惌娆撴煕閳规儳浜炬俊鐐€栫敮濠囨嚄閸洖鐓″鑸靛姈閻撳啴鎮峰▎蹇擃仼闁诲繑鎸抽弻鈩冩媴閸濄儛褏鈧娲滈崰鏍€侀弴銏℃櫖闁告洦鍓欑粈瀣攽閻樺灚鏆╁┑顕呭弮楠炲繘宕橀鐓庣獩濡炪倖姊婚弲顐ャ亹瑜忕槐鎾诲磼濞嗘垼绐楅梺鍝ュУ椤ㄥ﹪骞冮悜鑺ユ櫆闁绘劦鍓欐惔濠傗攽閻樼粯娑фい鎴濇嚇閹瑦绻濋崶銊у帾婵犵數鍊崘銊︽缂備胶濮锋晶妤冩崲濠靛鍋ㄩ梻鍫熷垁閻愮儤鐓曢柕濞у啫浠橀梺宕囩帛閺屻劑鍩ユ径鎰潊闁斥晛鍟悵鏍⒒娓氣偓濞佳囨偋閸℃蛋鍥敍濮e灕鍥ц摕闁靛濡囬崢鎼佹⒑閹肩偛鍔楅柡鍛洴瀵悂骞嬮敂鐣屽幈濠电偛妫楃换鎺旂不婵犳碍鐓涚€光偓鐎n剛袦闂佽鍠撻崹浠嬪箖閳╁啯鍎熸繝闈涙閻庡啿鈹戦悩娈挎毌婵℃彃鎳樺畷褰掝敂閸繄锛涢梺鐟板⒔缁垶鍩涢幒鎳ㄥ綊鏁愰崼婢捇鏌曢崱妤€鏆熺紒杈ㄥ浮閸┾偓妞ゆ帊鐒︽刊鎾煣韫囨挻璐¢柣鎾愁儔濮婃椽宕烽鈩冾€楅梺鍝ュУ椤ㄥ﹤鐣烽幋锕€鐓涢柛灞剧矌閻﹀牓姊婚崒姘卞濞撴碍顨婂畷鏇$疀閺囩姷锛滈梺閫炲苯澧寸€规洘锕㈤崺鐐村緞閸濄儳娉垮┑锛勫亼閸婃牠骞愭ィ鍐ㄧ獥閹兼番鍔嶉崐鍫曟煥濠靛棭妲归柣鎾寸懇閹鎮介惂璇茬秺瀹曘垻鈧稒蓱閸欏繐鈹戦悩鎻掝伀閻㈩垱鐩弻鐔风暋閻楀牆娅х紓渚囧枤閺佹悂鍩€椤掑﹦绉靛ù婊勭箞閹偓瀵肩€涙ǚ鎷绘繛杈剧到閹诧繝骞夌粙搴撴斀妞ゆ梻鍋撻弳顒侇殽閻愭潙鐏寸€规洘鍎奸ˇ鎾煛閸☆參妾紒缁樼箞濡啫鈽夐崘鎻掝潬闂備焦鐪归崐鎰板磻閹惧绡€闁汇垽娼ф禒锕傛煕閵娿儱鎮戦柕鍥ㄦ楠炴牗鎷呴崫銉︾叄闂備焦瀵х换鍌毭洪妸褍顥氬ù鐘差儐閻撴洟鎮橀悙鎻掆挃闁瑰啿鎳橀弻娑㈠棘鐠恒剱銏ゆ煟閿濆棛绠為柡浣瑰姍瀹曘劑顢欓梻瀛樻▕闂傚倷鑳剁涵鍫曞棘閸岀偛鍨傞柦妯侯樈閸ゆ洘銇勯弴妤€浜鹃悗瑙勬礀閵堟悂骞冮姀銈呬紶闁告洦鍋嗛鎴︽⒒閸屾艾鈧兘鎳楅崼鏇炵疇闁圭偓妞块弫瀣亜閹惧崬鐏╃痪鎯ф健閺岀喓鈧數枪娴犙囨煟閻旀椿娼愮紒缁樼洴楠炲鈻庤箛鏇氱棯闂備胶绮幐璇裁洪悢鐓庤摕闁跨喓濮寸壕鍏兼叏濡搫缍侀柧蹇撻叄濮婃椽宕崟顒婄川缂傚倸绉崇欢姘嚕婵犳艾鐏崇€规洖娲﹀▓鏇㈡煟鎼搭垳绉甸柛鎾寸洴閹線宕奸妷锕€鈧敻鎮峰▎蹇擃仾缂佲偓閸愵喗鍋ㄦい鏍ㄧ☉濞搭噣鏌ㄥ┑鍫濅粶闁宠鍨归埀顒婄到閻忔岸寮查敐澶嬧拺缂備焦锕懓鎸庣箾娴e啿娲ㄥ畵渚€鏌嶈閸撴稓妲愰幘璇茬<婵﹩鍏橀崑鎾诲箹娴e摜锛欓梺缁樺灱婵倝宕愰崸妤佺叆闁哄洨鍋涢埀顒€鐖奸崺鈧い鎺嗗亾闁哥喐娼欓悾鐑藉Ω閵夘喗妗ㄩ梺闈涚墕閹冲繘宕板鈧弻锛勪沪閼恒儺妫炲銈嗘尭閵堢ǹ鐣烽妸鈺婃晬婵﹩鍎烽弴鐐╂斀闁挎稑瀚禍濂告煕婵犲啰澧遍柡渚囧櫍楠炴帒螖閳ь剛绮eΔ浣瑰弿婵妫楅崢宕囩磽閸屾稑鍝洪柡宀€鍠栭弻鍥晝閳ь剟寮搁妶鍥╂/闁诡垎鍐╁€梺闈涙搐鐎氭澘顕i鈧畷鎯邦槺婵顨堢槐鎾存媴閸濆嫅銏°亜椤撶姴鍘寸€殿喖顭锋俊鎼佸Ψ閵忊剝鏉搁梻浣稿閸嬪懐鎹㈤埀顒佷繆閸欏濮嶆慨濠勫劋濞碱亪骞嶉鍛滈梻浣告憸閸犳劕岣垮▎鎾村仼闁绘垼妫勭粻鎶芥煙閹碱厼骞楅柛宥囨暬濮婃椽宕ㄦ繝搴㈢暭闂佺ǹ顑囬崑銈夌嵁婢舵劕顫呴柕鍫濇閹风粯绻涙潏鍓хК婵☆偄瀚板畷銉╂偡閹佃櫕鏂€闂佺粯鍔忛弲娑欑閸撗呯=闁稿本鑹鹃埀顒佹倐瀹曟劘銇愰幒鎾虫疄婵°倧绲介崯顐︽偂濠靛牃鍋撻獮鍨姎妞わ缚鍗冲鏌ュ蓟閵夛妇鍘遍梺闈涱槶閸ㄥ搫鈻嶉崶顒佺厽闁圭偓鍓氶崕鏃€鎱ㄦ繝鍐┿仢鐎规洘绮撻幊鐘活敆閳ь剛鏁妷鈺傗拺闁告縿鍎辨牎闂佸湱鎳撳ú顓炵暦濞差亜鐒垫い鎺嶉檷娴滄粓鏌熼崫鍕ゆい锔煎缁辨帒螖娴e摜鐟查梺闈涙搐鐎氫即銆侀弴銏℃櫜闁搞儮鏅濋弶鑺ヤ繆閻愵亜鈧垿宕瑰ú顏呮櫇闁靛繈鍊曠粻鏍煃閸濆嫭濯奸柡浣稿暣閺屻劌鈹戦崱妤婂妷缂傚倸绉撮惌鍌氼潖婵犳艾纾兼慨姗嗗厴閸嬫挻顦版惔銏╁仺濠殿喗锕╅崗妤冪磽濮樿鲸鍠愮€广儱娲ゅ鏌ユ⒒娓氣偓濞佳囨偋閸℃稑绠栭柛顭戝晹濞差亶鏁傞柛鏇炵岸閺呯娀寮诲鍡樺闁瑰嘲鑻崢锟犳⒑閸涘﹥顥栫紒鐘冲灴閳ユ棃宕橀鍢壯囨煕閳╁喚娈橀柣鐔村姂濮婅櫣绮欏▎鎯у壈濠碘槅鍋呯换鍌烆敋閿濆惟闁冲搫鍊告禍閬嶆⒑閸撴彃浜栭柛銊ヮ煼椤㈡瑩寮撮悢铏圭槇闂侀潧楠忕徊鍓ф兜閻愵兙浜滈柟瀛樼箖閹兼劙鎮¢妶澶嬪€甸柣銏㈡暩閵嗗﹪鏌涚€n偅灏甸柟鍙夋尦瀹曠喖顢楅崒锔惧枠闂傚倷鐒﹂幃鍫曞礉韫囨梹鍙忛柕鍫濇閳瑰秴鈹戦悩鍙夋悙闂佸崬娲弻锟犲炊閳轰椒鎴烽梺绋匡功閸嬨倕顫忓ú顏咁棃婵炴番鍔岀紞濠傜暦閺囥垺鍊绘俊顖欒閸ゃ倝姊洪崫鍕偍闁搞劍妞介幃锟犳偄閸忚偐鍘介梺鍝勫€圭€笛囧箟閹间焦鐓熸い鎾跺仩鐎氱増銇勯鈥冲姷妞わ箒娅曢妵鍕Ω閵壯冣叺閻庢鍣崑濠囥€佸▎鎾冲簥濠㈣泛顑嗛ˉ鍫⑩偓瑙勬处娴滎亜鐣峰鈧、鏃堝幢韫囨棑绱欐繝鐢靛Х閺佸憡鎱ㄦ导鏉戝瀭濠靛倻枪閺勩儵鏌″搴″箹缂佲偓婢舵劖鐓涚€广儱楠搁獮鏍棯閹岀吋闁哄被鍔岄埞鎴﹀幢閳哄倐褔姊虹紒妯诲鞍婵炶尙鍠栧濠氬即閻旈绐炲┑鈽嗗灥濞咃絾绂掗埡渚囨富闁靛牆鍊瑰▍鎾绘煕閹惧鎳囬柛鈹垮劜瀵板嫰骞囬鍌ゅ晪闂備浇宕甸崰鎰珶閸℃ぅ鎺楀閵忋垻锛濇繛杈剧导缁瑩宕ú顏呭仺妞ゆ牗渚楀▓鏂跨暆閿濆牆鍔电紒鐘崇☉閳藉螣濠х偓娅囬柟鍙夌摃缁犳稑鈽夊▎蹇庢偅濠电娀娼ч崐鎼佸箟閿熺姴鐓曢柟瀵稿У閸犳劙鏌eΔ鈧悧鍡樼┍椤栨稐绻嗘い鎰剁到閻忋儵鏌嶇憴鍕伌闁诡喗鐟╅崺鈩冩媴瀹勯偊妫滈梻鍌氬€搁崐宄懊归崶顒夋晪闁哄稁鍘肩粣妤佷繆閵堝懎鏆欓柛銊︾箞閺岀喖骞戦幇闈涙缂備胶濮靛姗€鍩為幋锔藉亹闁圭粯甯楀▓顓犵磽娴gǹ顣抽柛瀣洴閳ワ妇鎹勯妸锕€纾梺鎯х箰濠€杈ㄥ閸パ€鏀芥い鏃傘€嬮崝鐔兼煕鐎n偅灏甸柟骞垮灩閳规垹鈧綆鍋勬禒娲⒒閸屾氨澧涚紒瀣姉閸掓帞鎹勬笟顖涘瘜闂侀潧鐗嗗Λ娆愬緞閸曨垱鐓曟俊顖氭贡閻瑦顨ラ悙鑼ⅵ濠碘剝鎮傞弫鍌炲传閸曨亞鍑归梻鍌欑閹诧繝宕濋幋锕€绀夌€广儱鎲橀敐澶樻晢闁告洦鍏橀幏娲⒑閸涘﹦绠撻悗姘煎櫍閸┿垺寰勯幇顓犲幈濠碘槅鍨辨禍浠嬪磻閵忊懇鍋撶憴鍕缂傚秴锕ら悾鐑藉箳濡や礁鈧兘鎮楅悽鐧诲湱鏁幆褉鏀介柣妯虹仛閺嗏晠鏌涚€n剙鏋涚€规洘鍨块獮姗€寮妷锔锯偓娲⒑缁洖澧茬紒瀣浮閹繝寮撮姀鈥斥偓鐢告煥濠靛棗鏆欏┑锛勫帶閳规垿鍨鹃悙钘変划濠碘槅鍋傞悞锕€顕ラ崟顖氱疀妞ゆ挾濮村铏節閻㈤潧鈻堟繛浣冲吘娑樷枎閹炬娊妫烽悷婊呭鐢鎮″☉銏$厱闁斥晛鍟伴幊鍐煛鐎n偄鐏ラ懣鎰版煕閵夋垵绉烽崥顐㈩渻閵堝啫鐏紒瀣灴閿濈偛鈹戠€n亞顢呴梺缁樺姀閺呮稑顕ュ鍛斀闁绘ɑ鍓氶崯蹇涙煕閻樺磭澧甸柡浣稿暣婵$兘濡烽钘夌槣濠电偛顕慨鎾敄閸愵喖鐒垫い鎴f硶缁愭梻鈧鍠楅幐铏叏閳ь剟鏌嶉埡浣告殲闁绘繃濞婂缁樻媴閾忓箍鈧﹪鏌涢幘瀵哥疄闁诡喚鍏橀弫鍐磼閵堝棙娅嶅┑鐘垫暩婵數鍠婂澶婄厱闁圭儤鍤氳ぐ鎺撴櫜闁割偆鍣ユ禒鈺呮煟閵忊晛鐏¢柣鈺婂灦瀵鎮㈤崫鍕€抽梺鍛婎殘閸嬫ḿ鑺辨禒瀣拺闁荤喐澹嗛悾顓㈡煕閵娿儳绉洪柛鈺冨仱楠炲鎮╅顫闂佹寧绻傛鍛婃櫠椤斿墽纾煎〒姘功閻g敻鏌″畝瀣ɑ闁诡垱妫冮、娑樷堪閸涘拑缍佸娲传閵夈儛锝夋煟濡や胶鐭岄柛鎺撳笚缁绘繂顫濋鐔哥彸闂備胶纭堕崜婵嬫偡瑜旈幆渚€宕奸悢铏圭槇闂佹眹鍨藉ḿ褍鐡梻浣呵归敃銉╁箖閸岀偑鈧線寮崼顐f櫇闂佹寧姊婚弲顐﹀储閻㈠憡鈷戦柛婵嗗閸庢盯鏌涚€n亜顏柟渚垮姂瀹曟帒鈽夊▎蹇撲紟濠电姷鏁告慨鎾疮椤栫偛桅婵犻潧娴傚▓浠嬫煟閹邦厽缍戝┑顔肩墦閺岀喖顢涘☉娆樻闂佽桨鐒﹂崝娆忕暦閹烘垟妲堥弶鍫涘妿缁夐箖姊婚崒姘偓鎼佸磹妞嬪海鐭嗗〒姘e亾妤犵偞鐗犻、鏇㈡晜閽樺缃曟繝鐢靛Т閿曘倝鎮ч崱娆戠焼闁割偁鍎查悡鐔兼煛閸愩劎澧辨俊顐e灴閺岋繝宕遍鐘垫殼闂佸搫鐭夌紞渚€骞冮姀銏㈢煓婵炲棛鍋撻ˉ鎾绘⒒娴gǹ顥忛柛瀣笚缁傚秹顢旈崼婵婃憰濠电偞鍨崹褰掑础閹惰姤鐓忓┑鐐茬仢閸旀岸鎮楀☉宕囧埌闁宠鍨块幃娆戔偓娑欋缚缁嬪洭鏌f惔銏犲毈闁告挾鍠栭崹楣冨籍閸繄顦ㄥ銈嗘煥濡插牐顦归柡灞剧洴閸╁嫰宕橀悙顒傛毉闂備浇宕甸崰鎰板礉濞嗗浚娼栭柧蹇撴贡绾惧吋淇婇娑欍仢闁搞倕顑囩槐鎾存媴閸濆嫅锝夋煙閸涘﹥鍊愭い銏″哺閺佹劖寰勬繝鍕靛敶缂傚倷鐒︾粙鎴︻敄閸℃稑鐤炬繝濠傜墛閳锋垿鏌涢幘鏉戠祷濞存粎鍋ら弻娑㈡偐閾忣偄纾抽悗瑙勬磸閸ㄤ粙寮婚崱妤婂悑闁糕€崇箲鐎氬ジ姊绘担钘夊惞闁哥姵鎸婚弲璺何旈崨顓犵暰闂佸壊鍋侀崹鑽ゅ閻撳寒鐔嗛悹杞拌閸庢劗绱撳鍡楃伌闁哄矉缍侀弫鎰板炊閵婏附鐦撻柣搴ゎ潐濞叉牕鐣烽鍕叀濠㈣泛艌閺嬪