WW Domain–Containing Proteins, WWOX and YAP, Compete for Interaction with Erbb-4 and Modulate Its Transcriptional Function

Research Article

WW Domain–Containing Proteins, WWOX and YAP, Compete for Interaction with ErbB-4 and Modulate Its Transcriptional Function

Rami I. Aqeilan,1 Valentina Donati,1 Alexey Palamarchuk,1 Francesco Trapasso,1 Mohamed Kaou,1 Yuri Pekarsky,1 Marius Sudol,2,3 and Carlo M. Croce1

1Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, Ohio; 2Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania; and 3Department of Medicine, Mt. Sinai School of Medicine, New York, New York

Abstract and AP2g transcription factors (2, 3). WWOX inhibits nuclear g The WW domain–containing oxidoreductase, WWOX,isa translocation of p73 and AP2 hence suppressing their trans- tumor suppressor that is deleted or altered in several cancer activating ability. types. We recently showed that WWOX interacts with p73 and WWOX was originally cloned as a putative tumor suppressor AP-2; and suppresses their transcriptional activity. Yes- gene that spans one of the most common active fragile sites in the associated protein (YAP), also containing WW domains, was human genome, FRA16D (4–6). WWOX is located on chromosome shown to associate with p73 and enhance its transcriptional 16q23.3 and exhibits genomic alterations in several cancer types (7) activity. In addition, YAP interacts with ErbB-4 receptor and a recent study showed a possible involvement of methylation tyrosine kinase and acts as transcriptional coactivator of the in the regulation of WWOX expression (8). WWOX expression is COOH-terminal fragment (CTF) of ErbB-4. Stimulation of frequently altered in tumor tissues (9–13) and introduction of ErbB-4–expressing cells with 12-O-tetradecanoylphorbol-13- WWOX into WWOX-negative tumor cells resulted in tumor acetate (TPA) results in the proteolytic cleavage of its suppression and apoptosis (10, 14). The tandem WW domains of cytoplasmic domain and translocation of this domain to the WWOX (Fig. 1A) play an important role in WWOX function. Both nucleus. Here we report that WWOX physically associates WW domains of WWOX contain the central core of two with the full-length ErbB-4 via its first WW domain. consecutive aromatic amino acids and therefore belong to the Coexpression of WWOX and ErbB-4 in HeLa cells followed class I specificity of domains, which recognize ligands with the by treatment with TPA results in the retention of ErbB-4 in PPxY consensus motif (where P is proline, Y is tyrosine, and x is the cytoplasm. Moreover, in MCF-7 breast carcinoma cells, any amino acid; refs. 2, 15–19). Recent mapping of the WW domain expressing high levels of endogenous WWOX, endogenous in the human proteome identified a repertoire of PPxY-containing ErbB-4 is also retained in the cytoplasm. In addition, our ligands that bind to individual domains of WWOX (20). The first results show that interaction of WWOX and ErbB-4 sup- WW of WWOX bound 18 and the second WW bound 16 ligands, all presses transcriptional coactivation of CTF by YAP in a dose- with PPxY consensus. The mapping data clearly documented that dependent manner. A mutant form of WWOX lacking although the second WW domain of WWOX contains a tyrosine in interaction with ErbB-4 has no effect on this coactivation of the place of the second conserved tryptophan, the signature ErbB-4. Furthermore, WWOX is able to inhibit coactivation of residue directly involved in ligand binding (21), the specificity of p73 by YAP. In summary, our data indicate that WWOX the ‘‘WY domain’’ toward PPxY core was not changed (20). antagonizes the function of YAP by competing for interaction Yes-associated protein (YAP) was originally cloned as a partner with ErbB-4 and other targets and thus affect its transcrip- of Yes protein tyrosine kinase, which formed a complex with the tional activity. (Cancer Res 2005; 65(15): 6764-72) SH3 domain of Yes (22). Detailed characterization of YAP lead to the identification of the WW domain as a modular protein domain Introduction with predilection toward proline-rich ligands (15, 23). Two WW domains of YAP (Fig. 1B) also belong to the class I of domain Gene expression can be regulated through a variety of specificity that recognizes proteins containing PPxY motifs. YAP mechanisms including the modulation of transcription factor has been characterized as a transcriptional coactivator of several localization (1). Transcription factors may associate with different transcription factors including Runx factors and p73 (24–26). Two cofactors that may repress or enhance their transcriptional activity. major isoforms of YAP were identified (27); YAP1 contains one WW Modulation of subcellular localization of transcription factors in domain, whereas YAP2 harbors two WW domains and is a more the nucleus or cytoplasm plays an important role in the integration potent transcriptional coactivator when compared with YAP1 (28). of signal transduction and control of transcriptional machinery (1). Intracellular localization of YAP is regulated by Akt kinase Recently, we showed that WW domain–containing oxidoreductase phosphorylation at Ser127 and subsequent binding of phosphory- (WWOX) functions as a regulator of subcellular localization of p73 lated YAP to 14-3-3 protein (29). Recently, YAP was shown to interact with ErbB-4 receptor acting as a transcriptional coactivator of the COOH terminus domain of Requests for reprints: Rami I. Aqeilan, Department of Human Cancer Genomics, ErbB-4 (28, 30). The HER4/ERBB4 gene is a member of type I The Ohio State University, Room 455E, Wiseman Hall, 410 West 12th Avenue, receptor tyrosine kinase subfamily that includes epidermal growth Columbus, OH 43220. Phone: 614-292-3120; Fax: 614-292-4053; E-mail: rami.aqeilan@ osumc.edu. factor receptor ERBB1, ERBB2, and ERBB3 (reviewed in ref. 31). I2005 American Association for Cancer Research. ErbB-4 regulates cell proliferation and differentiation (32). After

