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Merlin Differs from Moesin in Binding to F-Actin and in Its Intra- and Intermolecular Interactions
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
248, 548–553 (1998)
RC989009
Merlin Differs from Moesin in Binding to F-Actin and in Its Intra- and Intermolecular Interactions Laiqiang Huang,* Eiji Ichimaru,* Kersi Pestonjamasp,† Xiangmin Cui,* Hiroaki Nakamura,* Grace Y. H. Lo,* Frank I. K. Lin,* Elizabeth J. Luna,† and Heinz Furthmayr* *Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324; and †Department of Cell Biology, University of Massachusetts Medical Center, Worcester Foundation Campus, Shrewsbury, Massachusetts 01545
Received June 17, 1998
The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a protein with homology to the cell membrane/F-actin linking proteins, moesin, ezrin and radixin. Unlike these closely related proteins, merlin lacks a C-terminal F-actin binding site detectable by actin blot overlays, and the GFP-tagged merlin C-terminal domain co-distributes with neither stress fibers nor cortical actin in NIH3T3 cells. Merlin also differs from the other three proteins in its inter- and intramolecular domain interactions, as shown by in vitro binding and yeast two-hybrid assays. As is true for ezrin, moesin and radixin, the N- and C-terminal domains of merlin type 1 bind to each other. However, full-length merlin and its N- and C-terminal domains, as well as the C-terminal domain of ezrin, interact with other full-length merlin type 1 molecules, and its C-terminal domain interacts with itself. Merlin 1 function in cells may thus depend on intra- and intermolecular interactions and their modulation, which include interactions with other members of this protein family. q 1998 Academic Press
Mutations in the gene coding for merlin are responsible for the predisposition to benign tumors of the autosomal dominant inherited disorder neurofibromatosis 2 (1). This protein also is required for normal embryonic development. Disruption of the merlin gene in the mouse leads to failed development between embryonic days 6.5 and 7, marked by collapse of extraembryonic tissue probably due to absence of ectoderm (2). Merlin expression is tightly regulated during development and is also made in many differentiated tissues in the adult (3). The mechanism by which merlin exerts its tumorsuppressive effects remains obscure. However, several intriguing possibilities have emerged from recent work. In cultured cells and tissues, merlin is variably associated with plasma membrane and cytoplasmic struc0006-291X/98 $25.00
tures (4,6,7), ruffles (5), filopodia and cell adhesion points (6). Thus, merlin has been suggested to redistribute to membrane sites during cell activation (7). Merlin also has been suggested to associate with the cytoskeleton because it co-localizes with stress fibers in smooth muscle cells (8), because it redistributes during treatment with the actin filament disrupting drug, cytochalasin D (9), and because in vitro translated merlin or N-terminal fragments can co-sediment with F-actin and/or microtubules (10). Some of the variability in subcellular localization and function may be explained by the existence of multiple alternatively-spliced products from the merlin gene (11-14). In contrast to merlin type 1 (15,16), merlin type 2 is truncated by a premature stop codon in the 45-bp exon 16. This splice variant contains an alternative C-terminus and does not inhibit schwannoma cell growth (17). Also in contrast to isoform 1, C- and N-terminal domains of merlin type 2 do not interact in vitro (17). Regulation of inter- and intramolecular domain interactions of merlin with itself or with cytoskeletal proteins could therefore be necessary for merlin to function as a tumor suppressor. Similar interactions have been suggested by recent experiments with ezrin, radixin, and moesin domains (18,19), and phosphorylation has been proposed to regulate both, F-actin binding properties and intramolecular interactions of N- and C-terminal domains of moesin and radixin (20-22). In spite of these advances, the relationship of F-actin binding to merlin function in cells remains unclear. In this study, we describe extensive intra- and intermolecular self-associations between merlin type 1 and its major domains, but we found that it lacks the high-affinity F-actin binding site present in the C-terminal region of ezrin, radixin, and moesin (23,24). We also found that the merlin N-terminal domain can interact with the actin-binding C-terminal domain of ezrin, suggesting that merlin function in cells may involve interactions with other members of this protein family.
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MATERIALS AND METHODS Plasmids. pMAL-NF2 (MBP-merlin) was created by subcloning full-length NF2 cDNA as an Eco RI-Sal I fragment excised from pGBT-NF2 (see below) into pMAL-c2 vector (New England BioLabs, Beverly, MA); and pGEX-NF2 (GST-merlin) was constructed by subcloning full-length NF2 cDNA as an Xma I-Eco RI fragment from NF2/pBSSK- (generously provided by V. Ramesh, MGH, Boston, MA) into pGEX-3X (Pharmacia Biotech, Piscataway, N.J.). pGEX-NF2 was digested with Bam HI and Bgl II to delete the N-terminal sequence of NF2, and self-ligated to generate pGEX-NFc (GST-merlin C-terminal region, residues 254–595). pGhuMo (GST-moesin) was as described previously (27). NF2/pCR3 was created by subcloning full-length NF2 cDNA as an Eco RI-Eco RV fragment from pGBT-NF2 into pCR3 (Invitrogen, San Diego, CA). NF2/pCR3 was digested with Xho I to delete the Cterminal sequence of NF2, and recircularized to generate NFn/pCR3 (residues 1-341). HA-NF2c/pCR3 (residues 254-595) was created by replacing the GFP-encoding Bam HI-Xba I fragment in HA-GFP/ pCR3 with the Bgl II-Xba I fragment encoding the merlin C-terminal region (from NF2/pCR3). GFP-NF2c/pCR3 was created by replacing the NF2 N-terminal Bam HI-Bgl II fragment in NF2/pCR3 with a Bgl II-Bam HI fragment encoding a mutated green fluorescent protein (GFP) (26). The Bam HI-Eco RI fragment encoding full-length merlin from pGEX-NF2 was ligated into pBSKS- (Stratagene, San Diego, CA) resulting in NF2/pBSKS-. The merlin-encoding sequence was then excised from NF2/pBSKS- as a Bam HI-Sal I fragment and subcloned into pGBT9 (GBT), pGAD424 (GAD4, gift of Stanley Fields, SUNY at Stony Brook, N.Y.), pAS2 (AS2, gift of Stephen Elledge, Baylor College of Medicine, Houston, TX), and pACT2 (ACT2, digested with Bam HI and Xho I), to create pGBT-NF (G4BD-merlin, residues 1595), pGAD4-NF (G4AD-merlin, residues 1-595), pAS2-NF (G4BDHA-merlin, residues 1-595), and pACT2-NF (G4AD-HA-merlin, residues 1-595), respectively. The merlin-encoding sequence was excised