酣鏌熺€涙ḿ绠撶紒妤佸哺濮婃椽鎳¢妶鍛€剧紓渚囧枛缁夊爼宕氶幒妤€绠荤€规洖娲﹀▓鎯р攽閻樿宸ラ柛鐘宠壘宀e潡鍩¢崒妯圭盎闂佸湱鍎ら崺鍫澪hぐ鎺撶厱鐎广儱娲﹂弳顒佹叏婵犲啯銇濇俊顐㈠暙闇夐柕澶堝劤婢э妇鈧鍠楄ぐ鍐偑娴兼潙閱囨繝闈涚墳缁遍亶姊绘担鍛靛綊鎯夋總绋跨;闁绘劗鍎ら崑鍌炴煏婢跺棙娅嗛柣鎾跺枛閺岋繝宕掑☉姗嗗殝闂佽鍨伴悧蹇涘焵椤掍胶鈯曠紒璇茬墦瀵鏁愭径瀣簻闂佸憡绺块崕鏌ュ吹閸屾粎纾藉ù锝呮惈鏍¢梺鍦嚀濞差參骞冩导鎼晪闁逞屽墮椤曪絾绂掔€e灚鏅i梺缁樺姈椤旀牠宕崶銊ょ箚闁绘劦浜滈埀顒佺墵楠炴劖銈i崘銊э紱闂佺粯鏌ㄩ幗婊堛€呴柨瀣ㄤ簻闁哄秲鍔庨惌宀€鐥幑鎰棄闂囧鏌ㄥ┑鍡橆棞缂佽尪顕ч湁婵犲﹤鎳庢禍鍓х磼缂佹ḿ绠炲┑顔瑰亾闂佹寧绻傚Λ娑㈠Υ閹扮増鈷戦柟棰佺閻忊剝绻涢崣澶岀疄濠碉紕鏁婚獮鍥级鐠侯煈鍞洪梻浣烘嚀椤曨厽鍒婇鐐嶏綁顢涢悙绮规嫽婵炶揪绲介幊娆撳捶椤撶喎搴婂┑掳鍊曢幊搴e婵犳碍鐓欓梺顓ㄧ畱閸樻挳鏌$€n偅顥堥柡灞炬礋瀹曠厧鈹戦幇顓壯囨⒑閹惰姤鏁遍柛銊ユ贡濡叉劙骞樼€涙ê顎撻柣鐘冲姦閸ㄥ磭妲愰幍顔剧=濞达絽鎼牎闂佺粯顨堟繛鈧€殿喖顭烽弫鎰緞婵犲嫬骞愬┑鐘垫暩婵挳宕愮紒妯碱浄闁冲搫鎳忛埛鎴澝归崗鑲╂噮闁诡喖銈搁弻娑滅疀閺冩捁鈧法鈧鍣崑濠囩嵁濡偐纾兼俊顖濇〃濮规姊绘担钘変汗闁冲嘲鐗撳畷婊堟晝閳ь剙宓勬繛瀵稿帶閻°劑鍩涢幒鎳ㄥ綊鏁愰崼鐕佹婵炲瓨绮岀紞濠囧蓟瀹ュ懐鏆嬮柟娈垮枛閳敻鎮楃憴鍕缂侇喖鐭傞敐鐐测攽閸ラ鐭楀┑鐘绘涧濞诧箑鈻嶉幘缁樷拻濞达絼璀﹂弨浼存煙濞茶閭慨濠佺矙瀹曠喖顢涘鍗炲箞闂備胶顢婇幓顏堟⒔閸曨垰纾婚柨婵嗩槹閻撴洟鏌曟径妯虹仯鐎光偓濞戙垺鐓曢悗锝庡亝瀹曞矂鏌熼悡搴gШ妞ゃ垺娲熸俊鍫曞礋椤撶喎鐦婚梻鍌氬€搁崐鎼佸磹妞嬪海鐭嗗〒姘e亾妤犵偛顦甸弫鎾绘偐閼艰埖鎲伴梻渚€娼чオ鐢碘偓闈涜嫰閳绘挻绂掔€n偆鍘甸梻鍌氬€搁顓⑺囬敃鍌涚厽闁规儳顕粻鐐烘煛瀹€鈧崰鏍嵁閹达箑绠涢梻鍫熺⊕椤斿嫮绱撻崒娆掑厡濠殿垰顕槐鐐寸瑹閳ь剙顕f繝姘櫜濠㈣泛锕﹂悿鈧俊鐐€栭幐楣冨窗閹惧墎鐭欓柛銉戝本瀵岄梺闈涚墕閹冲酣顢楅姀銈嗙厵闁告稑锕ラ崐鎰版煕閳哄啫浠辨鐐差儔閺佸倿鎸婃径澶嬬潖闂備浇宕垫慨鏉戔枖瑜斿畷妯款槾闁活厽顨婂濠氬磼濞嗘埈妲梺纭呭Г缁挸鐣烽幎钘壩ㄩ柍杞扮閻庮參姊洪崜鎻掍簼婵炲弶绮岄悾鍨瑹閳ь剟寮诲☉銏犵労闁告劦浜栧Σ鍫㈢磼閻愵剙鍔ら柛姘儔楠炲牓濡搁埡鍌涙珳闁硅偐琛ラ埀顒€纾鎰磽閸屾艾鈧摜绮旈幘顔芥櫇妞ゅ繐鐗忓畵渚€鏌涢幇闈涙灈闁绘挻鐩弻娑樷槈閸楃偛绠诲銈嗘煥閻倸顫忓ú顏勬嵍妞ゆ挾鍋涙俊铏圭磽娴g瓔鍤欓柛濠咁潐缁岃鲸绻濋崶銊у弳闁诲函缍嗛崜娑㈡儊閸儲鈷戞慨鐟版搐閻忓弶绻涙担鍐叉閸欐挳姊婚崒娆掑厡閺嬵亝銇勯幋婵囶棦妤犵偞鍨垮畷鍫曨敆閳ь剟鎷戦悢鍏肩厽闁哄倽娉曞▓閬嶆煛鐎n偅顥堥柡灞剧洴閳ワ箓骞嬪┑鍥╀憾闂備浇顕х换鎴﹀箰閸撗勵潟闁圭儤鎸堕崑鍛存煕閹板吀绨介柡鍡╁亞缁辨挻鎷呴崫鍕戯綁鏌eΔ鍐ㄐ㈡い鏇稻缁绘繂顫濋鐐扮盎闂佽崵鍋炵粙鍫ュ焵椤掆偓閸樻牗绔熼弴銏♀拺婵懓娲ら悘顔界箾閸涱偄鍔﹂柟顔惧厴閹囧醇閻旂儤顫栭梻鍌氬€峰ù鍥х暦閻㈢ǹ绐楅柟閭﹀枛閸ㄦ繈骞栧ǎ顒€鐏繛鍛У娣囧﹪濡堕崨顔兼闂佺ǹ顑呴崐鍧楀蓟閵娾晜鍋勯柛婵嗗珔閵忋倖鐓㈤柛鎰典簻閺嬫垹绱掔紒妯兼创妤犵偛顑呴埢搴ょ疀閺囨碍鍋呯紓鍌氬€烽懗鑸垫叏閸偆绠惧┑鐘叉搐閽冪喖鏌ㄩ悢鍝勑㈢痪鎯у悑閹便劌螣閸ф鎽甸梺姹囧妽閸ㄥ灝顫忓ú顏勪紶闁告洟娼ч崜浼存⒑閻熸澘鏆遍梺甯秮瀹曟椽鎮欓崫鍕吅闂佹寧妫佸Λ鍕焵椤掑倸鍘撮柡灞剧☉閳诲氦绠涢敐鍠帮箑鈹戦悙鍙夊珔缂傚秳绀侀~蹇曠磼濡顎撶紓浣割儓濞夋洟鎯勬惔锝囩=濞达綀妫勯惃娲煕鐎n偅灏电紒顔界濞煎繘濡歌閻﹀牓姊洪幖鐐插妧闁告粈绀侀弲顏勨攽閿涘嫬浜奸柛濠冪墪铻炲ù锝堫潐閸欏繘鏌曢崼婵囧櫝闁哄绉归弻鏇$疀鐎n亖鍋撻弽顓炵厱闁硅揪闄勯崑锝夋煙椤撶喎绗掑┑鈥冲悑缁绘盯宕ㄩ鐓庮潚闂佸搫鐬奸崰鏍х暦濞嗘挸围闁糕剝顨忔导锟�Correspondence to: Monique Arpin, Laboratoire de Morphogénèse et Signalisation Cellulaires, UMR 144 CNRS/Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France. Tel:33 1 42 34 63 72 Fax:33 1 42 34 63 77 E-mail:marpin@curie.fr.
Abstract
ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH2- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.
Key Words: ERM, head-to-tail interaction, conformation, actin, cytoskeleton
Introduction
ERM (ezrin, radixin, moesin)1 proteins act as linkers between the plasma membrane and the actin cytoskeleton. Inactivation studies indicated that these proteins play a role in the formation of microvilli, cell–cell junctions, and membrane ruffles, and also regulate substrate adhesion and motility (Takeuchi et al. 1994 ; Crepaldi et al. 1997 ; Lamb et al. 1997 ). Regulation of ERM linker function is thought to occur through conformational changes (Bretscher 1999 ).
ERM proteins possess two conserved domains. The NH2-terminal domain is responsible for membrane targeting, whereas the COOH-terminal domain contains an F-actin binding site (Algrain et al. 1993 ; Turunen et al. 1994 ). These two domains interact strongly with each other, and have been termed N- and C-ERMADs, standing for ERM association domain (Gary and Bretscher 1995 ; Magendantz et al. 1995 ). In ezrin, N-ERMAD has been mapped to the first 296 amino acids and C-ERMAD to the last 107 amino acids. Because of the intramolecular N/C-ERMAD interaction, most ERM proteins are in a cytosolic dormant form, in which binding sites for membrane components and F-actin are masked. In the NH2-terminal domain, cryptic binding sites have been identified for Rho-GDI and EBP-50 proteins (Reczek et al. 1997 ; Takahashi et al. 1997 ; Reczek and Bretscher 1998 ). Recently, the crystal structure of the moesin N-ERMAD bound to the C-ERMAD revealed a globular conformation for the N-ERMAD domain and an extended conformation for the C-ERMAD, which mutually mask binding sites (Pearson et al. 2000 ).
