Journal of Cell Science 113, 1481-1490 (2000) 1481 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1224

ARVCF localizes to the nucleus and adherens junction and is mutually exclusive with p120ctn in E-cadherin complexes

Deborah J. Mariner, Jue Wang and Albert B. Reynolds* Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2175, USA *Author for correspondence (e-mail: [email protected])

Accepted 12 February; published on WWW 21 March 2000

SUMMARY

ARVCF is a novel Armadillo repeat domain that is previous results indicating that the branching activity of closely related to the catenin p120ctn. Using new ARVCF p120 maps to its Armadillo repeat domain. Surprisingly, monoclonal antibodies, we have found that ARVCF the preferential localization of ARVCF to the nucleus associates with E-cadherin and competes with p120 for required sequences in the amino-terminal end of ARVCF, interaction with the E-cadherin juxtamembrane domain. suggesting that the sequences directing nuclear ARVCF also localized to the nucleus in some cell types, translocation of ARVCF are distinct from the predicted however, and was significantly more nucleophilic than bipartite nuclear localization signal located between p120. Surprisingly, despite apparently ubiquitous repeats 6 and 7. The dual localization of ARVCF to expression, ARVCF was at least tenfold less abundant than junctions and to nuclei suggests activities in different p120 in a wide variety of cell types, and was difficult to cellular compartments, as is the case for several other detect by immunofluorescence unless overexpressed. Armadillo repeat including β-catenin, p120 and Consequently, it is not likely to be abundant enough in the plakophilins. adult tissues to functionally compete with p120. ARVCF also completely lacked the ability to induce the cell- branching phenotype associated with overexpression of Key words: ARVCF, p120ctn, E-cadherin, Catenin, Adherens p120. Expression of ARVCF/p120 chimeras confirmed junction, Armadillo repeat