from pGBT-NF as an Eco RI-Eco RV fragment and subcloned into pAS1 (AS1, from S. Elledge), digested with Eco RI and Sma I, to create pAS1-NF (G4BD-merlin, residues 1-595). The NF2 N-terminal Bam HI-Xho I fragment from pGBT-NF was subcloned into pACT2, and pAS1-NF with NF2 deleted by predigestion with Bam HI and Sal I, to make pACT2-NFn (G4AD-HA-merlin N-terminal region, residues 1-341) and pAS1-NFn (G4BD-merlin N-terminal region, residues 1-341), respectively. pAS1-NFn was unsuitable for the twohybrid assay because of high background and was digested with Nco I to delete the NF2 N-terminal 28 codons and then re-ligated to make pAS1-NFnD, (residues 29-341). The NF2 C-terminal Bgl II-Sal I fragment from pGBT-NF was subcloned into pACT2 cut with Bam HI and Xho I to generate pACT2-NFc (G4AD-HA-merlin C-terminal region, residues 254-595), and pAS1-NF cut with Bam HI and Sal I to generate pAS1-NFc (G4BD-merlin C-terminal region, residues 254595). The NF2 mid-region Xmn I-Xho I fragment from NF2/pBSSKwas subcloned into pGBT cut with Sma I and Sal I to create pGBTNFm (G4BD-merlin mid region, residues 169-341), and pACT2 cut with Sma I-Xho I to create pACT2-NFm (G4AD-merlin mid region, residues 169-341). The NF2 N-terminal Eco RI-Xmn I fragment from pGBT-NF was subcloned into pAS1 to make pAS1-NFsn (G4BDmerlin short N-terminal region, residues 1-168), from which the Eco RI-Sal I fragment was then subcloned into pGAD4 to make pGAD4NFsn (G4AD-merlin short N-terminal region, residues 1-168). pAS1NFsc (G4BD-merlin short C-terminal region, residues 520-595) and pACT2-NFsc (G4AD-HA-merlin short C-terminal region, residues 520-595) were made by digesting pAS1-NF and pACT2-NF, respectively, with Nco I followed by self-ligation. Recombinant proteins and antibodies. MBP-merlin was expressed in E. coli harboring pMAL-NF2, extracted, and partially purified through an amylose column (New England BioLabs) according to the manufacturer’s instructions. GST fusions of merlin and moesin polypeptides were expressed in E. coli transformed with
the corresponding plasmids, and when indicated, extracted and partially purified through Glutathione Sepharose 4B (Pharmacia Biotech) according to the manufacturer’s instructions. Rabbit anti-MBP polyclonal antibody was from New England BioLabs, and goat antiGST antibody from Pharmacia Biotech. Rabbit anti-merlin peptide (residues 508-533) polyclonal antibody 1398NF2 was generously provided by Dr. Michael A. den Bakker (Department of Pathology, Erasmus University Rotterdam, The Netherlands). Rabbit anti-moesin polyclonal antibody pAs90-7 was as previously described (27). F-actin blot overlay assays. Protein samples were resolved by SDS-PAGE under reducing conditions, and electrotransferred to nitrocellulose membranes. The blots were incubated overnight at 47C in TTBS (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 0.005% sodium azide, and for a second overnight incubation at 47C in blocking buffer (TTBS containing 5% dry milk and 0.005% sodium azide) to renature proteins. After washing with TTBS, the blots were probed with 50 mg/ml gelsolin-capped, phalloidin-stabilized, 125I-labeled F-actin (23,25), washed four times again with TTBS and exposed to film. Cell culture, transfection and microscopy. NIH3T3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO BRL), supplemented with 10% fetal calf serum (GIBCO BRL), at 377C, under 5% CO2 atmosphere. One day before transfection, the cells were split and plated at 30-40% confluency. On the following day, the cells were harvested and resuspended in growth medium at a concentration of 1.5-21107 cells/ml. 0.2 ml of the cell suspension and 10 mg of Qiagen-column-purified plasmid DNA in 50 ml of phosphatebuffered saline, pH 7.4 (PBS), were mixed in a 0.4 cm electroporation cuvette (Bio-Rad, Richmond, CA), and incubated at room temperature for 10 minutes. Electroporation was carried out at 300 V, 960 mF and 100 V using a Bio-Rad Gene Pulser. The cells were immediately collected into 4 ml growth medium, and 0.5 ml of the diluted cell suspension was plated onto a 22140 mm glass coverslip contained in a culture dish, and placed in the incubator. One hour later the transfected cells were rinsed with growth medium, received fresh growth medium, and returned to the incubator. After additional 45 hours the cells grown on the coverslips were imaged live or after fixation and staining with TRITC-conjugated phalloidin by fluorescence and differential interference contrast (DIC) microscopy as described (26). In vitro translation and protein binding assay. pCR3 plasmids carrying merlin inserts were transcribed and translated in vitro using a TNT coupled rabbit reticulocyte lysate system (Promega, Madison, WI) in the presence of T7 RNA polymerase and L-[35S]methionine according to the manufacturer’s instructions. In vitro translation products were incubated with GST fusion proteins (immobilized on Glutathione Sepharose beads and washed 4 times with PBS) in binding buffer (200 mM NaCl, 20 mM Tris pH 8, 0.2% Triton X-100) at 47C. After 1.5 hours the beads were washed 6 times in binding buffer. Bound proteins were boiled in SDS sample buffer, resolved by SDS-PAGE and subjected to autoradiography. Yeast two-hybrid protein-protein interaction assay. Pairs of hybrid plasmids were transformed (28) into the yeast reporter strain Y190 (29) and co-transformants were selected on SC media lacking Trp and Leu at 307C for 4 days. Single colonies were patched onto selective plates, incubated at 307C for 3 days, and subjected to a filter lift assay for b-galactosidase activity essentially as described (29,30); and onto selective plates lacking His and containing 25 mM 3-amino-1,2,4-triazole (3-AT), and incubated at 307C for 7 days to assay for growth (29). While results of both assays were in good agreement in indicating protein interactions, those of b-galactosidase assay are presented.
RESULTS AND DISCUSSION A high affinity F-actin binding site in ezrin and moesin has been mapped to the extreme C-terminal se-
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FIG. 1. Sequence similarities in the C-terminal F-actin-binding region of moesin, ezrin radixin and merlin. Deduced protein sequence alignment of human moesin, porcine radixin, human ezrin, and human merlin, types 1 and 2. As compared with the C-terminal sequences that contain the high affinity F-actin binding site of moesin, ezrin and radixin (23,24), merlin type 1 is 40% identical and 57% similar. Merlin type 2 is 30% identical and 43% similar over this same region. Identical residues (black boxes) and conservative replacements (gray boxes) are shaded; The arrow denotes the conserved threonine residue that is the major phosphorylation site of moesin (20–22).