Intermolecular N/C-ERMAD interactions also form ERM oligomers. In purified placental microvilli, ezrin dimers, trimers, tetramers, and higher order oligomers were identified, suggesting a head-to-tail assembly (Berryman et al. 1995 ). These oligomers are proposed to be associated with the cytoskeleton and to be involved in microvillar morphogenesis. However, soluble ezrin dimers were also detected (Bretscher et al. 1995 ). Oligomerization is not specific to ezrin, since mixed oligomers containing different ERM members were observed (Gary and Bretscher 1993 ; Andreoli et al. 1994 ). To engage ERMADs in intermolecular interactions, cytosolic dormant monomers are thought to be subjected to a gross conformational change. This phenomenon does not occur spontaneously in vitro with purified ezrin, and probably requires an activation step (Bretscher et al. 1995 ).
Phosphorylation has been proposed to regulate ERM activation, since phosphorylation of ERM proteins correlates with their cytoskeletal association (Chen et al. 1994 ; Kondo et al. 1998 ; Simons et al. 1998 ). Ezrin is phosphorylated on tyrosine residues upon growth factor stimulation (Gould et al. 1989 ; Fazioli et al. 1993 ; Crepaldi et al. 1997 ). In response to EGF, ezrin phosphorylation on tyrosines 145 and 353 is concomitant with an increase in dimer formation, suggesting a causal relationship between phosphorylation and oligomerization (Krieg and Hunter 1992 ; Berryman et al. 1995 ). However, mutations of these tyrosines into phenylalanines does not alter ezrin localization in microvilli, and production of this mutated ezrin does not affect cell morphology (Crepaldi et al. 1997 ). Rather than controlling its cytoskeletal association, tyrosine phosphorylation of ezrin appears to transduce signals. For example, phosphorylation of tyrosine 353 was found to signal cell survival during epithelial differentiation (Gautreau et al. 1999 ).
Another phosphorylation site is a better candidate to activate ERM cytoskeletal linkage. A phosphothreonine residue, originally identified in moesin (Nakamura et al. 1995 ), is localized in a conserved COOH-terminal region of ERM proteins (T567 in ezrin, T564 in radixin, and T558 in moesin). Using phosphospecific antibodies, this phosphorylated residue was detected in ezrin, radixin, and moesin from a variety of cells and tissues, and phosphorylated ERM proteins were shown to be present in actin-rich membrane structures (Nakamura et al. 1996 ; Matsui et al. 1998 ; Oshiro et al. 1998 ; Hayashi et al. 1999 ). Two kinases, protein kinase C-theta (PKC-) and Rho-kinase, and two phosphatases, myosin phosphatase and type 2C protein phosphatase (PP2C), were found in different systems to regulate the phosphorylation status of this conserved threonine in ERM proteins (Fukata et al. 1998 ; Matsui et al. 1998 ; Pietromonaco et al. 1998 ; Hishiya et al. 1999 ).