INTRODUCTION terminal regions flanking the Arm repeats, the intron-exon structure of the is nearly identical (Keirsebilck et al., ARVCF (Armadillo repeat deleted in Velo-cardio-facial 1998), indicating an ancient evolutionary relationship. syndrome) is a candidate gene for the related developmental Alternative splicing results in the cell type-specific expression abnormalities Velo-cardio-facial syndrome (VCFS) and of multiple p120 isoforms whose individual roles are unknown DiGeorge syndrome (DGS) (Sirotkin et al., 1997), (Keirsebilck et al., 1998; Mo and Reynolds, 1996). The cloned characterized by a wide spectrum of phenotypes such as human ARVCF cDNA most closely resembles p120 isoform conotruncal heart defects, cleft palate and facial p120ctn1A (Reynolds and Daniel, 1997; Sirotkin et al., 1997). dysmorphology (Shprintzen et al., 1981; Young et al., 1980). p120 interacts with a variety of cadherins, including E-, The gene maps to a region of human 22q11 N-, P-, C- and VE-cadherins (Lampugnani et al., 1997; (Bonne et al., 1998; Sirotkin et al., 1997) that is hemizygous Reynolds et al., 1996; Yap et al., 1998). Cadherins are a large in 80-85% of VCFS/DGS patients (Desmaze et al., 1993; superfamily of transmembrane glycoproteins that mediate Driscoll et al., 1993, 1992; Kelly et al., 1993; Morrow et al., Ca2+-dependent cell-cell adhesion and participate in multiple 1995; Scambler et al., 1992; Wilson et al., 1992a,b). Other aspects of development, morphogenesis and malignancy (for genes also map to this region and the contribution of the loss reviews, Takeichi, 1991; Yap, 1998). Of the classical cadherins, of ARVCF to these syndromes has not yet been fully E-cadherin has been studied most extensively, in part because established. its expression is frequently downregulated or turned off in ARVCF is the closest identified relative to the catenin metastatic cancer (reviewed in Birchmeier and Behrens, 1994). p120ctn (‘p120’), suggesting that they have related functions It is the major cell-cell adhesion molecule in most carcinomas, (Sirotkin et al., 1997). Both proteins contain an amino-terminal and its downregulation in late-stage tumors is widely coiled-coil domain, as well as a central Armadillo repeat considered to be an important factor in the transition to domain (Arm domain). The Arm domains are 56% identical to malignancy. Indeed, restoration of E-cadherin expression in one another and consist of 10 imperfect 42 amino acid repeats. such cells restores adhesiveness and reduces invasive Although the homology decreases in the amino- and carboxy- phenotypes both in vitro and in vivo (Frixen et al., 1991; Perl 1482 D. J. Mariner, J. Wang and A. B. Reynolds et al., 1998; Vleminckx et al., 1991). Cadherin function is they compete for the same binding site on E-cadherin. regulated by catenins (α, β-catenins, plakoglobin/γ-catenin and Unexpectedly, analysis of multiple cell types revealed that the p120), proteins that bind directly or indirectly to the cadherin expression level of ARVCF in adult tissues was at least tenfold cytoplasmic tail (reviewed in Nollet et al., 1999; Steinberg and less than that of p120. Therefore, it is unlikely under normal McNutt, 1999). β-catenin links E-cadherin to α-catenin, which circumstances that ARVCF functions simply as a competitive in turn links the complex to the actin cytoskeleton either binding protein. Instead, it is likely to bring to the junction its directly or indirectly through other proteins such as α-actinin own activity, one that is distinct from that of p120 and does not or vinculin (Hazan et al., 1997; Herrenknecht et al., 1991; require high level expression. Additionally, ARVCF showed a Knudsen et al., 1995; Nagafuchi et al., 1991; Nieset et al., strong cell type-specific preference for localization to the 1997). p120 does not interact with α-catenin (Daniel and nucleus and completely lacked the strong branching activity Reynolds, 1995), suggesting that it does not participate directly associated with overexpression of p120 in fibroblasts. in the linkage to the actin cytoskeleton. ARVCF/p120 chimeras were generated and used to map the Alterations in α- and β-catenins have been reported in sequences responsible for the branching phenotype to the metastatic cell lines and may also contribute to the invasive carboxy terminus of p120. Despite the presence of a classic phenotype (Hirano et al., 1992; Kawanishi et al., 1995; Morton bipartite nuclear localization signal in the central region of et al., 1993; Oyama et al., 1994). β-catenin has well- ARVCF, the nuclear translocation preference of this protein characterized roles in both adhesion and transcription required sequences residing in the amino-terminal end of the (reviewed in Peifer, 1995). Less is known about the role of molecule. These data indicate dual functions for ARVCF in p120 and its family members in these processes. Though adherens junctions and the nucleus, and highlight significant originally characterized as a prominent substrate for the Src differences relative to p120 in activity, abundance and protein tyrosine kinase, there is recent evidence suggesting localization. roles for p120 in cadherin clustering (Yap et al., 1998), cell motility (Chen et al., 1997), neuronal outgrowth (Riehl et al., MATERIALS AND METHODS 1996), and inhibition or dismantling of the cadherin complex (Aono et al., 1999; Ohkubo and Ozawa, 1999). In addition, Cell culture p120 binds to a transcription