quence of these proteins (23,24), and the last 34 amino acid residues of ezrin have been shown to be sufficient for binding (24). This region is highly conserved across species and among ezrin, moesin and radixin, with 94% (32/34) of the amino acids being similar. The region includes an identical sequence of 24 amino acid residues (31). Both isoforms of human merlin, but not Drosophila merlin (4), contain an additional serine residue (Ser576), but otherwise they are quite similar (15,16). In optimal sequence alignments, merlin type 1 is 40% identical and 57% similar to the identified F-actin binding region, and merlin type 2 is 30% identical and 43% similar (Fig. 1). By contrast, merlin type 2 is quite dissimilar from the other proteins at the extreme Cterminus, which is required for F-actin binding (23,24). It is also interesting that the threonine residue, corresponding to Thr558 of moesin, is conserved in all members of this protein family. Phosphorylation of this residue increases F-actin binding to moesin, ezrin and radixin, perhaps by disruption of the intramolecular interaction between N- and C-domains (18-21). The relatively high structural similarity in this region thus suggests that at least merlin type 1 shares some of the properties of the other proteins and we therefore tested for high affinity binding to F-actin and for its ability to undergo inter- and intramolecular associations. As reported previously (23), endogenous (not shown) and recombinant moesin (Fig. 2) bind F-actin after electrotransfer to nitrocellulose blots. This binding is specific because it is inhibited by excess unlabeled F-actin (23) and because it is independent of the nature of the radiolabel on the F-actin probe, binding to both 125Ilabeled F-actin and also to F-actin labeled with [a-32P]ATP [32] or [a-35S] (data not shown). By contrast, merlin type 1 exhibits no detectable binding to F-actin under identical conditions (Fig. 2). This result is not in disagreement with the report that in vitro transcribed and translated full-length merlin type 1 and 2 co-sediment with polymerized actin due to a binding site within the N-terminal domain because this site, located between residues 178-367 (10), may be either dena-
tured by SDS-PAGE or of lower affinity. Also, our results are not inconsistent with the report that ezrin contains a G-actin binding site within residues 288310 (33), a region that contains a perfectly conserved cluster of positively charged residues (RRRK), a sequence similar to actin-binding motifs in myosin (reviewed in 34), a protein that competes with moesin and ezrin for binding to F-actin (23). Thus, while merlin may contain one or more internal actin-binding sites, it lacks the high affinity F-actin binding site in the Cterminal domain. Depending on the assay, different results have been obtained also for ezrin. While only the F-actin binding site in the C-terminal domain is detectable with GST-fusions of ezrin (24), binding of For G-actin was seen to a fragment comprising the Nterminal peptide sequence 1-333 (33). A larger question is which, if any, of the actin binding activities observed in different in vitro assays, are functional and utilized in cells. Recent studies have shown endogenous or de novo expressed full-length merlin to localize to the plasma membrane or to uncharacterized cytoplasmic granules (4-7). Although merlin may interact with actin filaments at the cytoplasmic surface of these membranes, with but one exception (8), co-localization with stress fibers has not been seen. In agreement with reports that merlin with deletions in the N-terminal domain lacks both membrane binding and co-localization with actin filaments (4,36), the GFPlabeled merlin-1 C-terminal domain is exclusively as-
FIG. 2. Recombinant merlin does not bind F-actin in vitro. A, Coomassie blue-stained gels of moesin (thrombin-cleaved GST-moesin, lanes 1–4), and partially purified MBP-merlin (lanes 5–8) at a range of concentrations varying 10-fold. Additional bands migrating below moesin in lanes 1–4 are due to partial proteolysis. Positions of moesin and MBP-merlin are indicated by arrows. B, blot overlay with 125I-labeled F-actin of a duplicate gel transferred onto nitrocellulose. C, blot from B after washing.
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FIG. 3. Intermolecuclar and intradomain interactions of merlin in vitro. Full-length (Merlin), C-terminal domain (Merlin C) and HRX (negative control; ref. 46) were expressed as GST-fusion proteins in bacteria and bound to glutathione affinity matrix. Labeled in vitro transcribed and translated full-length (Merlin), N-terminal (Merlin N), or C-terminal domain of merlin (Merlin C) were added to the affinity beads and after extensive washing, bound proteins were dissolved in SDS buffer, separated by SDS-PAGE and analysed by autoradiography. Two lanes are shown for each experiment: left lane, starting material (In), right lane, bound protein. No binding is observed of any of the merlin products to GST-HRX. To exclude effects of GST on the binding interaction, merlin and merlin C were tested in both combinations.
sociated with coarse granules in living NIH3T3 fibroblasts (data not shown). By contrast, GFP- or epitopetagged C-terminal domain fusion proteins of moesin, ezrin or radixin, co-localize with stress fibers and subcortical actin filaments (37,38; Litman, Amieva, Huang and Furthmayr, unpublished). Thus, merlin type 1 lacks the high affinity C-terminal actin binding site in vivo, as well as in vitro. Converging lines of evidence suggest that the actinbinding activities of moesin, ezrin and radixin may require additional factors (reviewed in 35). For instance, tight binding of the N-terminal to the C-terminal domain is proposed to regulate interactions of these proteins with the cytoskeleton and with each other (18,19). Phosphorylation of a single conserved threonine residue apparently disrupts this intramolecular interaction between domains of moesin or radixin, exposing the C-terminal actin binding site (20-22). Since merlin type 1 lacks this binding site, we asked whether merlin type 1 exhibits intra- or interamolecular interactions that differ as well and that may have other functions. As a first approach, we looked for binding of radiolabeled in vitro translated full-length merlin type 1 and its N-(amino acid residues 1-341) and C-terminal fragments (amino acid residues 254-595) to glutathione columns containing bound GST-merlin fusion proteins. As shown in Fig. 3, full-length merlin binds to full-length molecules, as well as to both C- and N-merlin domain fragments. The C-terminal domain apparently interacts both, homotypically with other C-terminal domains and heterotypically, with the merlin N-terminal domain. We did not test for self-association of the merlin N-domain. Our second approach was to test the binding interactions of merlin type 1 domains using the yeast twohybrid system and several different vectors (Table I). The results confirm the homotypic binding of fulllength merlin to itself (Table I, A1, A2; B1, B2; C1, C2), and interactions between full-length merlin and both N- (C3) and C-terminal merlin fragments (C4,E2,H2). The N-terminal domain in the AS1-vector (AS1-NFn, residues 1-341) gave high background and we tested
instead truncated fragments prepared from it by deleting either the first 28 amino acid residues (AS1-NFnD, residues 29-341) or C-terminal sequences (AS1-NFsn, residues 1-168). Both truncations result in inactive peptides that are neither capable of interaction with full-length merlin nor with the C-domain (Table I, rows D, G; column 6). By contrast, the C-terminal 75 amino acid long fragment (AS1-NFsc, residues 520-595) retains the binding activity of the longer C-terminal region in that it interacts both with full-length merlin (Table I, C7,H2) and with the N-terminal domain (H3). However, sequences required for homotypic binding of the C-terminal domain to itself are lost (E7,H4,H7). No interaction of the mid-region of merlin (residues 169341) was detected with any other parts of merlin (Table I, row F; column 5). Our results suggest that merlin type 1 (and isoforms) shares binding properties with moesin, ezrin and radixin and has capabilities for unique interactions. First, like the three homologous proteins, merlin type 1 is capable of self-association between sequences at the extreme C- and N-terminal regions. The loss of binding upon truncation of the first 28 amino acid residues in the NFnD fragment is similar to results reported for the head-to-tail association and oligomerization sequences in ezrin (18,39). Although the observation that the merlin C-terminal fragment binds to itself, an interaction that requires amino acid residues 254-520, is currently unique to merlin, this region does contain one of the two sequences in ezrin (residues 283310) that have been implicated in the regulation of the head-to-tail interaction (40). Similarly, the absence of the high-affinity F-actin binding site from the merlin Cterminus, a sequence that is highly conserved between human and Drosophila merlins (4) suggests that domain has an important function unique to merlin. This conclusion is consistent with the observation that the intracellular distribution of merlin overlaps, but appears to be distinct from that of moesin, ezrin and radixin (4-9). One type of interaction that the merlin isoforms may share with moesin, ezrin and radixin is binding to
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Interactions of Full-Length and Domains of Merlin in Yeast Two-Hybrid System GAL4AD fusion
GAL4BD fusion
1 GAD4-NF FL 1–595
2 ACT2-NF HA FL 1–595
3 ACT2-NFn HA N 1–341
4 ACT2-NFc HA C 254–595
5 ACT2-NFm HA 169–341
6 GAD4-NFsn N 1–168
7 ACT2-NFsc HA C 520–595
A
GBT-NF
FL 1–595
/////
/////
B
AS2-NF
HA FL 1–595
/////
/////
C
AS1-NF
FL 1–595
/////
/////
/////
///
—
—
///
D
AS1-NFnD
N 29–341
—
—
—
—
—
—
E
AS1-NFc
C 254–595
////
/////
////
—
—
—
F
GBT-NFm
M 169–341
—
—
—
—
G
AS1-NFsn
N 1–168
—
/
—
H
AS1-NFsc
C 520–595
///
///
—
I
GBT
GAL4BD
—
—
—
—
— —
—
—
8 ACT2 GAL4AD — — — — — — — — — — — — — — — — —
Note. Filter lift b-galactosidase assays were performed on yeast Y190 cells co-expressing pairs of GAL4-merlin fusions as indicated in the Table and the Materials and Methods to assess intermolecular and interdomain interactions of merlin. b-galactosidase activity was scored visually, with ‘‘/////’’ denoting dark blue and ‘‘—’’ white colonies. NF, full-length merlin (residues 1–595); NFn, N-terminal domain (residues 1–341); NFnD, N-terminal domain with deletion at the N-terminal end (residues 29–341); NFc, C-terminal domain (residues 254–595); NFm, middle part (residues 169–341); NFsn, short N-terminal domain (residues 1–168); NFsc, short C-terminal domain (residues 520–595). GBT, AS1, AS2, GAD4, and ACT2 refer to different vectors as detailed in Materials and Methods.