The primary consequence of this phosphorylation event is to impair N/C-ERMAD interaction. In an overlay assay, phosphorylation of T564 in radixin COOH-terminal domain impairs its association with the NH2-terminal domain (Matsui et al. 1998 ). Similarly, the T558D mutation of moesin, which mimics the phosphorylated state, was shown to affect the N/C-ERMAD interaction (Huang et al. 1999 ). From the crystal structure, it appears that the phosphorylation of moesin T558 weakens the N/C-ERMAD interaction due to both electrostatic and steric effects (Pearson et al. 2000 ). The phosphorylation of an isolated COOH-terminal fragment of ERM proteins does not affect its association with F-actin (Matsui et al. 1998 ; Huang et al. 1999 ). However, in full-length ERM proteins, phosphorylation of this conserved threonine is required to bind to F-actin (Simons et al. 1998 ; Hishiya et al. 1999 ; Nakamura et al. 1999 ). These results suggest that phosphorylation of this residue activates ERM cytoskeletal association by unmasking the cryptic F-actin binding site. Furthermore, expression of T into D mutant forms of ezrin or moesin potentiates the formation of microvilli-like dorsal projections by growth factors (Oshiro et al. 1998 ; Yonemura et al. 1999 ), whereas transfection of the nonphosphorylatable T558A moesin inhibits RhoA-induced formation of these structures (Oshiro et al. 1998 ; Shaw et al. 1998 ).
Although phosphorylation of this conserved threonine residue regulates the activation of ERM cytoskeletal linkers, the mechanism of this regulation is still poorly understood. By disrupting N/C-ERMAD interaction, this phosphorylation event could trigger the opening of dormant monomers, could impair oligomerization, or both. To clarify the mechanism of ERM conformational regulation, we analyzed the role of ezrin T567 phosphorylation in LLC-PK1 epithelial cells. We found that T567D ezrin exhibited a drastic reduction in the amount of oligomers at the plasma membrane. Monomeric T567D ezrin was associated with the actin cytoskeleton and induced actin-rich membrane projections. Production of T567D ezrin strongly affected epithelial morphology and differentiation. In contrast, T567A ezrin exhibited a level of membrane oligomers similar to wild-type (wt), but was poorly associated with the actin cytoskeleton. These results suggest that phosphorylation of this conserved threonine regulates a membrane-specific transition from oligomers to monomers, which are active plasma membrane–actin cytoskeleton linkers.
Materials and Methods
Cells and Recombinant Proteins
LLC-PK1 cells (CCL 101; American Type Culture Collection) were cultured in DME containing 10% FCS and maintained at 37°C in 10% CO2. Recombinant NH2-terminal fragment 1–309 of ezrin was produced and purified as a GST fusion as previously described (Gautreau et al. 1999 ). GST moiety was cleaved off by thrombin digestion. Recombinant NH2-terminal fragment was biotinylated with NHS-LC-biotin (Pierce Chemical Co.) according to the manufacturer's instructions.
cDNA Constructs and Transfection
To substitute T567 with A567 or D567, PCR reactions were performed with oligonucleotides in which the codon ACG was replaced by GCG or GAC, respectively. The amplified fragments were subcloned into the pCB6 vector containing VSV G-tagged ezrin cDNA (Algrain et al. 1993 ). Myc-tagged ezrin was cloned in pCDNA 3.1 vector (Invitrogen). All PCR fragments were verified by sequencing.
For transfection, trypsinized cells were resuspended at a concentration of 2.5 x 107 cells/ml in 15 mM Hepes, pH 7.4, buffered medium. 200 μl of cell suspension was added to 50 μl of a solution containing 210 mM NaCl, 5 μg of plasmid DNA, and 30 μg of salmon sperm DNA carrier (Sigma-Aldrich). LLC-PK1 cells were electroporated with a BioRad Gene Pulser at 950 μF and 240 V using 4-mm width cuvettes. Transiently transfected cells were analyzed after 48 h of cDNA expression. Clones producing T567A and T567D ezrin were established as previously described, and were compared with the previously obtained clones transfected with the empty plasmid or producing wt ezrin (Crepaldi et al. 1997 ).
Cytosol/Membrane Fractionation and Gel Filtration Analysis
Cells from a confluent 10-cm dish (for standard immunoprecipitation), or from ten confluent 10-cm dishes (for the Coomassie blue-stained immunoprecipitation experiment or gel filtration analysis), were rinsed once with cold PBS, once with cold cyt buffer (10 mM Hepes, 1 mM EDTA, 150 mM NaCl, pH 7.4), and scraped off with a rubber policeman in 1 ml of cold cyt buffer supplemented with protease inhibitors (200 μg/ml pefabloc, 15 μg/ml benzamidine, 1 μg/ml pepstatin, 1 μg/ml antipain). Cells in suspension were mechanically disrupted by 10 strokes of a cell cracker. Debris and nuclei were pelleted by a 10-min centrifugation at 600 g at 4°C. The supernatant was then subjected to a 20-min centrifugation at 100,000 g using a TLA-120.2 rotor in an optima TLX ultracentrifuge (Beckman Coulter). This ultracentrifugation pellets crude membranes, whereas the supernatant is the cytosolic fraction.
For gel filtration analysis, membrane pellets were further extracted by a 15-min incubation in 200 μl of mbn buffer (10 mM Hepes, 1 mM EDTA, 600 mM KCl, 1% Triton X-100, pH 7.4) supplemented with protease inhibitors, and then ultracentrifuged again. 200 μl of cytosol or membrane extracts were loaded onto a superose-6 HR10/30 gel filtration column (Amersham Pharmacia Biotech), preequilibrated with cyt or mbn buffer respectively, and run at a flow rate of 300 μl/min. 250 μl fractions were collected. The column was calibrated with thyroglobulin, ferritin, aldolase, and BSA (Sigma-Aldrich) as standards. Thyroglobulin (Stokes radius of 85 ?), ferritin (61 ?), aldolase (48 ?), and BSA (35.5 ?) peaked at fractions 24, 32, 41, and 43, respectively. Void volume of the column emerged in fraction 3.
When endogenous ERM phosphorylation was examined, cyt and mbn buffers were also supplemented with 2 mM of sodium pyrophosphate and 1 μM of calyculinA (Upstate Biotechnology). When indicated, LLC-PK1 cells were pretreated for 10 min at 37°C with 300 nM of calyculinA. For oligomer and monomer samples of Fig 2, 150 μl of fractions 24–27 and 150 μl of fractions 35–38 were pooled and precipitated by 2 min at 100°C, followed by 10 min at 4°C. For efficient precipitation of the oligomeric fraction, 15 μg of BSA was added before boiling, because this fraction contained a low amount of proteins. The precipitates were pelleted by a 10-min centrifugation at 20,000 g at 4°C, and resuspended in SDS-loading buffer.
Figure 1. T567D ezrin has a reduced N-ERMAD binding activity in vitro, and does not oligomerize with moesin at the membrane in vivo. Throughout this study, clones producing VSV G-tagged wt ezrin, T567A ezrin, T567D ezrin (E, A, and D cells, respectively) or clones obtained after transfection of the empty plasmid (P cells) were compared. (A) Denatured extracts from P, E, A, and D clones were used to immunoprecipitate ezrin or VSV G-tagged wt, T567A, and T567D ezrin. The immunoprecipitates were probed with ezrin antibodies or with biotinylated ezrin N-ERMAD (1–309). (B) Ezrin from P, E, A, or D cell membrane extracts was immunoprecipitated with either anti-ezrin antibodies or anti-VSV G antibodies as indicated. After SDS-PAGE, the immunoprecipitates were stained by Coomassie blue to reveal ezrin (top), or immunoblotted with moesin-specific antibodies (bottom). Moesin associated with ezrin can be seen in the Coomassie blue–stained gel as a faint band just below the strong ezrin band.
Figure 2. T567D ezrin and endogenous phosphorylated ERM proteins are preferentially monomeric at the plasma membrane. A, Membrane or cytosolic extracts from P, E, A, and D cells were resolved by gel filtration chromatography on a superose-6 column. Fractions 15–41 were analyzed by SDS-PAGE and immunoblotted with antiezrin antibodies (P) or anti-VSV G antibodies (E, A, and D). T567D ezrin exhibited a strongly reduced amount of oligomers at the membrane, but not in the cytosol. B, Total, cytosolic, and membrane extracts were immunoblotted with 297S mAb, recognizing all three ERM proteins when phosphorylated on this conserved threonine (pERM), or with a mixture of antibodies specific for ERM. Ezrin and radixin comigrated at 80 kD, and moesin migrated at 75 kD. Phosphorylated ERM proteins were strongly enriched in the membrane fraction. C, LLC-PK1 cells were pretreated with calyculinA, a protein phosphatase inhibitor, and the membrane extract was resolved by gel filtration chromatography. Oligomeric and monomeric fractions were pooled, and immunoblotted as in B. Monomers were preferentially phosphorylated over oligomers.
Immunoprecipitations
Cytosol or total extracts were adjusted to 1 ml of RIPA buffer (50 mM Hepes, 10 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4). Membrane pellets were resuspended in RIPA buffer. RIPA extracts were supplemented with protease inhibitors. For the experiment in which denatured extracts are used, cells were lysed in 1 ml of 50 mM Hepes, 10 mM EDTA, 150 mM NaCl, 1% NP-40, pH 7.4, buffer. The extracts were adjusted to 1% SDS and boiled for 2 min. Denatured extracts were then put on ice for 10 min and diluted to 10 ml with a cold buffer reconstituting RIPA composition.