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Figure 1. Schematic illustration of WWOX and ErbB-4 proteins. A, structure of full-length of human WWOX. WWOX contains two WW domains and short dehydrogenase domain (SDR). Myc-WWOX, Myc-WWOXY33R, and WWOX-GFP constructs were used to express WWOX protein. B, structure of human YAP. There are two YAP isoforms. YAP2 contains two WW domains compared with one WW domain in YAP1 but both share the same Akt phosphorylation site and transcription activation domain. C, structure of human ErbB-4. All isoforms of ErbB-4 contain cysteine-rich domains, domains I to IV in the ectodomain, transmembrane region (TM), and the tyrosine kinase domain. In the CYT-2 isoform, the PI3K-binding motif is deleted. There are three PPxY motifs that are possible binding sequences for the class I WW domains. The second PPxY motif is included in the PI3K binding domain site that is deleted in the CYT-2 isoform. The COOH-terminal fragment (residues, 676-1292) that is known to translocate to the nucleus is indicated as CTF. CTF-DK (residues, 988-1292) lacks the tyrosine kinase domain but still have better transactivation ability than CTF. binding its ligand heregulin or following the activation of protein Because WWOX and YAP have the similar tandems of WW kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA), the ErbB- domains and interact with common protein targets but have 4 ectodomain is cleaved by a metalloprotease tumor necrosis different effects on transactivation, we decided to investigate factor-a–converting enzyme followed by g-secretase cleavage (33). whether WWOX and YAP expression have opposite effects on The cleavage by g-secretase facilitates the translocation of the their target proteins. Here we report that WWOX physically COOH-terminal fragment of ErbB-4 (CTF) to the nucleus where it associate with the cytoplasmic region of ErbB-4 via its first WW may affect the transcription of target genes (34). CTF by itself does domain and suppresses transactivation activity of ErbB-4 medi- not possesses transactivating activity; however, in the presence of ated by YAP. YAP, its transcriptional activity is revealed (28, 30). Different isoforms of ErbB-4 were previously described (Fig. 1C). JM-a isoform is sensitive to cleavage, whereas the JM-b isoform is Materials and Methods insensitive to the cleavage because of the sequence difference in Cell culture and transient transfection. Human HEK293, HeLa, and the juxtamembrane region (35). CYT-1 and CYT-2 are two other MCF-7 cells were grown in RPMI supplemented with 10% fetal bovine isoforms of ErbB-4. CYT-1 contains a binding motif for the serum (Invitrogen, Carlsbad, CA) and Gentamicin. Cells were maintained at phosphatidylinositol 3-kinase (PI3K), whereas CYT-2 lacks this 37jC in a water saturated atmosphere of 5% CO2 in air. Overexpression of sequence (36, 37). proteins was achieved by transient transfections using FuGENE 6 www.aacrjournals.org 6765 Cancer Res 2005; 65: (15). August 1, 2005

