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MGH Center for Cancer Research and Harvard Medical School, Department of Pathology, Charlestown, Massachusetts 02129,1 MGH Department of Radiation Oncology, Harvard Medical School, Boston, Massachusetts 021142
Received 26 June 2007/ Returned for modification 15 August 2007/ Accepted 4 December 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Merlin is closely related to the ERM (ezrin, radixin, moesin) proteins, which are thought to organize specific membrane domains by providing regulated anchorage between the membrane and underlying cortical actin cytoskeleton (reviewed in references 4 and 33). All four proteins contain an amino (N)-terminal FERM (four-point one, ezrin, radixin, moesin) domain, which mediates membrane association, are regulated by conformation-dependent changes in localization, and share common interacting partners. Nevertheless, the ERM proteins cannot functionally compensate for merlin, which is required for embryonic development in mice, flies, and worms (10, 31; J. Gervais, J. Satterlee, and A. I. McClatchey, unpublished data); similarly, in contrast to merlin, the ERM proteins are not known to function as tumor suppressors and loss of ERM function in mammals, flies, and worms is not associated with altered proliferation. While the subcellular distributions of merlin and the ERM proteins can overlap, they are often distinct. For example, in cultured cells, merlin and the ERM proteins are often concentrated in dynamic membrane structures, such as lamellipodia, filopodia, and sites of cell-cell adhesion (8, 14, 17, 25, 43). However, in highly polarized cells the localization of merlin and the ERM proteins is more discrete. In Drosophila melanogaster epithelial tissues, the single ERM orthologue moesin exhibits a uniform, apical and apicojunctional distribution while merlin exhibits a more punctate localization to the apical junction region and adjacent cytoplasm (30). In Caenorhabditis elegans ERM-1 distributes uniformly along the apical surfaces of non-cuticle-expressing tubular organ epithelia while NFM-1 (the product of the worm NF2 orthologue) is enriched along the basolateral surfaces (extending to the apical junction) of tubular epithelia (13). Furthermore, unlike ezrin, which is largely detergent soluble, a large fraction of merlin is insoluble (7, 48). Together these observations suggest that the distinct activities of merlin and the ERM proteins may be in part due to their partitioning within the cell.
The phenotypic consequences of loss of merlin or ERM function are consistent with functional partitioning of their activities. Thus, in mammalian, Drosophila, and C. elegans epithelial tissues ERM function is required for establishing or maintaining the integrity of the apical epithelial surface (2, 6, 13, 21, 42, 47). In contrast, accumulating evidence suggests that merlin mediates contact-dependent inhibition of proliferation (7, 18, 25, 35, 45), and we have found that merlin localizes to and stabilizes adherens junctions between cells (25). Thus, the ERM proteins appear to be dedicated to providing apical membrane-cytoskeleton linkage while merlin may perform a similar role in stabilizing the apical junction region.
Recent studies suggest that merlin and the related tumor suppressor, expanded, can control the surface abundance of certain membrane receptors including the epidermal growth factor receptor (EGFR) in Drosophila tissues (29). Similarly, we have found that, in contacting mammalian cells, merlin physically associates with the EGFR via the tandem PDZ domain-containing adaptor NHE-RF1 (Na+ hydrogen exchanger regulatory factor 1) and sequesters the EGFR into an insoluble membrane compartment from which it can neither internalize nor signal (7). Although these two studies seem to reach differing conclusions as to how merlin regulates membrane receptor surface availability—in one case stabilizing surface levels and the other promoting turnover—the primary function of merlin in both cases could be to retain receptors in a certain membrane compartment. To better delineate the molecular mechanism whereby merlin controls the surface availability of certain membrane receptors, we sought to understand how the membrane distribution of merlin itself is controlled. We have found that the N-terminal 17 amino acids of merlin, which form an extension not found in the ERM proteins, confer the distinct insoluble-membrane distribution of merlin relative to the ERM proteins. We also found that the ability of merlin to inhibit cell proliferation and to negatively regulate EGFR internalization and signaling depends on its insoluble-membrane distribution and its ability to stably decorate the cortical actin network. We propose a model wherein local activation of merlin from the cortical actin cytoskeleton controls the lateral mobility of and signaling from certain surface receptors (23, 24).