Extracts were clarified by 10 min centrifugation at 20,000 g at 4°C, and incubated with 10 μl of protein A–Sepharose fast-flow beads (Amersham Pharmacia Biotech) and 5 μg of affinity-purified ezrin or VSV G rabbit polyclonal antibodies for 2 h (1 ml vol) or overnight (10 ml vol). Beads were washed five times with 1 ml of RIPA buffer, and boiled 2 min in SDS loading buffer.
Overlay and Western Blotting
All blots were performed on nitrocellulose membranes (Protran Hybond). P5D4 anti-VSV G mAb, 9E10 anti-myc mAb, phosphospecific 297S mAb (a kind gift of Dr. S. Tsukita, Kyoto University, Japan), anti-moesin affinity-purified mouse polyclonal antibodies, anti-radixin affinity-purified guinea pig polyclonal antibodies (both kind gifts of Dr. P. Mangeat, University of Montpellier, France), anti-ezrin affinity-purified rabbit polyclonal antibodies (Algrain et al. 1993 ), or biotinylated NH2-terminal domain of ezrin were used as primary reagents, and alkaline phosphatase-coupled immunoglobulins or streptavidin, as secondary reagents (Promega). Blots were developed with nitroblue tetrazolium/5-bromo, 4-chloro, 3-indolyl phosphate as substrates (Promega). The intensities of bands were quantitated by scanning densitometry on a Bio-Profil station (Vilbert-Lourmat).
Immunofluorescence and Microscopy
Cell morphology was examined by phase-contrast optics or by Nomarski optics on a Leica microscope. For scanning EM, cells grown at confluence on 10-mm 0.2 μm anopore membrane filters (Nunc) were dehydrated in graded ethanol baths, dried by the critical point method using liquid CO2, coated with gold palladium, and observed with a JEOL microscope (JSM 840A).
Immunolocalization of exogenous ezrin was achieved with VSV G affinity-purified polyclonal antibodies at 10 μg/ml and Cy-2–conjugated goat anti–rabbit secondary antibodies (Jackson ImmunoResearch Laboratories) on fixed and permeabilized cells using the paraformaldehyde/Triton X-100 protocol previously described (Crepaldi et al. 1997 ). Samples were examined with a Leica confocal laser-scanning microscope, using the same settings for all acquisitions.
Analysis of Cytoskeletal Fraction
Confluent cultures from 6 well plates were rinsed quickly with PBS at room temperature. The soluble fraction was extracted by a 1-min incubation with 500 μl of a Triton X-100 buffer that preserves cytoskeleton-associated material (csk buffer: 50 mM MES, 3 mM EGTA, 5 mM MgCl2, 0.5% Triton X-100, pH 6.4) at room temperature. For immunofluorescence analysis, the insoluble material was immediately fixed in 3% paraformaldehyde. We verified that actin and microtubule cytoskeletons were not affected by this treatment. For immunoblot analysis, the insoluble material was quickly rinsed with 500 μl of csk buffer, and further extracted by a 1-min incubation with 500 μl of ice-cold RIPA buffer. When endogenous ERM phosphorylation was examined, csk and RIPA buffers were also supplemented with 2 mM of sodium pyrophosphate and 1 μM of calyculinA.
Development of Multicellular Epithelial Structures
For the suspension cyst assay, 25-ml siliconized erlenmeyers containing 5 ml of a cell suspension at 106 cells/ml in 15 mM Hepes, pH 7.4, buffered medium were rocked in a shaking incubator at 75 rpm (ROSI 1000; Thermolyne). Cultures were analyzed after 48 h. For the tubulogenesis assay, cells were seeded in a collagen type I gel, as previously described (Gautreau et al. 1999 ). 1-wk cultures, in DMEM containing 10% FCS and 100 U/ml of HGF, were examined. Importantly, in these two assays, all clones isolated in each category behaved similarly.
Results
T567D Ezrin Exhibits a Low Amount of Oligomers at the Membrane
We chose to study ezrin function in LLC-PK1 epithelial cells, which are derived from kidney proximal tubule. This cell line retains many features of proximal tubule cells, since it harbors numerous microvilli at its apical surface, and coexpresses ERM proteins (Berryman et al. 1993 ). To analyze the role of ezrin T567 phosphorylation, cDNAs encoding a nonphosphorylatable variant, T567A ezrin, and a pseudophosphorylated variant, T567D ezrin, were expressed in LLC-PK1 cells. We derived stably transfected clonal cell lines producing wt, T567A, or T567D ezrin, called E, A, or D cells, respectively, and we compared them to control clones transfected with the empty plasmid, called P cells. The exogenous proteins were tagged at the COOH terminus with a VSV G epitope so they could be readily distinguished from the endogenous ezrin. The amount of exogenous ezrin relative to endogenous ezrin was estimated to be about tenfold higher in the isolated clones, by immunoblotting serially diluted extracts with the antiezrin antibody (data not shown).
To assess if the T567D mutation of ezrin impairs the N/C-ERMAD interaction, we analyzed wt, T567A, and T567D ezrin for their binding to biotinylated N-ERMAD in a blot overlay assay. We immunoprecipitated endogenous ezrin or VSV G-tagged ezrin variants from denatured lysates derived from P, E, A, and D cells. This allowed the isolation of ezrin without associated proteins. An equivalent amount of ezrin and VSV G-tagged wt, T567A, and T567D ezrin was immunoprecipitated as revealed by ezrin immunoblotting (Fig 1 A). The endogenous ezrin and VSV G-tagged wt ezrin bound strongly to the N-ERMAD, indicating that the VSV G tag has little influence, if any, on the N/C-ERMAD interaction. In contrast to wt and T567A ezrin, which bind efficiently to the ezrin N-ERMAD, the ability of T567D ezrin to interact with N-ERMAD was substantially diminished. This result suggests that the T567D mutation of ezrin mimics the phosphorylation of the C-ERMAD, at least in its disruptive effect on the N/C-ERMAD interaction (Matsui et al. 1998 ; Huang et al. 1999 ; Pearson et al. 2000 ).
In our current view of ERM activation, a discrepancy remains unresolved. On one hand, the active ERM protein, which is bound to the plasma membrane and the actin cytoskeleton, is expected to be oligomeric (Bretscher 1999 ). On the other hand, an ERM protein requires the phosphorylation of this conserved threonine of the COOH-terminal domain to bind to F-actin (Nakamura et al. 1999 ). However, phosphorylation might disrupt the oligomeric form, through its impairment of intermolecular N/C-ERMAD interaction. To examine ERM oligomerization, we looked at whether the ezrin variants were associated with endogenous moesin. We immunoprecipitated ezrin from the membrane pool to enrich for the active form. The immunoprecipitates were either stained with Coomassie blue or immunoblotted with moesin-specific antibodies (Fig 1 B). A high amount of ezrin was precipitated with anti-ezrin or anti-VSV G antibodies, as seen by Coomassie blue staining. Moesin coprecipitated with endogenous ezrin, exogenous wt, and T567A ezrin, but not with T567D ezrin. This lack of hetero-oligomerization of T567D ezrin with moesin in the membrane fraction suggests that the T567D mutation of ezrin impairs intermolecular N/C-ERMAD interactions. An association with radixin could not be determined, because the immunoprecipitated ezrin gave a high background at the position of radixin, which migrates only slightly faster than ezrin in SDS-PAGE.
To examine directly the oligomeric status of T567 mutant forms of ezrin, we used a procedure that resolves ezrin oligomers from monomers (Berryman et al. 1995 ). First, we examined the distribution of endogenous ezrin in P cells. Ninefold less ezrin is found in the membrane fraction compared with the cytosolic fraction (Table 1). Membrane and cytosolic extracts from the P cells were applied to a superose-6 gel filtration column. Oligomers were detected in the cytosol, as well as in the membrane fraction (Fig 2 A). Even if most oligomers were cytosolic (Table 1), the relative level of oligomers over the total ezrin at the membrane was about twofold that in the cytosol (28% of oligomers at the membrane vs. 15% of oligomers in the cytosol). Then, we examined the oligomer profile for the ezrin variants (Fig 2 A). In the membrane extracts, ezrin oligomers were observed with wt and T567A ezrin. However, consistent with its lack of association with moesin, T567D ezrin from the membrane fraction was eluted essentially as monomers, with only a trace amount of oligomers. Interestingly, in the cytosol, no differences between ezrin variants were noted. Thus, T567D ezrin exhibited a strong reduction in the amount of oligomers exclusively at the membrane.
Table 1. Quantification of the Ezrin Content in the Different Pools
Phosphorylated ERM Proteins Are Preferentially Monomeric at the Membrane
The analysis of T567D ezrin indicated that the effect of phosphorylation occurs at the membrane. To get insight into the distribution of phosphorylated endogenous ERM proteins from LLC-PK1 cells, we used 297S, an mAb recognizing this conserved phosphorylated threonine of ERM COOH-terminal domain (Matsui et al. 1998 ). We examined the distribution of phosphorylated ERM proteins between the membrane and the cytosol. When a similar amount of ERM proteins from total, cytosol, and membrane fractions was blotted with 297S, phosphorylated ERM proteins appeared highly enriched in the membrane fraction (Fig 2 B). This result is consistent with the membrane-dependent effect of the ezrin T567D mutation.
Because the T567D mutation reduced the amount of ezrin oligomers at the membrane, we hypothesized that the phosphorylation of this threonine dissociates ezrin oligomers into monomers. If this hypothesis is correct, monomers should be more phosphorylated than oligomers. Our initial attempts to determine which of the monomers or the oligomers were preferentially phosphorylated failed because of dephosphorylation during the gel filtration procedure. To overcome this problem, we pretreated LLC-PK1 cells with calyculinA, a serine/threonine protein phosphatase inhibitor known to affect moesin phosphorylation (Nakamura et al. 1995 ). This treatment enhanced the phosphorylation signal and preserved it during gel filtration. Consistent with the preferential distribution of T567D ezrin in the monomeric fraction, ERM monomers were found to be preferentially phosphorylated over oligomers (Fig 2 C).