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Research transfection reagent, according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN). Plasmid construction. The mammalian expression plasmids encoding MYC epitope-tagged WWOX and pCMV-MYC-WWOXY33R were previously described (ref. 2; Fig. 1A). An additional Myc Tag was added in 3Vterminus of both WWOX constructs. Full-length WWOX cDNA was cloned into a pEGFP- N1 vector (Clontech, Palo Alto, CA). Full-length cDNA of YAP2 were cloned into a pCDNA4-HisMaxB vector (Omni-YAP2; Invitrogen) using standard protocols (Fig. 1B). Full-length ErbB-4 with COOH-terminal HA epitope tag, ErbB-4 CTF, and CTF lacking the kinase domain (CTF-DK) were previously described (ref. 28; Fig. 1C). Full-length ErbB-4PY1-HA, ErbB-4PY3-HA and ErbB-4Py1,3-HA mutants were generated using Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to manufacturer’s instructions. The reporter plasmid pFR-luc expressing Firefly luciferase and contains a synthetic promoter with five tandem repeats of the yeast GAL4-binding sites and pTK-RL expressing Renilla luciferase as an internal control were purchased from Stratagene and Promega (Madison, WI), respectively. The reporter p53RE-Luc (Stratagene) carrying the p53-responsive element derived from p53 promoter was used to assay p73 transactivation. Immunoprecipitation and immunoblot analysis. Cells were lysed and immunoprecipitations were carried out as previously described (2). Anti- bodies used were mouse monoclonal anti-HA (Covance, Princeton, NJ), mouse monoclonal anti-Omni (Santa Cruz Biotechnology), mouse monoclonal anti-Myc (Zymed, South San Francisco, CA) and mouse monoclonal anti-WWOX (10). Antibodies used for immunoblot were anti- HA-horseradish peroxidase (HRP, Roche Diagnostics), anti-Myc-HRP, and mouse monoclonal anti-Omni (Santa Cruz Biotechnology). Immunofluorescence. Cells were seeded on fibronectin-covered cell culture slides (Becton Dickinson, San Jose, CA). HeLa cells were transfected with expression vectors encoding WWOX-GFP, Omni-YAP2, and ErbB-4-HA using FuGENE 6 (Roche Diagnostics). After 24 hours, cells were treated with TPA (100 ng/mL) or DMSO for 90 minutes and then cells were prepared as previously described (2). MCF-7 cells were treated with TPA and prepared as above. Antibodies used were mouse monoclonal anti-HA (Covance), rabbit polyclonal anti-ErbB-4 (Santa Cruz Biotechnology), mouse monoclonal anti- WWOX, anti-Rabbit Flouro-conjugated antibody and anti-mouse Texas red– conjugated antibody (Invitrogen). Cells were visualized using Zeiss LCM 510 confocal microscope. Figure 2. WWOX binds ErbB-4 via its first WW domain. A, 293 cells were Luciferase assays. In these experiments, we mainly used the CTF transiently transfected with the expression plasmids encoding ErbB-4-HA and Myc-WWOX or Myc-WWOXY33R. Twenty-four hours after transfection, whole isoform that lacks kinase domain (CTF-DK) because it shows a better cell lysates were immunoprecipitated (IP) with anti-Myc (lanes 2 and 5), anti-IgG transactivation activity when compared with the entire CTF (28, 33). We (lanes 3 and 6), or anti-HA (lanes 4 and 7) antibodies. The immunoprecipitates also used pFR-luc reporter construct (Stratagene) containing five copies of were analyzed by immunoblotting (IB) with anti-HA (top) or anti-Myc Gal4 DNA binding sequences. 