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MATERIALS AND METHODS |
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Plasmids. Generation of pCDNA3 (Invitrogen), encoding wild-type and L64P-containing mouse Nf2 cDNAs, was previously described (25, 45). Ezrin cDNA in pCDNA3.1 was a kind gift from Reuben Shaw. Both Nf218-595 and EzNf21-18 were created via PCR amplification of the mouse Nf2 and Ezrin coding regions and then cloned into pCDNA3 and the pBMN-GFP retroviral vector (Orbigen), respectively. For EzNf21-18, Nf2 residues 1 to 18 were appended to the N terminus of full-length ezrin in place of the N-terminal methionine. Nf215-17A, Nf2S7-13A, and Nf2S7-13D were generated via site-directed mutagenesis of a mouse Nf2wt pCDNA3 construct, according to the manufacturer's instructions (Stratagene). Finally, Nf2wt, Nf218-595, Nf2L64P, Nf215-17A, Nf2S7-13A, Nf2S7-13D, and Ezrin were subcloned into pBMN. Retroviral expression of pBMN-Nf2 and -Ezrin constructs was performed essentially according to the manufacturer's instructions (Orbigen). Nf2wt and Nf218-595 were also cloned into the adenoviral vector pAdEasy-1 (MP Biomedicals), and adenoviral infection with Ad-Nf2wt and -Nf218-595 was performed as described in reference 25.
Subcellular fractionation. Subcellular fractionation was performed as previously described (25) with the final insoluble pellet boiled in modified radioimmunoprecipitation assay buffer containing 0.5% sodium dodecyl sulfate.
Antibodies. Primary antibodies were from Abcam (NHE-RF1, ab3452), Babco (ezrin, MMS-143R; 1:500), Becton Dickinson (5-bromo-2-deoxyuridine [BrdU], 347580), Biosource International (Src-pY418, 44-660; 1:1,000), Calbiochem (cyclin D1, CC12; 1:500), Cell Signaling (EGFR-pY845, 2231; EGFR-pY992, 2235; EGFR-pY1068, 2234; Raf-pS259, 9421; STAT3-pY705, 9138; all at 1:1,000), NeoMarkers (ezrin, Ab-1; EGFR, Ab-17), Santa Cruz (NF2, sc-331 and sc-332; 1:20,000; anti-EGFR, sc-03; 1:1,000), Sigma (actin, A-4700; 1:5,000), Transduction Labs (p120ctn-pY228, 612537; 1:2,000; caveolin-pY14, 611338; 1:1,000; E-cadherin, 610182; 1:1,000; paxillin, P13520). Horseradish peroxidase-conjugated secondary anti-mouse and anti-rabbit antibodies were from Amersham. All dilutions are for immunoblotting.
Indirect immunofluorescence and confocal imaging. Coverslips containing confluent LDCs were prepared in three ways. For fix 1, cells were fixed for 10 min in 3.7% formaldehyde in cytoskeletal buffer [CB; 10 mM 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), pH 6.1, 138 mM KCl, 3 mM MgCl2, and 2 mM EGTA] and subsequently permeabilized for 5 min in 0.5% Triton X-100 in phosphate-buffered saline (PBS). For fix 2, cells were simultaneously fixed and permeabilized in 1% formaldehyde and 2% Triton X-100 in CB for 15 min. For fix 3, cells were washed in 1% Triton X-100 lysis buffer for 30 min on ice, washed once in PBS, and then fixed for 10 min in 3.7% formaldehyde in CB. All coverslips were processed by fix 1, unless noted otherwise. Coverslips were blocked in 10% goat serum in PBS for 30 min and incubated overnight at 4°C with anti-NF2 (sc-332; 1:1,000), -ezrin (Ab-1; 1:200), -NHE-RF1 (ab3452l; 1:100), or -paxillin (P13520; 1:100) antibodies in 10% goat serum in PBS. Coverslips were rinsed five times in PBS, incubated for 1 h at room temperature in fluorescein isothiocyanate- or Cy3-conjugated secondary antibodies (Jackson Immunoresearch Laboratories), and mounted (Vectashield; Vector). Nuclei were stained with DAPI (4',6'-diamidino-2-phenylindole dihydrochloride) or TOTO-3 iodide (Molecular Probes). Two-dimensional images were acquired using a Zeiss Plan Fluor 63x, 1.4-numerical-aperture (NA) oil objective lens on a Zeiss Axioplan microscope with IP Lab software and a Sony charge-coupled device camera. Z sections were acquired using a Zeiss Plan Fluor 40x, 1.3-numerical-aperture oil objective lens on a Zeiss LSM 510 microscope. Final images were prepared using Adobe Photoshop 7.0. For cell surface concanavalin A binding, fixed confluent LDCs were labeled with 50 µg/ml of concanavalin A prior to permeabilization (Molecular Probes; C11253) for 30 min at room temperature.