Membrane/Cytosol Distribution of T567D Ezrin Is Regulated through its Functional C-ERMAD Domain
These results suggest that phosphorylation of ezrin occurs at the membrane, and dissociates oligomers, through an impairment of intermolecular N/C-ERMAD interaction at the membrane. However, we were intrigued by the fact that T567D ezrin was not defective in cytosolic oligomerization. This result is compatible with the hypothesis that the C-ERMAD of T567D ezrin is functional for oligomerization in the cytosol, but not at the membrane. To confirm in vivo that T567D ezrin has a functional C-ERMAD in the cytosol, we compared T567D ezrin to 29 ezrin, a form in which the 29 COOH-terminal amino acids were eliminated. Such a deletion completely abrogates C-ERMAD activity, i.e., N-ERMAD binding (Gary and Bretscher 1995 ; data not shown). We devised a sensitive assay to study specifically ezrin homo-oligomerization. We transiently cotransfected LLC-PK1 cells with two ezrin cDNAs, one of them being tagged by the VSV G epitope, the other by the myc epitope, and examined the amount of ezrin–myc coprecipitating with ezrin–VSV G.
We analyzed the distribution of produced proteins between the membrane and cytosol pools (Fig 3). Half of the transfected cell sample was analyzed directly (total), and the other half was separated in cytosolic and membrane fraction before analysis, so that the three pools were comparable. In total lysates, similar amounts of wt, T567A, T567D, and 29 ezrin were detected. In all transfections, a similar amount of ezrin–myc was produced. Analysis of cytosolic and membrane fractions revealed that wt, T567A, and T567D ezrin were similarly distributed between cytosol and membrane, most of ezrin being in the cytosol. In contrast, 29 ezrin was highly enriched in the membrane fraction. VSV G-tagged proteins were immunoprecipitated, and the immunoprecipitates were analyzed by immunoblotting with either anti-VSV G or antimyc antibodies. In total lysates, wt, T567A, and T567D ezrin oligomerized to roughly the same extent, whereas 29 ezrin was completely unable to form oligomers. T567A ezrin exhibited no difference with wt ezrin in its oligomerization ability in the cytosol and at the membrane. Consistent with gel filtration analysis, oligomers of T567D ezrin were present in the cytosol, but only as a trace amount at the membrane.
Figure 3. In the cytosol, T567D ezrin has a functional C-ERMAD. Various VSV G-tagged cDNAs, wt, T567A, and T567D ezrin, or an ezrin construct lacking a C-ERMAD due to the deletion of the 29 COOH-terminal amino acids (29), were cotransfected with myc-tagged wt ezrin cDNA into LLC-PK1 cells to detect oligomerization. Total, cytosol, or membrane lysates (top) or VSV G immunoprecipitates of these lysates (bottom) were analyzed by immunoblotting with anti-VSV G or anti-Myc antibodies as indicated on the right of each panel. 29 ezrin was strongly enriched in the membrane fraction, and completely defective in oligomer formation. In contrast, T567D ezrin was correctly distributed between the cytosolic and the membrane fraction, and exhibited a strongly reduced amount of oligomers specifically at the membrane.
These data suggest that a functional C-ERMAD is needed both to mask membrane binding sites in the NH2-terminal domain, and to form oligomers. T567D ezrin has a functional C-ERMAD in the cytosol, since T567D ezrin forms cytosolic oligomers and is correctly distributed between the cytosol and the membrane. However, upon membrane recruitment, the N/C-ERMAD interaction is abolished by the T567D mutation, and membrane oligomers of T567D ezrin are dissociated.
Dramatic Morphological Changes of LLC-PK1 Cells Producing T567D Ezrin
We asked whether there was a consequence of producing monomeric T567D ezrin on actin-rich membrane structures. We examined the morphology of LLC-PK1 clones producing wt, T567A, or T567D ezrin and the control clones transfected with the empty plasmid. E clones formed typical epithelial islets in sparse cell culture (Fig 4 A), as did A and P clones (data not shown). These islets were composed of cells adhering to each other. The islet periphery was regular. In sharp contrast, all the D clones had an altered morphology. Some space between cells could be distinguished. The edges of D colonies were not smooth, but interrupted by wide lamellipodia. Those lamellipodia were sometimes at the tip of long extensions. By phase contrast, a refractile relief was also prominent at the position of the nucleus in most D cells. By scanning EM, this relief was shown to be due to extensive membrane ruffling (Fig 4 B).
Figure 4. Morphology of LLC-PK1 cells producing T567D ezrin. (A) Clones were examined by phase-contrast optics. P, E, and A cells grew in typical LLC-PK1 islets (only the E control is presented). D colonies exhibited a number of morphological changes. In D colonies, cells were not always adherent to each other. The periphery of D colonies was irregular with wide lamellipodia (arrowheads). Those lamellipodia were occasionally formed at the extremity of long extensions (arrow). In most D cells, the membrane area around the nuclei was highly refractile. Bar, 25 μm. (B) Scanning EM examination of E and D cell morphology. Extensive membrane ruffling was observed in the cell central area, probably corresponding to the refractility observed by phase-contrast optics. Bars: (A) 25 μm; (B) 1 μm.
We observed confluent cultures of P, E, A, and D cells for optimal epithelial polarization and development of microvilli. Microvilli containing apical surface of these cells were analyzed by scanning EM. Production of wt or T567A ezrin did not alter microvillar density, length or organization, which were similar to those of control P cells (Fig 5). In these confluent cultures, D cells formed a less organized layer of cells. The layer of flat cells was often interrupted by holes that exposed the filter surface, and some round cells attached above this flat cell layer were frequently observed. We verified that these round structures were indeed cells by fluorescent staining of nuclei (data not shown). Flat cells were covered with microvilli similar to controls, whereas round cells were covered with denser and longer microvilli. Occasionally, on the flat cell layer, microvilli developed aberrantly into tufts (Fig 5). Such a tree-like organization of microvilli was never observed in P, E, or A cultures. In conclusion, production of monomeric T567D ezrin in LLC-PK1 cells induces numerous actin-rich membrane structures, such as lamellipodia, ruffles, and projections that are covered with microvilli.
Figure 5. Scanning EM analysis of microvilli in clones producing T567A and T567D ezrin. Monolayers of P, E, and A cells exhibited comparable microvilli in density and length. In D cultures, above a layer of flat cells, which contain comparable microvilli to P, E, and A cells, some round cells containing numerous and long microvilli were frequently observed. In addition, tufts of microvilli occasionally emerged from flat D cells (right). Bars, 1 μm.
Ezrin Association with the Actin Cytoskeleton Requires Phosphorylation of T567
Because these ezrin variants displayed differential capacity to affect membrane morphogenesis, we compared their ability to associate with the actin cytoskeleton. Localization of exogenous ezrin by immunofluorescence was performed with anti-VSV G antibodies on confluent cultures of E, A, and D cells. Wt and T567A ezrin were detected in microvilli (Fig 6 A), in a pattern similar to the one of endogenous ezrin in LLC-PK1 cells (data not shown). In the D cultures, T567D ezrin was found in microvilli of both flat and round cells and in ruffles. To evaluate whether these ezrin variants were associated with the actin cytoskeleton, we used an extraction procedure with a Triton X-100 buffer, which preserves the cytoskeleton and cytoskeleton-associated proteins. After extraction, wt ezrin and T567D ezrin were still detected in microvilli. In sharp contrast, T567A ezrin was almost completely extracted. Consistently, when Triton X-100 buffer–extracted material and cytoskeleton-associated material were compared by immunoblotting, T567A ezrin was highly extracted (Fig 6 B). After densitometry of the signals, insoluble to soluble ratios were calculated. For exogenous ezrin, as well as endogenous ezrin, the insoluble pool was always less than the soluble pool. However, T567A ezrin was significantly less insoluble than endogenous ezrin, exogenous wt ezrin, or T567D ezrin (P < 10-3, ANOVA followed by a Bonferroni t test). Therefore, T567A ezrin binds inefficiently to the actin cytoskeleton. Then we examined the distribution of endogenous phosphorylated ERM proteins. Consistently, phosphorylated ERM proteins are enriched in the Triton X-100–insoluble fraction. Taken together, these results indicate that the phosphorylation of the C-ERMAD is required for the association of ERM proteins with the actin cytoskeleton.
Figure 6. Ezrin association with the actin cytoskeleton requires phosphorylation of T567. (A) Localization of ezrin variants with anti-VSV G antibodies by immunofluorescence and confocal microscopy in E, A, or D cells. Cells were also stained after extraction with a Triton X-100 buffer that preserves cytoskeleton-associated material (csk). A single apical section is shown. Wt, T567A, and T567D ezrin were observed in microvilli. T567D ezrin was also present in the membrane ruffles it induced. After extraction of ezrin-soluble pool, wt and T567D ezrin staining were preserved, whereas T567A ezrin staining was strongly reduced. Bar, 5 μm. (B) Western blot analysis of ezrin cytoskeletal association. Similar fractions of soluble material (Sol), extracted with the Triton X-100 buffer, and insoluble material (Ins), were immunoblotted with anti-ezrin antibodies for P cell extract or with anti-VSV G antibodies for E, A, and D cell extracts. A densitometric analysis was performed and the Ins/Sol ratio was calculated from data obtained from two to four independent experiments with three different A and D clones (mean ± SEM). (C) Soluble and insoluble fractions from LLC-PK1 cells were equalized for their ERM content and immunoblotted with either 297S mAb (pERM) or ERM antibodies. Phosphorylated ERM proteins are enriched in the insoluble fraction.
Effect of T567A and T567D Ezrin on the Development of Multicellular Epithelial Structures
Organization of the actin cytoskeleton is a crucial point for the establishment and the maintenance of epithelial polarity. Ezrin has been implicated in the development of multicellular epithelial structures (Crepaldi et al. 1997 ; Gautreau et al. 1999 ). Therefore, we investigated whether production of T567A ezrin or T567D ezrin affects the morphogenesis of LLC-PK1 cells into suspension cysts and into tubules (Fig 7).