membrane receptors via the homolgous N-terminal domains (9,37,38; Litman, Amieva, Huang and Furthmayr, unpublished). In fact, both ezrin and merlin bind to CD44 and to NHE-RF, a regulatory factor that mediates protein kinase A inhibition of the plasma membrane Na/H/ exchanger (20,41). Merlin’s greater tendency for self-interaction may potentiate receptor clustering and/or the anchorage of other proteins to these membrane proteins. Other binding functions of merlin are under investigation and several candidate proteins have been identified, including a variety of as yet uncharacterized proteins (42; Huang and Furthmayr, unpublished). Whether putative merlin/receptor complexes or merlin oligomers interact with actin remains unknown. An actin linking function could also be isoform specific and may involve additional proteins. For instance, merlin type 2 forms a complex with the actin-binding protein fodrin (43) and this isoform also binds actin at an internal site in vitro (10). Rapid changes in expression and phosphorylation of merlin accompany conditions of cell confluency, serum deprivation, loss of cell adhesion and cell growth (44,45). Phosphorylation could thus provide an additional mechanism for activation of binding functions. Preliminary data suggest that the C-domains of ezrin and radixin interact with full-length merlin (data not shown). This further suggests that these proteins might influence merlin function during cell activation. In summary, we hypothesize that the tumor sup-
pressive effect of merlin is mediated by intra- and intermolecular interactions of its isoforms that may include actin only indirectly. Merlin’s normal function could involve interplay or competition with substrates of signaling molecules, including moesin, ezrin and radixin, to form regulated and potentially transient interactions at the membrane/cytoskeletal interface. ACKNOWLEDGMENTS We thank John Kim and Shellie Yamashita for assistance in making the constructs and yeast two-hybrid experiments. This work was supported by California Tobacco-Related Disease Research Program grant 4RT-0316. E. Ichimaru (on leave from Nagasaki University School of Dentistry) and H. Nakamura (on leave from Niigata University School of Dentistry) were supported in part by the Japanese Ministry of Education. This work was also supported in part by NIH grants AR41045 to HF and GM33048 to EJL.
REFERENCES 1. Lutchman, M., and Rouleau, G. A. (1996) Trends Neurosci. 19, 373–377. 2. McClatchey, A. I., Saotome, I., Ramesh, V., Gusella, J. F., and Jacks, T. (1997) Genes Dev. 11, 1253–1265. 3. Huynh, D. P., Tran, T. M., Nechiporuk, T., and Pulst, S. M. (1996) Cell Growth Diff. 7, 1551–1561. 4. McCartney, B. M., and Fehon, R. G. (1996) J. Cell Biol. 133, 843–852. 5. Gonzalez-Agosti, C., Xu, L., Pinney, D., et al. (1996) Oncogene 13, 1239–1247.
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6. Schmucker, B., Ballhausen, W. G., and Kressel, M. (1997) Eur. J. Cell Biol. 72, 46–54. 7. Stemmer-Rachamimov, A. O., Gonzalez-Agosti, C., Xu, L., et al. (1997) J. Neuropathol. Exp. Neurol. 56, 735–742. 8. Den Bakker, M. A., Tascilar, M., Riegmen, P. H., et al. (1995) Am. J. Pathol. 147, 1339–1349. 9. Sainio, M., Zhao, F., Heiska, L., et al. (1997) J. Cell Sci. 110, 2249–2260. 10. Xu, H. M., and Gutmann, D. H. (1998) J. Neurosci. Res. 51, 403– 415. 11. Bianchi, A. B., Hara, T., Ramesh, V., et al. (1994) Nature Genet. 6, 185–192. 12. Jacoby, L. B., MacCollin, M. M., Louis, D. N., et al. (1994) Hum. Mol. Genet. 3, 413–419. 13. Pykett, M., Murphy, M., Harnish, P. R., and George, D. (1994) Hum. Mol. Genet. 3, 559–564. 14. Arakawa, H., Hayashi, N., Nagase, H., Ogawa, M., and Nakamura, Y. (1994) Hum. Mol. Genet. 3, 565–568. 15. Trofatter, J. A., MacCollin, M. M., Rutter, J. J., et al. (1993) Cell 72, 791–800. 16. Rouleau, G. A., Merel, P., Lutchman, M., et al. (1993) Nature 363, 515–521. 17. Sherman, L., Xu, H. M., Saporito-Irwin, S., et al. (1997) Oncogene 15, 2505–2509. 18. Gary, R., and Bretscher, A. (1995) Mol. Biol. Cell 6, 1061–1075. 19. Berryman, M., Gary, R., and Bretscher, A. (1995) J. Cell Biol. 131, 1231–1242. 20. Matsui, T., Maeda, M., Doi, Y., Yonemura, S., Amano, M., Kaibuchi, K., Tsukita, S., and Tsukita, S. (1998) J. Cell Biol. 140, 647–658. 21. Nakamura, F., Huang, L., Amieva, M. R., Pestonjamasp, K., Luna, E., and Furthmayr, H. (1998), submitted. 22. Nakamura, F., Amieva, M. R., and Furthmayr, H. (1995) J. Biol. Chem. 270, 31377–31385. 23. Pestonjamasp, K., Amieva, M. R., Strassel, C. P., Nausseef, W. M., Furthmayr, H., and Luna, E. J. (1995) Mol. Biol. Cell 6, 247–259. 24. Turunen, O., Wahlstrom, T., and Vaheri, A. (1994) J. Cell Biol. 126, 1445–1453. 25. Chia, C. P., Hitt, A. L., and Luna, E. J. (1991) Cell Motil. Cytoskel. 18, 164–179.