BrdU incorporation and Tr-EGF internalization. For BrdU experiments, coverslips containing confluent LDCs were labeled with 20 µM BrdU for 12 h and DNA was denatured and prepared for immunofluorescence. To monitor Texas Red-labeled epidermal growth factor (Tr-EGF) internalization, coverslips containing confluent LDCs were serum starved in 1% bovine serum albumin in Dulbecco's modified Eagle medium for 4 h, incubated for 1 h at 37°C with 2 µg/ml Tr-EGF (Molecular Probes; E3480), and prepared for immunofluorescence.
IP. Immunoprecipitation (IP) of late confluent LDCs was performed as described in reference 7. To facilitate the coimmunoprecipitation of insoluble membrane proteins, we included n-octyl-β-D-glucopyranoside (Calbiochem) in the extraction buffer to further extract insoluble-membrane domains. Total membrane extracts and total cell lysates were isolated via lysis of total membrane pellets and total cell pellets, respectively, in Triton X-100 lysis buffer containing 60 mM n-octyl-β-D-glucopyranoside. The following amounts of antibody were used for each IP: 3 µg anti-NHE-RF1 (ab3452), 2 µg anti-E-cadherin (610182), 6 µg anti-EGFR (Ab-17), and 5 µl antiezrin (ascites MMS-143R).
Cell surface biotinylation. Late confluent LDCs were cooled at 4°C for 15 min and then incubated with 0.5 mg/ml of biotin {EZ-Link sulfo-NHS-LC-biotin [sulfosuccinimidyl-6-(biotinamido)hexanoate]; Pierce} in PBS for 1 h at 4°C. After LDCs were washed in PBS, the remaining biotin was quenched in 50 mM NH4Cl, 1 mM MgCl2, and 0.1 mM CaCl2 buffer in PBS for 10 min, washed with PBS, and either harvested immediately (time zero) or shifted to 37°C in full serum for 30 min and harvested on ice. Biotin pull-downs were performed with total membrane extracts by first preclearing extracts in 20 µl of protein G beads and then using 25 µl immobilized neutravidin biotin binding protein beads (Pierce).