Figure 7. Production of T567A and T567D ezrin affects the development of multicellular epithelial structures. (A) Morphogenesis of suspension cysts examined by phase-contrast optics. Aggregates of LLC-PK1 cells in suspension are able to form hollow epithelial cysts. P, E, and A cells were not affected in this process, whereas D cells formed loose aggregates in which individual cells could still be distinguished at the periphery. (B) Tubulogenesis assay examined by Nomarski optics. In three-dimensional collagen type I, in the presence of HGF, P cells are able to differentiate into multicellular tubules. Production of wt ezrin potentiated growth and branching morphogenesis of tubules. Production of both T567A and T567D ezrin impaired tubulogenesis. A cells exhibited a growth defect, whereas D cells grew in disorganized colonies. Bars, 50 μm.
When isolated LLC-PK1 cells are put in suspension, they aggregate. These aggregates compact in an epithelial cyst with a smooth outline (Wohlwend et al. 1985 ). By two days, one or several cavities form in these cysts. Cavitation is a hallmark of the development of epithelial polarity. In the hollow cyst, the apical pole is in contact with the medium, whereas the basal pole is in contact with the cavity (Wohlwend et al. 1985 ). Consistent with the physiology of kidney proximal tubule, formation of this cavity is thought to reflect the vectorial transport of solutes and water from the medium. P, E, and A cells were similarly efficient in the development of hollow suspension cysts. In contrast to these cells, D cells were able to aggregate, but remained in clumps, in which individual round cells could still be distinguished at the periphery. Neither compaction nor cavitation occurred in D cells. This suggests that T567D ezrin impairs the development of epithelial polarity, probably by inducing constant membrane projections.
Consistent with the morphology of kidney proximal tubule, LLC-PK1 cells are able to develop into elongated polarized epithelial structures. Tubulogenesis occurs in a one-week culture in presence of HGF after seeding isolated cells in a three-dimensional collagen type I gel. P cells were able to differentiate into elongated tubules. Overproduction of wt ezrin in E cells potentiated this process and led to long branched tubules, as we previously reported (Crepaldi et al. 1997 ). In this sensitive assay for ezrin function, production of T567A ezrin impaired tubule formation by affecting clonal growth. By counting cell number, we observed that colonies formed by A cells were always composed of less than five cells, instead of tens of cells for P and E tubules (data not shown). In this assay also, production of T567D ezrin severely impairs epithelial organization of colonies. Colonies of D cells developed into loose aggregates with peripheral cells extending processes in the collagen matrix. D colonies never developed along a well-defined axis. In conclusion, we found that production of T567A ezrin affects tubule development, but not suspension cyst morphogenesis, whereas production of T567D ezrin impairs the establishment of epithelial polarity in both assays.
Discussion
In this report, we have examined in vivo the role of ezrin T567 phosphorylation by deriving stable clones producing wt, T567A, and T567D ezrin from the kidney epithelial cell line, LLC-PK1. Since ezrin is present in microvilli both as oligomers and as monomers, it was not known whether the active form of this plasma membrane–actin cytoskeleton linker is monomeric or oligomeric (Berryman et al. 1995 ; Bretscher 1999 ). Our study of T567A and T567D ezrin suggests that the active ezrin linker is a monomer. Given the high conservation of this threonine and of the amino acids forming the interface between the N- and C-ERMAD in ERM from vertebrates, and in homologues from invertebrates, the mechanism of conformational activation by phosphorylation of this conserved threonine from the C-ERMAD probably applies to all members of the ERM family (Pearson et al. 2000 ).
The Active Plasma Membrane–Actin Cytoskeleton Linker Is a Phosphorylated Monomer
T567A ezrin is poorly associated with microvillar cytoskeleton, as evidenced by the fact that this variant is almost completely extracted by a buffer preserving cytoskeleton-associated material, whereas a significant fraction of wt ezrin is not. This observation confirms in vivo the recent finding that phosphorylation of the homologous threonine 558 in moesin is required for F-actin binding in vitro (Hishiya et al. 1999 ; Nakamura et al. 1999 ). Although T567A ezrin is inactive as a cytoskeletal linker, its level of oligomers at the membrane is similar to that of wt ezrin. Thus, the oligomeric species are not sufficient to form active linkers.
The T567D mutation mimics the phosphorylation of this conserved threonine, since both disrupt the N/C-ERMAD interaction in vitro (our results; Matsui et al. 1998 ). In contrast to T567A ezrin, T567D ezrin is associated with the actin cytoskeleton. Moreover, T567D ezrin is a strongly morphogenic variant. It triggers the formation of wide lamellipodia, extensive membrane ruffles, and microvilli-rich projections, in which T567D ezrin is present. This finding is consistent with T567D ezrin being an active cytoskeletal linker. By three independent experiments, gel filtration analysis, assay of ezrin homo-oligomers, and ezrin-moesin hetero-oligomers, the level of T567D ezrin oligomers at the membrane was found to be strongly reduced. Thus, the oligomeric species are not necessary to form active linkers. Our study of these two mutant forms of ezrin, which uncouple oligomerization and cytoskeleton binding, provides strong evidence that the active form of this cytoskeletal linker is a phosphorylated monomer. However, it should be pointed out that phosphorylated active ezrin linkers might represent only a minor fraction of membrane monomers, since T567A ezrin exhibits as high a level of monomers at the membrane as wt ezrin.
To confirm this finding made with these ezrin variants, we have analyzed the endogenous phosphorylated ERM proteins. Consistently, we have found that the phosphorylated ERM proteins consist mainly of monomers at the membrane. Moreover, these phosphorylated ERM proteins are Triton X-100–insoluble, suggesting an association with the actin cytoskeleton. Therefore, it is likely that the T567D mutation mimics the phosphorylated state of ezrin in vivo, as well as in vitro, and thus, represents a useful tool to study ezrin function.
Phosphorylation Impairs N/C-ERMAD Interaction of Membrane-bound ERM Proteins
Since T567D ezrin shows a strongly reduced level of membrane oligomers and that phosphorylated ERM proteins are membrane monomers, it is likely that phosphorylation of this conserved threonine in ERM proteins dissociates membrane-bound oligomers into active monomers. It is striking that the phosphorylation-dependent impairment of N/C-ERMAD interaction occurs only on membrane-bound molecules, since the T567D mutation affects neither formation of cytosolic oligomers, nor the correct distribution between cytosol and membrane. Our in vivo analysis suggests the following model of ERM activation. The phosphorylation of a membrane-bound ERM molecule might disrupt intermolecular N/C-ERMAD interaction, thereby dissociating oligomers, and might prevent intramolecular N/C-ERMAD interaction from reforming, thereby exposing its F-actin binding site.
In vitro, phosphorylated moesin binding to F-actin was also found to be strongly dependent on the addition of phosphatidyl-inositol (4,5) bisphosphate or a charged detergent molecule (Nakamura et al. 1999 ). This result and our in vivo observations suggest that, for the maintenance of the active state, N/C-ERMAD interaction should be abrogated by both phosphorylation of the C-ERMAD and lipid binding to the N-ERMAD. This double regulation of ERM proteins is highly significant for these plasma membrane–actin cytoskeleton linkers. This double regulation might also explain that, despite the strong morphogenic effects of T567D ezrin, no more T567D ezrin than wt ezrin is Triton X-100–insoluble. This suggests that an activation factor other than phosphorylation is limiting the amount of cytoskeleton-bound ezrin. Since inactivation of ERM linkers occurs presumably through dephosphorylation of this conserved threonine (Fukata et al. 1998 ; Hishiya et al. 1999 ), T567D ezrin might be morphogenic, because, in contrast to wt ezrin, this variant cannot be dephosphorylated. The T567D mutation presumably locks the membrane pool of T567D ezrin in its active conformation.
What Is the Role of ERM Oligomers?
Since the dormant cytosolic form of ERM proteins is monomeric and the active plasma membrane–cytoskeleton linker is also monomeric, the role of oligomers in the activation pathway is rather intriguing. Formation of oligomers requires the conformational opening of monomers and the condensation of two, or more, opened monomers. The molecular components of the machinery required for oligomer formation are unknown. Membrane binding sites are cryptic in the dormant monomer, since a deletion of the C-ERMAD is sufficient to allow membrane recruitment of 29 ezrin. This truncated molecule is monomeric, indicating that oligomerization is not required, per se, for membrane recruitment. Also intriguing is the observation that 29 ezrin, despite having an exposed N-ERMAD, does not form oligomers, or at least dimers. One possible explanation is that the machinery for oligomer formation is only present in the cytosol, and is not accessible to the membrane-bound 29 ezrin. Since ezrin oligomers exist in cytosolic and membrane pools, asymmetric oligomers, having an exposed N-ERMAD (Bretscher 1999 ), might be in equilibrium between the cytosol and the membrane.
Phosphorylation of Ezrin on T567 Transduces Morphogenic and Growth Signals
ERM proteins are known to be essential for actin-rich membrane projections (Takeuchi et al. 1994 ; Crepaldi et al. 1997 ; Lamb et al. 1997 ; Bretscher 1999 ). Various stimuli that trigger the formation of these membrane structures induce ERM phosphorylation on this threonine residue (Nakamura et al. 1995 ; Matsui et al. 1999 ; Yonemura et al. 1999 ). Moreover, we showed that the production of T567D ezrin in LLC-PK1 cells induces a variety of actin-rich membrane projections, wide lamellipodia, membrane ruffling, and projections covered with microvilli, appearing as tufts of microvilli. Therefore, phosphorylation of this threonine residue on membrane ERM proteins appears to be critical for the generation of actin-rich membrane projections.