26. Amieva, M., Litman, P., and Furthmayr, H. (1998), submitted. 27. Amieva, M. R., and Furthmayr, H. (1995) Exp. Cell Res. 219, 180–196. 28. Schiestl, R. H., and Gietz, R. D. (1989) Curr. Genet. 16, 339– 346. 29. Durfee, T., Becherer, K., Chen, P. L., et al. (1993) Genes & Dev. 7, 555–569. 30. Breeden, L., and Nasmyth, K. (1985) Cold Spring Harbor Symp. Quant. Biol. 50, 643–650. 31. Lankes, W. T., Schwartz-Albiez, R., and Furthmayr, H. (1993) Biochim. Biophys. Acta 1216, 479–482. 32. Mackay, D. J. G., Esch, F., Furthmayr, H., and Hall, A. (1997) J. Cell Biol. 138, 927–938. 33. Roy, C., Martin, M., and Mangeat, P. (1997) J. Biol. Chem. 272, 20088–20095. 34. Vandekerckhove, J., and Vancompernolle, K. (1992) Curr. Opin. Cell Biol. 4, 36–42. 35. Bretscher, A., Reczek, D., Berryman, M. (1997) J. Cell Sci. 110, 3011–3018. 36. Deguen, B., Merel, P., Goutebroze, L., et al. (1998) Hum. Mol. Genet. 7, 217–226. 37. Algrain, M., Turunen, O., Vaheri, A., Louvard, D., and Arpin, M. (1993) J. Cell Biol. 120, 129–139. 38. Henry, M. D., Gonzalez Agosti, C., and Solomon, F. (1995) J. Cell Biol. 129, 1007–1022. 39. Martin, M., Roy, C., Montcourrier, P., et al. (1997) Mol. Biol. Cell. 8, 1543–1557. 40. Barthur, S. G., and Goldenring, J. R. (1998) Biochem. Biophys. Res. Commun. 243, 874–877. 41. Murthy, A., Gonzalez-Agosti, C., Cordero, E., et al. (1998) J. Biol. Chem. 273, 1273–1276. 42. Takeshima, H., Izawa, I., Lee, P. S., et al. (1994) Oncogene 9, 2135–2144. 43. Scoles, D. R., Huynh, D. P., Morcos, P. A., et al. (1998) Nature Genet. 18, 354–359. 44. Shaw, R. J., McClatchey, A. I., and Jacks, T. (1998) J. Biol. Chem. 273, 7757–7764. 45. Huynh, D. P., and Pulst, S. M. (1996) Oncogene 13, 73–84. 46. Cui, X., De Vivo, I., Slany, R., Miyamoto, A., Firestein, R., and Cleary, M. L. (1998) Nature Genet. 18, 331–337.
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RC989009
Merlin Differs from Moesin in Binding to F-Actin and in Its Intra- and Intermolecular Interactions Laiqiang Huang,* Eiji Ichimaru,* Kersi Pestonjamasp,† Xiangmin Cui,* Hiroaki Nakamura,* Grace Y. H. Lo,* Frank I. K. Lin,* Elizabeth J. Luna,† and Heinz Furthmayr* *Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324; and †Department of Cell Biology, University of Massachusetts Medical Center, Worcester Foundation Campus, Shrewsbury, Massachusetts 01545
Received June 17, 1998
The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a protein with homology to the cell membrane/F-actin linking proteins, moesin, ezrin and radixin. Unlike these closely related proteins, merlin lacks a C-terminal F-actin binding site detectable by actin blot overlays, and the GFP-tagged merlin C-terminal domain co-distributes with neither stress fibers nor cortical actin in NIH3T3 cells. Merlin also differs from the other three proteins in its inter- and intramolecular domain interactions, as shown by in vitro binding and yeast two-hybrid assays. As is true for ezrin, moesin and radixin, the N- and C-terminal domains of merlin type 1 bind to each other. However, full-length merlin and its N- and C-terminal domains, as well as the C-terminal domain of ezrin, interact with other full-length merlin type 1 molecules, and its C-terminal domain interacts with itself. Merlin 1 function in cells may thus depend on intra- and intermolecular interactions and their modulation, which include interactions with other members of this protein family. q 1998 Academic Press
Mutations in the gene coding for merlin are responsible for the predisposition to benign tumors of the autosomal dominant inherited disorder neurofibromatosis 2 (1). This protein also is required for normal embryonic development. Disruption of the merlin gene in the mouse leads to failed development between embryonic days 6.5 and 7, marked by collapse of extraembryonic tissue probably due to absence of ectoderm (2). Merlin expression is tightly regulated during development and is also made in many differentiated tissues in the adult (3). The mechanism by which merlin exerts its tumorsuppressive effects remains obscure. However, several intriguing possibilities have emerged from recent work. In cultured cells and tissues, merlin is variably associated with plasma membrane and cytoplasmic struc0006-291X/98 $25.00
tures (4,6,7), ruffles (5), filopodia and cell adhesion points (6). Thus, merlin has been suggested to redistribute to membrane sites during cell activation (7). Merlin also has been suggested to associate with the cytoskeleton because it co-localizes with stress fibers in smooth muscle cells (8), because it redistributes during treatment with the actin filament disrupting drug, cytochalasin D (9), and because in vitro translated merlin or N-terminal fragments can co-sediment with F-actin and/or microtubules (10). Some of the variability in subcellular localization and function may be explained by the existence of multiple alternatively-spliced products from the merlin gene (11-14). In contrast to merlin type 1 (15,16), merlin type 2 is truncated by a premature stop codon in the 45-bp exon 16. This splice variant contains an alternative C-terminus and does not inhibit schwannoma cell growth (17). Also in contrast to isoform 1, C- and N-terminal domains of merlin type 2 do not interact in vitro (17). Regulation of inter- and intramolecular domain interactions of merlin with itself or with cytoskeletal proteins could therefore be necessary for merlin to function as a tumor suppressor. Similar interactions have been suggested by recent experiments with ezrin, radixin, and moesin domains (18,19), and phosphorylation has been proposed to regulate both, F-actin binding properties and intramolecular interactions of N- and C-terminal domains of moesin and radixin (20-22). In spite of these advances, the relationship of F-actin binding to merlin function in cells remains unclear. In this study, we describe extensive intra- and intermolecular self-associations between merlin type 1 and its major domains, but we found that it lacks the high-affinity F-actin binding site present in the C-terminal region of ezrin, radixin, and moesin (23,24). We also found that the merlin N-terminal domain can interact with the actin-binding C-terminal domain of ezrin, suggesting that merlin function in cells may involve interactions with other members of this protein family.