293 cells in 6-well dishes were transfected (bottom)-HRP–conjugated antibodies. Lanes 1 and 8, lysate expression of ErbB-4 (top) and WWOX (bottom). B, 293 cells were transiently cotransfected with the plasmids indicated in the figure legends using FuGENE 6 with the expression plasmids encoding Myc-WWOX and ErbB-4-PY1-HA or transfection reagent. Cells were collected 24 hours later and Firefly and ErbB-4-PY3-HA. Immunoprecipitation were as follows: anti-HA (lanes 2 and 5), Renilla luciferase activities were assayed with the Dual Luciferase Assay anti-IgG (lanes 3 and 6), or anti-Myc (lanes 4 and 7) and immunoblotting were Reporter System (Promega). All experiments were done at least thrice. as in (A). Lanes 1 and 8, expression of ErbB-4 (top) and WWOX (bottom). C, 293 cells were transiently cotransfected with the expression plasmids encoding Myc-WWOX and ErbB-4-HA or ErbB-4-PY1,3-HA. Immunoprecipitation and Results immunoblotting were as in (B). WWOX associates physically with ErbB-4. Because the WW domain–containing protein YAP associates physically and func- WWOX, we generated a mutated form of WWOX in which the tionally with ErbB-4, we decided to investigate whether WWOX conserved Tyr33 (Y33) was substituted to arginine (R), WWOXY33R. also interacts with ErbB-4 through its WW domains. We did The Y33 is located within the central core of consecutive aromatics coimmunoprecipitation experiments to assess this interaction. In of the WW domain and was shown to be a site of phosphorylation all immunoprecipitation experiments, we used HA-tagged CYT-2 that regulates signaling of WWOX (2, 6). This Y-R mutation isoform of the full-length ErbB-4 that lacks the PI3K-binding motif abolished interaction between WWOX and p73 or AP2g proteins (Fig. 1C). We transiently cotransfected 293 cells with constructs (2, 3) and at the molecular level it could be explained as a expressing ErbB-4-HA and Myc-WWOX and cell lysates were substitution that compromises the ‘‘aromatic cradle’’ structure that immunoprecipitated with anti-HA or anti-Myc antibody followed is essential for the formation of WW domain-ligand complexes by immunoblotting with HRP-conjugated anti-HA or anti-Myc (38). Similarly, the WW domain mutation in WWOX completely antibodies. As shown in Fig. 2A, we were able to detect immune inhibited its binding to ErbB-4 (Fig. 2A, lanes 5, top and lane 7, complexes between ErbB-4 and WWOX in vivo (lane 2, top and lane bottom). These results indicate that the first WW domain of 4, bottom). As a control, there were no detectable complexes in WWOX is primarily responsible for the interaction with ErbB-4. anti-IgG immunoprecipitates (Fig. 2A, lanes 3 and 6). To determine The cytoplasmic region of ErbB-4 CYT-2 isoform contains two whether this interaction is mediated by the first WW domain of PPxY motifs at positions 1031 to 1040 (NIPPPIYTSR; PY1) and 1280