However, it is surprising that the mere production of an active linker tethering actin cytoskeleton to the plasma membrane induces such structures. Indeed, these membrane projections require several other coordinated processes, such as actin polymerization and cross-linking of actin filaments. Thus, T567D ezrin, in addition to being an active linker, induces a complete program for the formation of lamellipodia and ruffles. This is evidence of signaling events from active ezrin to the machineries controlling actin polymerization and cross-linking. These constitutive signaling events from T567D ezrin to actin dynamics and the consequent formation of membrane projections might explain why LLC-PK1 cells producing this ezrin variant fail to achieve epithelial polarity, as seen in suspension cyst and tubulogenesis assays.
In contrast, production of the inactive linker T567A ezrin did not affect epithelial polarity, as evidenced by the development of suspension cysts. In LLC-PK1 cells, production of the inactive T567A ezrin does not impair the formation of epithelial microvilli, whereas in transiently transfected COS7 cells, T558A moesin was reported to inhibit the RhoA-dependent formation of microvilli-like structures (Oshiro et al. 1998 ). However, T567A ezrin was found to be dominant negative in a tubulogenesis assay.
Differentiation of tubules in three-dimensional collagen matrix in the presence of HGF is a cellular assay critically dependent on ezrin function. Some phenotypes associated with expression of wt or mutant ezrin cDNAs are detected uniquely in these differentiation conditions. As reported previously, ezrin overproduction potentiates elongation and branching of tubules (Crepaldi et al. 1997 ). Production of Y353F ezrin specifically triggered apoptosis in this tubulogenesis assay (Gautreau et al. 1999 ). Here, production of T567A ezrin affected growth of LLC-PK1 cells in these conditions. One possible mechanism for this dominant negative effect is that overproduced inactive T567A ezrin is membrane-recruited instead of endogenous ezrin, and thereby impairs its function. In this tubulogenesis assay, phosphorylation of ezrin T567, and activation of its linker function, are needed for proliferation signaling. In 3T3 cells, T567A ezrin was also found to inhibit Ras- and Rho-dependent cellular transformation (Tran Quang, C., A. Gautreau, M. Arpin, and R. Treisman, manuscript submitted for publication). How ezrin signals proliferation is presently not known, but this signaling ability of ezrin relates to the activation of its linker function by phosphorylation of T567.
In the present study, we described the role of ezrin phosphorylation on T567 in vivo. This phosphorylation event regulates a membrane-specific transition of this actin cytoskeleton linker from inactive oligomers to active monomers. This unanticipated step of ezrin activation is critical for cell shape and growth during epithelial differentiation.
References
Algrain, M., Turunen, O., Vaheri, A., Louvard, D., and Arpin, M. 1993. Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane–cytoskeletal linker. J. Cell Biol. 120:129-139.
Andréoli, C., Martin, M., Le Borgne, R., Reggio, H., and Mangeat, P. 1994. Ezrin has properties to self-associate at the plasma membrane. J. Cell Sci. 107:2509-2521.
Berryman, M., Franck, Z., and Bretscher, A. 1993. Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J. Cell Sci. 105:1025-1043.
Berryman, M., Gary, R., and Bretscher, A. 1995. Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis. J. Cell Biol. 131:1231-1242.
Bretscher, A. 1999. Regulation of cortical structure by the ezrin-radixin-moesin protein family. Curr. Opin. Cell Biol. 11:109-116.
Bretscher, A., Gary, R., and Berryman, M. 1995. Soluble ezrin purified from placenta exists as stable monomers and elongated dimers with masked C-terminal ezrin-radixin-moesin association domains. Biochemistry. 34:16830-16837.
Chen, J., Doctor, R.B., and Mandel, L.J. 1994. Cytoskeletal dissociation of ezrin during renal anoxia: role in microvillar injury. Am. J. Physiol. 267:C784-C795.
Crepaldi, T., Gautreau, A., Comoglio, P.M., Louvard, D., and Arpin, M. 1997. Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells. J. Cell Biol. 138:423-434.
Fazioli, F., Wong, W.T., Ullrich, S.J., Sakaguchi, K., Appella, E., and Di Fiore, P.P. 1993. The ezrin-like family of tyrosine kinase substrates: receptor-specific pattern of tyrosine phosphorylation and relationship to malignant transformation. Oncogene. 8:1335-1345.
Fukata, Y., Kimura, K., Oshiro, N., Saya, H., Matsuura, Y., and Kaibuchi, K. 1998. Association of the myosin binding subunit of myosin phosphatase and moesin: dual regulation of moesin phosphorylation by rho-associated kinase and myosin phosphatase. J. Cell Biol. 141:409-418.
Gary, R., and Bretscher, A. 1993. Heterotypic and homotypic associations between ezrin and moesin, two putative membrane–cytoskeletal linking proteins. Proc. Natl. Acad. Sci. USA. 90:10846-10850.
Gary, R., and Bretscher, A. 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. 6:1061-1075.
Gautreau, A., Poullet, P., Louvard, D., and Arpin, M. 1999. Ezrin, a plasma membrane–microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway. Proc. Natl. Acad. Sci. USA. 96:7300-7305.
Gould, K.L., Bretscher, A., Esch, F.S., and Hunter, T. 1989. cDNA cloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1. EMBO (Eur. Mol. Biol. Organ.) J. 8:4133-4142.
Hayashi, K., Yonemura, S., Matsui, T., and Tsukita, S., Tsukita, S. 1999. Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues: application of a novel fixation protocol using trichloroacetic acid (TCA) as a fixative. J. Cell Sci. 112:1149-1158.
Hishiya, A., Ohnishi, M., Tamura, S., and Nakamura, F. 1999. Protein phosphatase 2C inactivates F-actin binding of human platelet moesin. J. Biol. Chem. 274:26705-26712.
Huang, L.Q., Wong, T.Y.W., Lin, R.C.C., and Furthmayr, H. 1999. Replacement of threonine 558, a critical site of phosphorylation of moesin in vivo, with aspartate activates F-actin binding of moesin: regulation by conformational change. J. Biol. Chem. 274:12803-12810.
Kondo, T., Takeuchi, K., Doi, Y., Yonemura, S., Nagata, S., and Tsukita, S. 1998. ERM (ezrin/radixin/moesin)-based molecular mechanism of microvillar breakdown at an early stage of apoptosis. J. Cell Biol. 139:749-758.
Krieg, J., and Hunter, T. 1992. Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin. J. Biol. Chem. 267:19258-19265.
Lamb, R.F., Ozanne, B.W., Roy, C., McGarry, L., Stipp, C., Mangeat, P., and Jay, D.G. 1997. Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts. Curr. Biol. 7:682-688.
Magendantz, M., Henry, M.D., Lander, A., and Solomon, F. 1995. Interdomain interactions of radixin in vitro. J. Biol. Chem. 270:25324-25327.
Matsui, T., Maeda, M., Doi, Y., Yonemura, S., Amano, M., Kaibuchi, K., and Tsukita, S. 1998. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J. Cell Biol. 140:647-657.
Matsui, T., Yonemura, S., and Tsukita, S., Tsukita, S. 1999. Activation of ERM proteins in vivo involves phosphatidylinositol 4-phosphate 5-kinase and not ROCK kinases. Curr. Biol. 9:1259-1262.
Nakamura, F., Amieva, M.R., and Furthmayr, H. 1995. Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets. J. Biol. Chem. 270:31377-31385.
Nakamura, F., Amieva, M.R., Hirota, C., Mizuno, Y., and Furthmayr, H. 1996. Phosphorylation of 558T of moesin detected by site-specific antibodies in RAW264.7 macrophages. Biochem. Biophys. Res. Comm. 226:650-656.
Nakamura, F., Huang, L., Pestonjamasp, K., Luna, E.J., and Furthmayr, H. 1999. Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and polyphosphatidylinositides. Mol. Biol. Cell 10:2669-2685.
Oshiro, N., Fukata, Y., and Kaibuchi, K. 1998. Phosphorylation of moesin by rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol. Chem. 273:34663-34666.
Pearson, M.A., Reczek, D., Bretscher, A., and Karplus, P.A. 2000. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell. 101:259-270.
Pietromonaco, S.F., Simons, P.C., Alman, A., and Elias, L. 1998. Protein kinase C-theta phosphorylation of moesin in the actin-binding sequence. J. Biol. Chem. 273:7594-7603.
Reczek, D., and Bretscher, A. 1998. The carboxy-terminal region of EBP50 binds to a site in the amino-terminal domain of ezrin that is masked in the dormant molecule. J. Biol. Chem. 273:18452-18458.
Reczek, D., Berryman, M., and Bretscher, A. 1997. Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family. J. Cell Biol. 139:169-179.
Shaw, R.J., Henry, M., Solomon, F., and Jacks, T. 1998. RhoA-dependent phosphorylation and relocalization of ERM proteins into apical membrane/actin protrusions in fibroblasts. Mol. Biol. Cell. 9:403-419.
Simons, P.C., Pietromonaco, S.F., Reczek, D., Bretscher, A., and Elias, L. 1998. C-terminal threonine phosphorylation activates ERM proteins to link the cell's cortical lipid bilayer to the cytoskeleton. Biochem. Biophys. Res. Comm. 253:561-565.
Takahashi, K., Sasaki, T., Mammoto, A., Takaishi, K., Kameyama, T., Tsukita, S., and Takai, Y. 1997. Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J. Biol. Chem. 272:23371-23375.
Takeuchi, K., Sato, N., Kasahara, H., Funayama, N., Nagafuchi, A., Yonemura, S., and Tsukita, S. 1994. Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J. Cell Biol. 125:1371-1384.
Turunen, O., Wahlstrom, T., and Vaheri, A. 1994. Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J. Cell Biol. 126:1445-1453.
Wohlwend, A., Montesano, R., Vassalli, J.D., and Orci, L. 1985. LLC-PK1 cysts: a model for the study of epithelial polarity. J. Cell. Physiol. 125:533-539.
Yonemura, S., and Tsukita, S., Tsukita, S. 1999. Direct involvement of ezrin/radixin/moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins. J. Cell Biol. 145:1497-1509.(Alexis Gautreaua, Daniel Louvarda, and M)