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Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Plasmids. pMAL-NF2 (MBP-merlin) was created by subcloning full-length NF2 cDNA as an Eco RI-Sal I fragment excised from pGBT-NF2 (see below) into pMAL-c2 vector (New England BioLabs, Beverly, MA); and pGEX-NF2 (GST-merlin) was constructed by subcloning full-length NF2 cDNA as an Xma I-Eco RI fragment from NF2/pBSSK- (generously provided by V. Ramesh, MGH, Boston, MA) into pGEX-3X (Pharmacia Biotech, Piscataway, N.J.). pGEX-NF2 was digested with Bam HI and Bgl II to delete the N-terminal sequence of NF2, and self-ligated to generate pGEX-NFc (GST-merlin C-terminal region, residues 254–595). pGhuMo (GST-moesin) was as described previously (27). NF2/pCR3 was created by subcloning full-length NF2 cDNA as an Eco RI-Eco RV fragment from pGBT-NF2 into pCR3 (Invitrogen, San Diego, CA). NF2/pCR3 was digested with Xho I to delete the Cterminal sequence of NF2, and recircularized to generate NFn/pCR3 (residues 1-341). HA-NF2c/pCR3 (residues 254-595) was created by replacing the GFP-encoding Bam HI-Xba I fragment in HA-GFP/ pCR3 with the Bgl II-Xba I fragment encoding the merlin C-terminal region (from NF2/pCR3). GFP-NF2c/pCR3 was created by replacing the NF2 N-terminal Bam HI-Bgl II fragment in NF2/pCR3 with a Bgl II-Bam HI fragment encoding a mutated green fluorescent protein (GFP) (26). The Bam HI-Eco RI fragment encoding full-length merlin from pGEX-NF2 was ligated into pBSKS- (Stratagene, San Diego, CA) resulting in NF2/pBSKS-. The merlin-encoding sequence was then excised from NF2/pBSKS- as a Bam HI-Sal I fragment and subcloned into pGBT9 (GBT), pGAD424 (GAD4, gift of Stanley Fields, SUNY at Stony Brook, N.Y.), pAS2 (AS2, gift of Stephen Elledge, Baylor College of Medicine, Houston, TX), and pACT2 (ACT2, digested with Bam HI and Xho I), to create pGBT-NF (G4BD-merlin, residues 1595), pGAD4-NF (G4AD-merlin, residues 1-595), pAS2-NF (G4BDHA-merlin, residues 1-595), and pACT2-NF (G4AD-HA-merlin, residues 1-595), respectively. The merlin-encoding sequence was excised from pGBT-NF as an Eco RI-Eco RV fragment and subcloned into pAS1 (AS1, from S. Elledge), digested with Eco RI and Sma I, to create pAS1-NF (G4BD-merlin, residues 1-595). The NF2 N-terminal Bam HI-Xho I fragment from pGBT-NF was subcloned into pACT2, and pAS1-NF with NF2 deleted by predigestion with Bam HI and Sal I, to make pACT2-NFn (G4AD-HA-merlin N-terminal region, residues 1-341) and pAS1-NFn (G4BD-merlin N-terminal region, residues 1-341), respectively. pAS1-NFn was unsuitable for the twohybrid assay because of high background and was digested with Nco I to delete the NF2 N-terminal 28 codons and then re-ligated to make pAS1-NFnD, (residues 29-341). The NF2 C-terminal Bgl II-Sal I fragment from pGBT-NF was subcloned into pACT2 cut with Bam HI and Xho I to generate pACT2-NFc (G4AD-HA-merlin C-terminal region, residues 254-595), and pAS1-NF cut with Bam HI and Sal I to generate pAS1-NFc (G4BD-merlin C-terminal region, residues 254595). The NF2 mid-region Xmn I-Xho I fragment from NF2/pBSSKwas subcloned into pGBT cut with Sma I and Sal I to create pGBTNFm (G4BD-merlin mid region, residues 169-341), and pACT2 cut with Sma I-Xho I to create pACT2-NFm (G4AD-merlin mid region, residues 169-341). The NF2 N-terminal Eco RI-Xmn I fragment from pGBT-NF was subcloned into pAS1 to make pAS1-NFsn (G4BDmerlin short N-terminal region, residues 1-168), from which the Eco RI-Sal I fragment was then subcloned into pGAD4 to make pGAD4NFsn (G4AD-merlin short N-terminal region, residues 1-168). pAS1NFsc (G4BD-merlin short C-terminal region, residues 520-595) and pACT2-NFsc (G4AD-HA-merlin short C-terminal region, residues 520-595) were made by digesting pAS1-NF and pACT2-NF, respectively, with Nco I followed by self-ligation. Recombinant proteins and antibodies. MBP-merlin was expressed in E. coli harboring pMAL-NF2, extracted, and partially purified through an amylose column (New England BioLabs) according to the manufacturer’s instructions. GST fusions of merlin and moesin polypeptides were expressed in E. coli transformed with
the corresponding plasmids, and when indicated, extracted and partially purified through Glutathione Sepharose 4B (Pharmacia Biotech) according to the manufacturer’s instructions. Rabbit anti-MBP polyclonal antibody was from New England BioLabs, and goat antiGST antibody from Pharmacia Biotech. Rabbit anti-merlin peptide (residues 508-533) polyclonal antibody 1398NF2 was generously provided by Dr. Michael A. den Bakker (Department of Pathology, Erasmus University Rotterdam, The Netherlands). Rabbit anti-moesin polyclonal antibody pAs90-7 was as previously described (27). F-actin blot overlay assays. Protein samples were resolved by SDS-PAGE under reducing conditions, and electrotransferred to nitrocellulose membranes. The blots were incubated overnight at 47C in TTBS (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 0.005% sodium azide, and for a second overnight incubation at 47C in blocking buffer (TTBS containing 5% dry milk and 0.005% sodium azide) to renature proteins. After washing with TTBS, the blots were probed with 50 mg/ml gelsolin-capped, phalloidin-stabilized, 125I-labeled F-actin (23,25), washed four times again with TTBS and exposed to film. Cell culture, transfection and microscopy. NIH3T3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO BRL), supplemented with 10% fetal calf serum (GIBCO BRL), at 377C, under 5% CO2 atmosphere. One day before transfection, the cells were split and plated at 30-40% confluency. On the following day, the cells were harvested and resuspended in growth medium at a concentration of 1.5-21107 cells/ml. 0.2 ml of the cell suspension and 10 mg of Qiagen-column-purified plasmid DNA in 50 ml of phosphatebuffered saline, pH 7.4 (PBS), were mixed in a 0.4 cm electroporation cuvette (Bio-Rad, Richmond, CA), and incubated at room temperature for 10 minutes. Electroporation was carried out at 300 V, 960 mF and 100 V using a Bio-Rad Gene Pulser. The cells were immediately collected into 4 ml growth medium, and 0.5 ml of the diluted cell suspension was plated onto a 22140 mm glass coverslip contained in a culture dish, and placed in the incubator. One hour later the transfected cells were rinsed with growth medium, received fresh growth medium, and returned to the incubator. After additional 45 hours the cells grown on the coverslips were imaged live or after fixation and staining with TRITC-conjugated phalloidin by fluorescence and differential interference contrast (DIC) microscopy as described (26). In vitro translation and protein binding assay. pCR3 plasmids carrying merlin inserts were transcribed and translated in vitro using a TNT coupled rabbit reticulocyte lysate system (Promega, Madison, WI) in the presence of T7 RNA polymerase and L-[35S]methionine according to the manufacturer’s instructions. In vitro translation products were incubated with GST fusion proteins (immobilized on Glutathione Sepharose beads and washed 4 times with PBS) in binding buffer (200 mM NaCl, 20 mM Tris pH 8, 0.2% Triton X-100) at 47C. After 1.5 hours the beads were washed 6 times in binding buffer. Bound proteins were boiled in SDS sample buffer, resolved by SDS-PAGE and subjected to autoradiography. Yeast two-hybrid protein-protein interaction assay. Pairs of hybrid plasmids were transformed (28) into the yeast reporter strain Y190 (29) and co-transformants were selected on SC media lacking Trp and Leu at 307C for 4 days. Single colonies were patched onto selective plates, incubated at 307C for 3 days, and subjected to a filter lift assay for b-galactosidase activity essentially as described (29,30); and onto selective plates lacking His and containing 25 mM 3-amino-1,2,4-triazole (3-AT), and incubated at 307C for 7 days to assay for growth (29). While results of both assays were in good agreement in indicating protein interactions, those of b-galactosidase assay are presented.