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Figure 3. Subcellular localization of WWOX and ErbB-4. A, HeLa cells were transfected with the indicated plasmids. Twenty-four hours later, cells (d-f) were treated with TPA (100 ng/mL) for 90 minutes. Cells then were fixed and permeabilized and immunostained with anti-HA antibodies followed by Texas red–conjugated anti-mouse IgG (red, CTF). Colocalization of WWOX and CTF is in yellow as a result of colors merging. B, MCF-7 cells were treated with TPA (100 ng/mL) for 90 minutes (c-d). Cells were prepared as in (A). Antibodies used were polyclonal anti-ErbB-4 (green) and monoclonal anti-WWOX (red). All cells were visualized by confocal microscopy using Â63 objective lens.

to 1288 (LPPPPYRHR; PY3). The PY3 motif in ErbB-4 has been WWOX and ErbB-4 colocalize in the cytoplasm. We next previously shown to mediate interaction with YAPs (28). To prove examined the effect of WWOX-ErbB-4 interaction on the that WWOX binds to PPxY motif of ErbB-4, we replaced the subcellular localization of WWOX and ErbB-4. To address this, tyrosine residues in the PPxY motif in the cytoplasmic region of we studied the localization of both proteins with the aid of ErbB-4 by alanine generating ErbB-4-PY1-HA and ErbB-4-PY3-HA. confocal microscopy. ErbB-4 receptor has a cytoplasmic domain To determine whether WWOX coimmunoprecipitates ErbB-4 that translocates to the nucleus following stimulation with TPA mutants, we cotransfected 293 cells with Myc-WWOX and either (33). To examine the effect of WWOX-ErbB-4 association on this ErbB-4-HA-PY1 or ErbB-4-HA-PY3 and extracts were immunopre- step of signaling in our experimental system, ErbB-4-HA alone or cipitated with anti HA or anti-Myc antibodies and detected using with WWOX-GFP was transiently expressed in HeLa cells. anti-HA (top) or anti-Myc (bottom) antibodies. Figure 2B shows Localization of the HA-tagged proteins was then determined by that WWOX coimmunoprecipitates with both ErbB-4 mutants at a immunofluorescent staining, as described in Materials and level comparable with its coimmunoprecipitation with wild-type Methods. As shown in Fig. 3A, ErbB-4 alone localizes in the cell ErbB-4 (Fig. 2B, lanes 4 and 7, top and lanes 2 and 6, bottom). We membrane and cytoplasm, whereas WWOX is mainly in the then examined whether the WW domain of WWOX interacts with cytoplasm (Fig. 3A, b and a). Coexpression of WWOX-GFP and both the PPxY motifs of ErbB-4 and generated a double-mutated ErbB-4-HA resulted in partial colocalization of WWOX and ErbB-4 form of ErbB-4 that harbor both mutations of PY1 and PY3, named in the cytoplasm (Fig. 3A, c). Figure 3A (bottom)shows ErbB-4-PY1,3-HA. Toward this end, 293 cells were transfected with translocation of CTF into the nucleus following treatment with Myc-WWOX and either wild-type ErbB-4-HA or ErbB-4-PY1,3-HA TPA; however, localization of WWOX-GFP was not affected (Fig. 3A, and the cell lysates were immunoprecipitated with anti-HA or anti- e and d). By contrast, cells coexpressing WWOX and ErbB-4 and Myc antibodies and probed using anti-HA (top) and anti-Myc treated with TPA showed a cytoplasmic colocalization of both (bottom) antibodies. As shown in Fig. 2C, mutation of both PPxY proteins (Fig. 3A, f ). These results indicate that expression of motifs in ErbB-4 inhibited interaction with WWOX (Fig. 2C, lane 7, WWOX causes retention of CTF in the cytoplasm and prevents its top and lane 5, bottom). These results imply that WWOX interacts translocation to nucleus. To show this effect on endogenous with both of the proline/tyrosine-containing motifs in ErbB-4.4 proteins, we used MCF-7 breast carcinoma cells that express high levels of endogenous WWOX and moderate levels of endogenous ErbB-4. We used confocal microscopy and localization of 4 Our results also confirm the in vitro data of WW domain mapping generated by AxCell Biosciences/Cytogen Corporation (20). Their protein-array data clearly showed endogenous proteins was then determined by immunofluorescent that all three PPxYs of ErbB4 formed complexes with WWOX. staining using polyclonal antibody against ErbB-4 (Santa Cruz www.aacrjournals.org 6767 Cancer Res 2005; 65: (15). August 1, 2005

Figure 4. A, association of WWOX-ErbB-4 suppresses transactivation of CTF mediated by YAP. 293 cells were cotransfected with the Gal4DNA binding domain (Gal4BD, residues 1-142) fused to the CTF-DK fragment (residues 988-1292) and with pFR-luc (0.3 Ag), Omni-YAP2 (0.2 Ag), Omni-YAP2-1stWW* [mYAP2] (0.2 Ag), Myc-WWOX (0.1-0.3 Ag), and Myc-WWOXY33R [mWWOX] (0.2-0.3 Ag) as indicated. Empty vector was cotransfected to normalize DNA amount where required. A reporter plasmid encoding Renilla luciferase was also cotransfected to normalize transfection efficiencies. Twenty-four hours posttransfection, cells were lysed and luciferase activity was determined. Results are shown as fold activation of the luciferase activity compared with control cells transfected with empty vector alone. Columns, averages of three experiments. B, HeLa cells were transfected with constructs expressing CTF-DK alone or together with WWOX-GFP. Cells were immunostained with polyclonal anti-ErbB-4 (red) and visualized as in Fig. 3. C, WWOX suppresses transactivation of p73 mediated through p73-YAP association. 293 cells were cotransfected with vectors expressing p53RE-Luc (0.1 Ag), HA-p73h (10 ng), Omni-YAP2 (0.2 Ag), Myc-WWOX (0.5 Ag), and Nuc-WWOX-Myc (0.5 Ag) as indicated. Cells were then treated as in (A). D, HeLa cells were transfected with pCMV-NUC-WWOX-MYC plasmid. Twenty-four hours later, cells were incubated with MitoTracker Red (red, mitochondria) and immunostained with anti-Myc antibodies followed by FITC-conjugated anti-mouse IgG (green, WWOX). Cells were visualized as in Fig. 3.