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RESULTS |
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Importantly, Nf218-595 also failed to associate with EGFR or with NHE-RF1, which mediates the association between merlin and EGFR by interacting directly with both (Fig. 3A and B) (7, 26, 37). The residues required for ERM-NHE-RF1 interaction have been mapped to the surface of the F3 lobe of the FERM domain (11, 49), indicating that residues 1 to 17 of merlin do not interact directly with NHE-RF1 but instead likely direct merlin to a membrane compartment where it can associate with NHE-RF1 and consequently with EGFR. We found that Nf218-595 also does not associate with E-cadherin, suggesting that the association between merlin and E-cadherin, which may precede the association of merlin with NHE-RF1-EGFR, normally occurs in the insoluble-membrane compartment (Fig. 3C). In fact, in contrast to Nf2wt, Nf218-595 failed to associate with any biotinylated surface proteins in confluent LDCs (Fig. 3D), suggesting that residues 1 to 17 are required for the association of merlin with any surface receptors. Notably, Nf218-595 does retain the ability to associate with ezrin, suggesting that Nf2-ezrin heterodimerization is not limited to the insoluble-membrane compartment and can occur in the absence of association with surface receptors (Fig. 3E). Regulated phosphorylation of serine 518 (S518) has been shown to control the localization and activity of merlin (22, 44, 45). However, we found that both hyper- and hypophosphorylated versions of merlin are associated with EGFR and with biotinylated surface proteins, suggesting that binary models of phosphorylation- or dephosphorylation-induced translocation to the membrane are oversimplified for merlin. Indeed, unlike Nf2L64P, Nf218-595 can be phosphorylated at residue S518 (hyperphosphorylated), indicating that neither localization to the insoluble membrane nor association with surface receptors is required for S518 phosphorylation. Together, these data are consistent with a model wherein recruitment of merlin to an insoluble surface membrane compartment via residues 1 to 17 facilitates interaction with NHE-RF1-EGFR, which, in turn, prevents the internalization of and signaling from EGFR.
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Confocal microscopy reveals the apical position of this network where Nf2wt colocalizes with both ezrin and concanavalin A (Fig. 4L). Notably, Nf2wt is markedly more concentrated in the apical junction region than ezrin in these cells (Fig. 4E, F, and L and 5B). The localization of ezrin to this network is not dependent on endogenous merlin or displaced by exogenous merlin, indicating that merlin is not required for network formation or integrity and that merlin and ezrin do not compete for network binding (Fig. 4E and F). Importantly, this network is distinct from the established localization of ezrin to apical microvilli as LDCs (and fibroblasts and keratinocytes) have few apical microvilli (not shown).
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The N-terminal residues of merlin are sufficient to confer insoluble membrane localization but not tumor suppressor function to ezrin. Given that removal of the N-terminal residues of merlin alters its membrane distribution and solubility, we asked whether those residues constitute a membrane localization determinant that is sufficient to alter the localization of ezrin. Subcellular fractionation reveals that both endogenous ezrin and exogenous ezrin are primarily soluble (Fig. 5A). However, expression of a version of ezrin onto which the N-terminal residues of merlin have been N-terminally appended, EzNf21-18, revealed that a substantial pool of this fusion protein is now present in the insoluble-membrane fractions and enriched at cell-cell boundaries (Fig. 5A and B), mirroring the distribution of Nf2wt (Fig. 1B and C). Importantly, EzNf21-18 was not able to restore contact-dependent inhibition of proliferation in Nf2–/– LDCs and immortalized Nf2–/– fibroblasts or prevent Tr-EGF internalization in Nf2–/– LDCs (Fig. 5C; not shown), suggesting that the growth-suppressing activity of merlin is mediated by additional features that are not conserved in the ERM proteins.
Membrane targeting via the N-terminal residues of merlin. The N-terminal residues of merlin could mediate association with proteins that reside in the insoluble membrane or confer direct association with the insoluble membrane. Close inspection reveals a cluster of positively charged residues and several serines within this sequence (Fig. 6A; see Fig. S1A in the supplemental material); both features are conserved among merlin orthologues. Notably, both features are also present in the N-terminal residues of Src that are known to contribute to its membrane association (see Fig. S1B in the supplemental material). Indeed, electrostatic interaction between N-terminal positively charged amino acids and negatively charged membrane phospholipids has been shown to mediate the association of several proteins with the membrane including Src and the MARCKS (myristoylated alanine-rich C kinase substrate) protein (reviewed in reference 39). Reasoning that these residues could similarly be important for localization of merlin to the insoluble membrane, we generated a version of merlin in which these residues have been changed to neutral alanines (Nf215-17A) and compared its biochemical distribution to that of Nf2wt in LDCs. We found that Nf215-17A, like Nf218-595, was markedly underrepresented in the Triton X-100-insoluble fractions (Fig. 6B), suggesting that electrostatic interactions may play a role in targeting merlin to the membrane.