RESULTS AND DISCUSSION A high affinity F-actin binding site in ezrin and moesin has been mapped to the extreme C-terminal se-
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FIG. 1. Sequence similarities in the C-terminal F-actin-binding region of moesin, ezrin radixin and merlin. Deduced protein sequence alignment of human moesin, porcine radixin, human ezrin, and human merlin, types 1 and 2. As compared with the C-terminal sequences that contain the high affinity F-actin binding site of moesin, ezrin and radixin (23,24), merlin type 1 is 40% identical and 57% similar. Merlin type 2 is 30% identical and 43% similar over this same region. Identical residues (black boxes) and conservative replacements (gray boxes) are shaded; The arrow denotes the conserved threonine residue that is the major phosphorylation site of moesin (20–22).
quence of these proteins (23,24), and the last 34 amino acid residues of ezrin have been shown to be sufficient for binding (24). This region is highly conserved across species and among ezrin, moesin and radixin, with 94% (32/34) of the amino acids being similar. The region includes an identical sequence of 24 amino acid residues (31). Both isoforms of human merlin, but not Drosophila merlin (4), contain an additional serine residue (Ser576), but otherwise they are quite similar (15,16). In optimal sequence alignments, merlin type 1 is 40% identical and 57% similar to the identified F-actin binding region, and merlin type 2 is 30% identical and 43% similar (Fig. 1). By contrast, merlin type 2 is quite dissimilar from the other proteins at the extreme Cterminus, which is required for F-actin binding (23,24). It is also interesting that the threonine residue, corresponding to Thr558 of moesin, is conserved in all members of this protein family. Phosphorylation of this residue increases F-actin binding to moesin, ezrin and radixin, perhaps by disruption of the intramolecular interaction between N- and C-domains (18-21). The relatively high structural similarity in this region thus suggests that at least merlin type 1 shares some of the properties of the other proteins and we therefore tested for high affinity binding to F-actin and for its ability to undergo inter- and intramolecular associations. As reported previously (23), endogenous (not shown) and recombinant moesin (Fig. 2) bind F-actin after electrotransfer to nitrocellulose blots. This binding is specific because it is inhibited by excess unlabeled F-actin (23) and because it is independent of the nature of the radiolabel on the F-actin probe, binding to both 125Ilabeled F-actin and also to F-actin labeled with [a-32P]ATP [32] or [a-35S] (data not shown). By contrast, merlin type 1 exhibits no detectable binding to F-actin under identical conditions (Fig. 2). This result is not in disagreement with the report that in vitro transcribed and translated full-length merlin type 1 and 2 co-sediment with polymerized actin due to a binding site within the N-terminal domain because this site, located between residues 178-367 (10), may be either dena-
tured by SDS-PAGE or of lower affinity. Also, our results are not inconsistent with the report that ezrin contains a G-actin binding site within residues 288310 (33), a region that contains a perfectly conserved cluster of positively charged residues (RRRK), a sequence similar to actin-binding motifs in myosin (reviewed in 34), a protein that competes with moesin and ezrin for binding to F-actin (23). Thus, while merlin may contain one or more internal actin-binding sites, it lacks the high affinity F-actin binding site in the Cterminal domain. Depending on the assay, different results have been obtained also for ezrin. While only the F-actin binding site in the C-terminal domain is detectable with GST-fusions of ezrin (24), binding of For G-actin was seen to a fragment comprising the Nterminal peptide sequence 1-333 (33). A larger question is which, if any, of the actin binding activities observed in different in vitro assays, are functional and utilized in cells. Recent studies have shown endogenous or de novo expressed full-length merlin to localize to the plasma membrane or to uncharacterized cytoplasmic granules (4-7). Although merlin may interact with actin filaments at the cytoplasmic surface of these membranes, with but one exception (8), co-localization with stress fibers has not been seen. In agreement with reports that merlin with deletions in the N-terminal domain lacks both membrane binding and co-localization with actin filaments (4,36), the GFPlabeled merlin-1 C-terminal domain is exclusively as-
FIG. 2. Recombinant merlin does not bind F-actin in vitro. A, Coomassie blue-stained gels of moesin (thrombin-cleaved GST-moesin, lanes 1–4), and partially purified MBP-merlin (lanes 5–8) at a range of concentrations varying 10-fold. Additional bands migrating below moesin in lanes 1–4 are due to partial proteolysis. Positions of moesin and MBP-merlin are indicated by arrows. B, blot overlay with 125I-labeled F-actin of a duplicate gel transferred onto nitrocellulose. C, blot from B after washing.
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FIG. 3. Intermolecuclar and intradomain interactions of merlin in vitro. Full-length (Merlin), C-terminal domain (Merlin C) and HRX (negative control; ref. 46) were expressed as GST-fusion proteins in bacteria and bound to glutathione affinity matrix. Labeled in vitro transcribed and translated full-length (Merlin), N-terminal (Merlin N), or C-terminal domain of merlin (Merlin C) were added to the affinity beads and after extensive washing, bound proteins were dissolved in SDS buffer, separated by SDS-PAGE and analysed by autoradiography. Two lanes are shown for each experiment: left lane, starting material (In), right lane, bound protein. No binding is observed of any of the merlin products to GST-HRX. To exclude effects of GST on the binding interaction, merlin and merlin C were tested in both combinations.