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Biotechnology) and our monoclonal antibody against WWOX (10). suppressed by WWOX, suggesting that WWOX may antagonize the As expected, MCF-7 cells express both ErbB-4 and WWOX in the function of YAP. cytoplasm (Fig. 3B, a-b). Treatment of MCF-7 cells with TPA To confirm interaction between WWOX and CTF in the resulted in partial translocation of CTF into the nucleus (Fig. 3B, transactivation assay, we cotransfected HeLa cells with WWOX- c-d). Interestingly, cells displaying nuclear CTF have significantly GFP and CTF-DK. As shown in Fig. 4B, CTF-DK when expressed reduced expression of WWOX (Fig. 3B, d), which might explain this alone showed clear localization in the nucleus (Fig. 4B, a). partial sequestration of endogenous ErbB-4. However, we cannot Coexpression of WWOX and CTF-DK caused partial colocalization exclude that there might be other factor(s) regulating ErbB-4 of WWOX and CTF-DK in the cytoplasm (Fig. 4B, b) suggesting that translocation into nucleus. Altogether, our results suggest that WWOX sequesters CTF in the cytoplasm and prevents its nuclear WWOX binds ErBb-4 in the cytoplasm and prevents CTF translocation leading to its reduced transactivation ability. translocation to the nucleus. Because it was shown that YAP could serve as a coactivator of WWOX antagonizes coactivation function of YAP. Stimula- p73 (25), we decided to check the effect of WWOX expression on tion of cells expressing ErbB-4 with TPA causes the translocation of this transactivation. We previously showed that WWOX sup- CTF to the nucleus where it may affect transcription of target presses p73-mediated transactivation (2). To assess the antago- genes. Although CTF by itself has no transactivation activity, nizing effect of WWOX on YAP function further, we used the Komuro et al. recently showed that YAP can act as a transcriptional previously described construct containing the luciferase gene coactivator of CTF in the Gal4 transactivation system (28). driven by a p53 cis-element from the p53 promoter (p53RE-Luc) Therefore, we used the same system to determine the effect of that can be used in p73h transactivation studies (2). As WWOX expression on this transactivation. YAP2 and CTF expected, p73h showed significant transactivation of luciferase transiently expressed in 293 cells as expected showed f35-fold reporter and expression of YAP2 increased this effect (Fig. 4C, activation compared with Gal4 control (Fig. 4A, column 8). In columns 2 and 6). By contrast, expression of p73h and WWOX contrast, coexpression of WWOX and CTF did not show any significantly suppressed the transactivation function of p73 transactivation (Fig. 4A, columns 2-6). Interestingly, WWOX (Fig. 4C, column 7). In addition, coexpression of YAP2, p73h, significantly suppressed YAP2-CTF-mediated transactivation in a and WWOX resulted in the suppression of p73 transactivation dose-dependent manner (Fig. 4A, columns 9-11). Furthermore, function (Fig. 4C, column 8) indicating that WWOX indeed WWOXY33R mutant lacking interaction with ErbB-4 had no effect antagonizes YAP function. on YAP2 transactivation (Fig. 4A, columns 12 and 13). In addition, a We previously showed that WWOX sequesters p73 in the YAP2 mutant harboring an inactivation mutation in the first WW cytoplasm explaining WWOX suppressive effect of p73 trans- domain and lacking interaction with CTF (28) did not show any activation (2). To further confirm that the effect of WWOX is due transactivation of the reporter (Fig. 4A, columns 7 and 14). These to the interaction with p73 and not solely to WWOX-p73 colo- data indicate that transactivation caused by YAP-CTF interaction is calization in the cytoplasm, we constructed a myc-tagged-nuclear

Figure 5. WWOX and YAP compete for interaction with ErbB-4. A, 293 cells were transiently transfected with the expression plasmids encoding ErbB-4-HA (6.0 Ag), Myc-WWOX (7.0 Ag), and Omni-YAP2 (1.0 or 2.0 Ag). Twenty-four hours after transfection, whole cell lysates were immunoprecipitated (IP) with anti-HA (lanes 2 and 6), anti-IgG (lanes 3 and 7), anti-WWOX (lanes 4 and 8), or anti-Omni (lanes 5 and 9) antibodies. The immunoprecipitates were analyzed by immunoblotting (IB) with anti-HA antibody (top). Lanes 1 and 10, expression of ErbB-4; bottom, expression of Myc-WWOX (left) and Omni-YAP2 (right). B, 293 cells were transiently transfected with the expression plasmids encoding ErbB-4ÀHA (6.0 Ag), Omni-YAP2 (1.0 Ag), and Myc-WWOX (7.0 Ag) or Myc-WWOXY33R (7.0 Ag). Cells were then treated as (A).