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several models could explain how the N-terminal 17 residues of merlin confer localization to cell-cell boundaries and stable association with the cortical actin network. First, these residues could mediate association with certain adhesion receptors/complexes, which, in turn, stabilizes cortical cytoskeleton association. Alternatively, the requirement of the N-terminal residues for stable association with the cortical cytoskeleton could be a prerequisite for association with junctional components given the continuum between the apical and junctional cortical cytoskeletons. Notably, the apical terminal web of intestinal enterocytes is a specialized cortical cytoskeleton that is firmly anchored to apical junctions; ezrin is required for proper maintenance of this structure (42). Importantly, endogenous ezrin and NHE-RF1 also decorate the cortical actin cytoskeleton but in a detergent-sensitive manner, suggesting that association with the cortical actin cytoskeleton per se does not render merlin insoluble. Finally, direct association between residues 1 to 17 and the lipid bilayer could direct merlin to the insoluble apical and junctional membrane compartment, where it stably associates with the cortical cytoskeleton and with surface receptors. In fact, this stretch of amino acids bears striking similarity to the N terminus of Src, which is known to contribute to its direct anchorage in the insoluble membrane (reviewed in reference 39) (see Fig. S1B in the supplemental material). We found that, as for Src, several charged residues within this segment are important for merlin localization to the insoluble-membrane compartment and that resident serines can be phosphorylated, providing a potential mechanism for regulating the membrane distribution of merlin.
The ERM proteins associate with the actin cytoskeleton via a conserved C-terminal motif that is not present in merlin. Indeed, the nature of the physical association between merlin and the actin cytoskeleton is unclear; notably, one study reported that a peptide representing the N-terminal 27 amino acids of merlin can cosediment with actin, as could polypeptides representing other portions of merlin (3). Neither merlin nor the ERM proteins localize to actin stress fibers; instead, the ERM proteins provide flexible anchorage between the membrane and cortical actin cytoskeleton (4, 6). Our observation that merlin also decorates the cortical actin network in epithelial cells is consistent with the reported localization of merlin in cultured meningioma cells by electron microscopy (17). Indeed the ability to visualize this structure by immunofluorescence localization of merlin/ERMs will facilitate an investigation of its properties given that the thin filaments of the cortical cytoskeleton are not easily detected by phalloidin.
Many studies support a key role for this cortical network in controlling the lateral mobility of certain membrane receptors (reviewed in references 23, 24, and 34 and references therein). However, it is not clear whether it does so by physically tethering the receptors or by forming a physical barrier to the lateral diffusion of untethered receptors or both. The compartment diameter outlined by merlin/ERMs in these cells is 
0.1 to 1 µM, which is within the range of sizes estimated by electron microscopy in other cell types (reviewed in references 6, 23, 24, and 34). Notably, it has been shown that the lateral mobility of fluorescently labeled lipids increases with jasplakinolide treatment in other cell types, and it was predicted that jasplakinolide treatment yielded aberrantly larger cortical network compartments (36). In perfect agreement with this, we discovered that jasplakinolide treatment led to markedly larger compartments within the merlin-decorated network (Fig. 4G). Jasplakinolide stabilizes actin filaments in vitro but exhibits more-complicated actin-disrupting properties in vivo (5); the cortical actin meshwork is particularly sensitive to the effects of jasplakinolide at the time point and concentration used here. Importantly, NHE-RF1 itself has been reported to slow the lateral diffusion of certain receptors, including the cystic fibrosis transmembrane conductance regulator, in a manner dependent upon its ERM (and merlin)-binding domain (1, 15). Perhaps by decorating the cortical network, merlin/ERMs are poised to transiently "trap" NHE-RF-associated receptors, preventing their lateral mobility and signaling. Local activation of merlin/ERMs would therefore be a powerful way to confer spatial and temporal regulation to certain membrane receptors in response to extracellular signals.
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ACKNOWLEDGMENTS |
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This work was supported by the National Institutes of Health (A.I.M.), Department of Defense Neurofibromatosis Research Program (A.I.M.), and the Mahoney Center for Neuro-Oncology, Charles A. Dana Foundation (A.W.C.).
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FOOTNOTES |
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Published ahead of print on 17 December 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
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