sociated with coarse granules in living NIH3T3 fibroblasts (data not shown). By contrast, GFP- or epitopetagged C-terminal domain fusion proteins of moesin, ezrin or radixin, co-localize with stress fibers and subcortical actin filaments (37,38; Litman, Amieva, Huang and Furthmayr, unpublished). Thus, merlin type 1 lacks the high affinity C-terminal actin binding site in vivo, as well as in vitro. Converging lines of evidence suggest that the actinbinding activities of moesin, ezrin and radixin may require additional factors (reviewed in 35). For instance, tight binding of the N-terminal to the C-terminal domain is proposed to regulate interactions of these proteins with the cytoskeleton and with each other (18,19). Phosphorylation of a single conserved threonine residue apparently disrupts this intramolecular interaction between domains of moesin or radixin, exposing the C-terminal actin binding site (20-22). Since merlin type 1 lacks this binding site, we asked whether merlin type 1 exhibits intra- or interamolecular interactions that differ as well and that may have other functions. As a first approach, we looked for binding of radiolabeled in vitro translated full-length merlin type 1 and its N-(amino acid residues 1-341) and C-terminal fragments (amino acid residues 254-595) to glutathione columns containing bound GST-merlin fusion proteins. As shown in Fig. 3, full-length merlin binds to full-length molecules, as well as to both C- and N-merlin domain fragments. The C-terminal domain apparently interacts both, homotypically with other C-terminal domains and heterotypically, with the merlin N-terminal domain. We did not test for self-association of the merlin N-domain. Our second approach was to test the binding interactions of merlin type 1 domains using the yeast twohybrid system and several different vectors (Table I). The results confirm the homotypic binding of fulllength merlin to itself (Table I, A1, A2; B1, B2; C1, C2), and interactions between full-length merlin and both N- (C3) and C-terminal merlin fragments (C4,E2,H2). The N-terminal domain in the AS1-vector (AS1-NFn, residues 1-341) gave high background and we tested
instead truncated fragments prepared from it by deleting either the first 28 amino acid residues (AS1-NFnD, residues 29-341) or C-terminal sequences (AS1-NFsn, residues 1-168). Both truncations result in inactive peptides that are neither capable of interaction with full-length merlin nor with the C-domain (Table I, rows D, G; column 6). By contrast, the C-terminal 75 amino acid long fragment (AS1-NFsc, residues 520-595) retains the binding activity of the longer C-terminal region in that it interacts both with full-length merlin (Table I, C7,H2) and with the N-terminal domain (H3). However, sequences required for homotypic binding of the C-terminal domain to itself are lost (E7,H4,H7). No interaction of the mid-region of merlin (residues 169341) was detected with any other parts of merlin (Table I, row F; column 5). Our results suggest that merlin type 1 (and isoforms) shares binding properties with moesin, ezrin and radixin and has capabilities for unique interactions. First, like the three homologous proteins, merlin type 1 is capable of self-association between sequences at the extreme C- and N-terminal regions. The loss of binding upon truncation of the first 28 amino acid residues in the NFnD fragment is similar to results reported for the head-to-tail association and oligomerization sequences in ezrin (18,39). Although the observation that the merlin C-terminal fragment binds to itself, an interaction that requires amino acid residues 254-520, is currently unique to merlin, this region does contain one of the two sequences in ezrin (residues 283310) that have been implicated in the regulation of the head-to-tail interaction (40). Similarly, the absence of the high-affinity F-actin binding site from the merlin Cterminus, a sequence that is highly conserved between human and Drosophila merlins (4) suggests that domain has an important function unique to merlin. This conclusion is consistent with the observation that the intracellular distribution of merlin overlaps, but appears to be distinct from that of moesin, ezrin and radixin (4-9). One type of interaction that the merlin isoforms may share with moesin, ezrin and radixin is binding to
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Interactions of Full-Length and Domains of Merlin in Yeast Two-Hybrid System GAL4AD fusion
GAL4BD fusion
1 GAD4-NF FL 1–595
2 ACT2-NF HA FL 1–595
3 ACT2-NFn HA N 1–341
4 ACT2-NFc HA C 254–595
5 ACT2-NFm HA 169–341
6 GAD4-NFsn N 1–168
7 ACT2-NFsc HA C 520–595
A
GBT-NF
FL 1–595
/////
/////
B
AS2-NF
HA FL 1–595
/////
/////
C
AS1-NF
FL 1–595
/////
/////
/////
///
—
—
///
D
AS1-NFnD
N 29–341
—
—
—
—
—
—
E
AS1-NFc
C 254–595
////
/////
////
—
—
—
F
GBT-NFm
M 169–341
—
—
—
—
G
AS1-NFsn
N 1–168
—
/
—
H
AS1-NFsc
C 520–595
///
///
—
I
GBT
GAL4BD
—
—
—
—
— —
—
—
8 ACT2 GAL4AD — — — — — — — — — — — — — — — — —
Note. Filter lift b-galactosidase assays were performed on yeast Y190 cells co-expressing pairs of GAL4-merlin fusions as indicated in the Table and the Materials and Methods to assess intermolecular and interdomain interactions of merlin. b-galactosidase activity was scored visually, with ‘‘/////’’ denoting dark blue and ‘‘—’’ white colonies. NF, full-length merlin (residues 1–595); NFn, N-terminal domain (residues 1–341); NFnD, N-terminal domain with deletion at the N-terminal end (residues 29–341); NFc, C-terminal domain (residues 254–595); NFm, middle part (residues 169–341); NFsn, short N-terminal domain (residues 1–168); NFsc, short C-terminal domain (residues 520–595). GBT, AS1, AS2, GAD4, and ACT2 refer to different vectors as detailed in Materials and Methods.
membrane receptors via the homolgous N-terminal domains (9,37,38; Litman, Amieva, Huang and Furthmayr, unpublished). In fact, both ezrin and merlin bind to CD44 and to NHE-RF, a regulatory factor that mediates protein kinase A inhibition of the plasma membrane Na/H/ exchanger (20,41). Merlin’s greater tendency for self-interaction may potentiate receptor clustering and/or the anchorage of other proteins to these membrane proteins. Other binding functions of merlin are under investigation and several candidate proteins have been identified, including a variety of as yet uncharacterized proteins (42; Huang and Furthmayr, unpublished). Whether putative merlin/receptor complexes or merlin oligomers interact with actin remains unknown. An actin linking function could also be isoform specific and may involve additional proteins. For instance, merlin type 2 forms a complex with the actin-binding protein fodrin (43) and this isoform also binds actin at an internal site in vitro (10). Rapid changes in expression and phosphorylation of merlin accompany conditions of cell confluency, serum deprivation, loss of cell adhesion and cell growth (44,45). Phosphorylation could thus provide an additional mechanism for activation of binding functions. Preliminary data suggest that the C-domains of ezrin and radixin interact with full-length merlin (data not shown). This further suggests that these proteins might influence merlin function during cell activation. In summary, we hypothesize that the tumor sup-
pressive effect of merlin is mediated by intra- and intermolecular interactions of its isoforms that may include actin only indirectly. Merlin’s normal function could involve interplay or competition with substrates of signaling molecules, including moesin, ezrin and radixin, to form regulated and potentially transient interactions at the membrane/cytoskeletal interface. ACKNOWLEDGMENTS We thank John Kim and Shellie Yamashita for assistance in making the constructs and yeast two-hybrid experiments. This work was supported by California Tobacco-Related Disease Research Program grant 4RT-0316. E. Ichimaru (on leave from Nagasaki University School of Dentistry) and H. Nakamura (on leave from Niigata University School of Dentistry) were supported in part by the Japanese Ministry of Education. This work was also supported in part by NIH grants AR41045 to HF and GM33048 to EJL.
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