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WWOX expression vector, named Nuc-WWOX-Myc, expressing Discussion WWOX exclusively in the nucleus (Fig. 4D). Coexpression of WW domain–containing proteins are widely expressed in all h Nuc-WWOX and p73 had the same suppressive effect on p73 eukaryotes and play a vital role in the regulation of a wide variety of transactivation as wild-type WWOX (Fig. 4C, column 9). cellular functions including regulation of gene expression (39, 40). Furthermore, expression of YAP2 did not affect this suppression Here we show that WWOX binds ErbB-4 and suppresses trans- (Fig. 4C, column 10) suggesting that the effect of WWOX activation of CTF mediated by YAP coactivation. We also report that expression is superior to that of YAP. Similar results were seen WWOX sequesters ErbB-4 in the cytoplasm and inhibits its nuclear for the CTF-YAP2 and Nuc-WWOX complex (data not shown). translocation following treatment with TPA. Our results show that This suggests that even when WWOX is in the nucleus together the association of WWOX and ErbB-4 in the cytoplasm might explain with p73 or CTF, it still inhibits its association with YAP and thus the suppressed transactivation ability of CTF, although a nuclear- prevents its coactivation. targeted WWOX still has the suppressive effect on CTF function. WWOX and YAP compete for interaction with ErbB-4. Our YAP is another WW domain–containing protein that functions as results may also suggest that WWOX and YAP compete for a modulator of several transcription factors. YAP has been interaction with ErbB-4 and modulate its transactivation activity. characterized as a cotransactivator for several transcription factors To check whether YAP and WWOX compete for interaction with including the Runx family proteins, the TEAD/TEF family of ErbB-4, we cotransfected 293 cells with fixed amounts of vectors transcription factors, and p73 (24, 25, 41). YAP activity typically expressing ErbB-4-HA and Myc-WWOX and variable amounts of involves either of the two WW domains that associate directly with Omni-YAP2. Cell lysates were then immunoprecipitated with anti- PPxY motifs within the transcription factors, although other mech- WWOX or anti-Omni antibodies and immunoblotted with anti-HA anisms of interaction may occur. Although YAP can modulate the antibody. Our results show that in the presence of reduced amount activity of multiple transcription factors, several reports suggest of YAP2, ErbB-4 interacts exclusively with WWOX (Fig. 5A, lane 4 that it localizes predominantly to the cytoplasm. In the cytoplasm versus 5). On the other hand, increased expression of YAP2 causes YAP interacts with EBp50 (ezrin/radixin/moesin-binding phospho- some ErbB-4-YAP2 association, although ErbB-4-WWOX interaction protein of 50 kDa), 14-3-3 and Yes protein tyrosine kinase (22, 29, 42). was still the predominant complex (Fig. 5A, lane 9 versus 8). To show Most recently, it was shown that 14-3-3 interacts with YAP phos- that WWOX expression indeed interfered with the formation of YAP- phorylated at Ser127 and that it prevents YAP from translocation ErbB-4 complex, we cotransfected 293 cells with ErbB-4-HA, Omni- into the nucleus (29). In our study, we used the alternatively spliced YAP2, and either Myc-WWOX or Myc-WWOXY33R. In this context, CYT-2 isoform of ErbB-4 that lacks the PI3K binding motif; expression of Myc-WWOXY33R resulted in significant rescue of therefore, Akt is not activated to phosphorylate YAP at Ser127; thus, ErbB-4-YAP2 interaction (Fig. 5B, lane 9 versus 8). Altogether, our YAP is retained in the cytoplasm. The subcellular localization of YAP results from the Gal4 transactivation assay (Fig. 4A) and immuno- and WWOX in the cytoplasm makes them accessible to bind the precipitation experiments (Fig. 5) indicate that WWOX and YAP may PPxY motif of ErbB-4. Our results imply that WWOX binds ErbB-4 compete for the association with ErbB-4 and that WWOX-ErbB-4 in the cytoplasm and makes it inaccessible for interaction with YAP. interaction is prevalent. Therefore, it is possible that WWOX binds CTF and prevents its

Figure 6. A proposed model of WWOX and YAP function competing for interaction with ErbB-4 and other target proteins. In this model, ErbB-4-CTF is cleaved following binding of its ligand heregulin or activation of PKC with TPA. CTF by itself or together with YAP translocates to the nucleus and transactivate target genes in the nucleus. In the presence of WWOX, CTF is sequestered in the cytoplasm and transactivation is suppressed. WWOX also interact with other transcription factors such as p73 and AP-2g and inhibit their translocation to the nucleus hence suppressing their transactivation activity.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. WWOX-ErbB-4 Association interaction with YAP; thus, CTF cannot translocate to the nucleus shown in Fig. 6, where both WW domain–containing proteins, YAP where it may transactivate target genes. A mutated form of WWOX, and WWOX, in the cytoplasm are competing for interaction with WWOXY33R, does not bind ErbB-4; therefore, when coexpressed PPxY-containing target proteins. Whereas WWOX inhibits nuclear with YAP, it does not prevent YAP from association with ErbB-4 and translocation of target proteins, YAP translocates with these targets transcriptional coactivation of the nucleus translocated ErbB4-YAP into the nucleus thus determining the transcriptional outcome of complex. Interestingly, WWOX binds ErbB-4 with better affinity these target proteins. than YAP, perhaps due to the interaction of WWOX with both the ErbB-4 regulates cell proliferation and differentiation (32); PPxY motifs of ErbB-4 rather than only with the most COOH- therefore, the impairment of its proper function may contribute terminal PPxY sequence in the case of YAP-ErbB-4 association (28). to cancer initiation and/or progression. ErbB-4 belongs to the type The functional importance of CTF translocation to the nuclear I receptor tyrosine kinase family which include ErbB-1 and ErbB-2 compartment is not yet clear. However, the fact that CTF was that are known proto-oncogenes in breast cancer and other detected in the nucleus in different tissues and expression of nuclear carcinomas (46). Indeed, ErbB-4 expression is altered in several CTF was reported in a number of tumors may indicate an important tumors including breast, prostate, ovary, and other carcinomas. role of this localization. Both the Notch receptor and Alzheimer’s Srinivasan et al. reported significant percentage of ErbB-4 nuclear precursor protein, are similarly cleaved by g-secretase and their staining in invasive breast cancer tumors (47). Interestingly, WWOX intracellular fragments translocate to the nucleus and directly is inactivated in invasive breast carcinoma (12) and YAP was regulate transcription of different genes (reviewed in ref. 43). documented to be differentially expressed with malignant trans- Moreover, the founding member of the epidermal growth factor formation and metastatic tumor progression of squamous cell receptor family, ErbB-1 has been shown to bind the cyclin D1 carcinoma (48). Moreover, the most recent work from the promoter and possibly to transactivate genes essential for laboratory of Elenius has documented a clear correlation between proliferation (44). In addition, ErbB-1 has been proposed to function a clinical outcome for estrogen receptor–positive breast cancer as a carrier for signal transducers and activators of transcription 3 to patients and the presence of cleavable and nucleus-localized ErbB4 the nuclear compartment (45). It is thus quite possible that ErbB4- isoforms (49). This may suggest that in WWOX-negative cells, CTF CTF can transactivate target genes in the nucleus or that it functions of ErbB-4 can translocate to the nucleus and transactivate target as a shuttle protein for different targets destined for the nucleus genes in the presence of YAP, estrogen receptor, or other (e.g., YAP; refs. 28, 31). Our findings show that in the presence of coactivators. Suppression of transcription factors such as ErbB-4- WWOX, CTF is retained in the cytoplasm; thus, CTF cannot CTF may have a great effect because these genes have been shown transactivate target genes or shuttle proteins into the nucleus. to be amplified and overexpressed in number of cancers. Recently, we showed that WWOX binds p73 transcription factor and suppresses its transcriptional function (2). Interestingly, YAP, via its WW domain also associates with p73 and this interaction Acknowledgments causes the enhanced transcriptional activity of p73 (25). When Received 4/4/2005; revised 5/24/2005; accepted 5/25/2005. coexpressed with YAP and p73, WWOX was able to reverse this Grant support: NIH grants CA77738 (C.M. Croce) and DK62345 (M. Sudol). The costs of publication of this article were defrayed in part by the payment of page effect. These results suggest that WWOX acts as a transcriptional charges. This article must therefore be hereby marked advertisement in accordance corepressor antagonizing the function of YAP. A proposed model is with 18 U.S.C. Section 1734 solely to indicate this fact.

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Rami I. Aqeilan, Valentina Donati, Alexey Palamarchuk, et al.

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