factor, Kaiso (Daniel and Cells were cultured in DMEM (Hyclone) containing 10% fetal bovine Reynolds, 1999), and has been reported to translocate to the serum and 1% penicillin (10,000 i.u./ml)-streptomycin (10 mg/ml) nucleus under some circumstances (van Hengel et al., 1999). (GibcoBRL). Human ARVCF (GenBank accession number U51269) The growing p120 family now includes ARVCF (Sirotkin et was stably overexpressed in Madin Darby Canine Kidney II cells al., 1997), δ-catenin/NPRAP/neurojungin (Paffenholz and (MDCK II) as described previously (Mariner et al., 1999). These cells Franke, 1997; Paffenholz et al., 1999; Zhou et al., 1997) and were maintained in the above medium supplemented with 250 µg/ml p0071 (Hatzfeld and Nachtsheim, 1996), and a related (active units) G418 (GibcoBRL). The MDCK II parent cell line was subgroup of proteins including plakophilin 1 (Hatzfeld et al., provided by Dr Robert Coffey (Vanderbilt University), and PANC-1 1994; Heid et al., 1994; Schmidt et al., 1997), plakophilin 2 cells (human pancreatic carcinoma) were provided by Dr Steven (Mertens et al., 1996) and plakophilin 3 (Bonne et al., 1999; Leach (Vanderbilt University). Other cell lines were obtained from the Schmidt et al., 1999). Whereas p120 and δ-catenin bind to type American Type Culture Collection, including MCF-7, human breast carcinoma; BeWo, human placental carcinoma; HepG2, human liver I and type II cadherins (Daniel and Reynolds, 1995; Lu et al., carcinoma; HCT 116, human colon carcinoma; 293, human 1999; Reynolds et al., 1996) in adherens junctions, the embryonic kidney; HeLa, human cervical carcinoma; Va-2, human plakophilins and possibly p0071 (Hatzfeld and Nachtsheim, fibroblasts; MOLT-4, human T-cells; NIH 3T3, murine fibroblasts. 1996) associate instead with desmosomal proteins (Hatzfeld et al., 1994; Mertens et al., 1996; Schmidt et al., 1997). In Transient transfection addition, most of these proteins localize to the nucleus to Transient transfections were performed with SuperFect Transfection varying degrees, and it has been suggested that this duality of Reagent (Qiagen) according to company protocol, except that DNA- localization may reflect a function common to most Armadillo dendrimer complexes were allowed to form for 15 minutes before repeat proteins (Fagotto et al., 1998). The emergence of p120 transfection, and cells were incubated in the presence of transfection family members, many of which are coexpressed in various reagent overnight. Cells were used for experiments 1-2 days post- transfection. cells, introduces a new level of complexity to the regulation of Human ARVCF (GenBank accession number U51269) and human cadherin function (see Commentary in this issue by p120-1A cDNA were expressed from a CMV promoter in pcDNA3.1 Anastasiadis and Reynolds, 2000). It is not clear, for example, plasmid (Invitrogen), while murine p120-1A was expressed from whether these different family members compete with one Rc/CMV plasmid (Invitrogen). another for binding to a particular cadherin, or what effect such competition might have on cadherin function. With the Immunoprecipitation, western blotting and Coomassie exception of β-catenin, the nuclear roles of these proteins are Blue staining largely unknown. ARVCF has not been characterized at the For immunoprecipitation, cells were washed once with phosphate- protein level, in part because antibodies have not been available buffered saline (PBS) (10 mM Tris, pH 7.4, 150 mM NaCl) at room until now. temperature, then lysed in ice-cold lysis buffer (0.5% Nonidet P-40 (NP-40), 50 mM Tris, pH 7.4, 150 mM NaCl) or RIPA (1% NP-40, We show here that despite the expected similarities between 0.1% sodium dodecyl sulfate, 0.5% deoxycholic acid, 50 mM Tris, ARVCF and p120, these proteins are functionally distinct in pH 7.4, 150 mM NaCl) containing 1 mM PMSF, 5 µg/ml leupeptin, several respects. Like p120, ARVCF colocalized and 2 µg/ml aprotinin, 1 mM EDTA and 1 mM Na3V04 (leupeptin from associated with E-cadherin. ARVCF and p120, however, were ICN; all other inhibitors from Sigma). Lysates were cleared by mutually exclusive in cadherin complexes, indicating that centrifugation at 14000 rpm in an Eppendorf microfuge at 4¡C for 5 ARVCF in the nucleus and adherens junction 1483 minutes. Immunoprecipitations were carried out as described 1A (p120-1A). First, 5′ ARVCF cDNA was amplified from previously (Reynolds et al., 1994). Briefly, primary antibody pcDNA3.1/hARVFC plasmid with the oligonucleotides (5′) CCTGA- incubations were performed with 4 µg purified monoclonal antibody, GCTGCCTGAGGTGCTGGC (3′) and (5′) GGATGAAAGATTCC- 100 µl hybridoma supernatant or 2 µl polyclonal antibody (pAb) for ACAGGGTGCCAGTGACAAGCTC (3′). Likewise, human p120-1A 1 hour at 4¡C. Complexes were collected by further incubation for 1 3′ cDNA was amplified from pcDNA3.1/hp120-1A plasmid with the hour in the presence of Protein A-Sepharose beads (Pharmacia) oligonucleotides (5′) ACTGGCACCCTGTGGAATCTTTCATCCC- coupled to rabbit anti-mouse bridge antibody (Jackson ATGACTCAATC (3′) and (5′) GCTTACATTCCTAAGGCAG- Immunoresearch Laboratories). Immunoprecipitates were washed 5× CCAGC (3′). These products served as template for a subsequent with ice-cold lysis buffer (without protease or phosphatase inhibitors), amplification reaction with the oligonucleotide primers (5′) CCTG- then boiled for 3 minutes in Laemmli sample buffer (Laemmli, 1970) AGCTGCCTGAGGTGCTGGC (3′) and (5′) GCTTACATTCCTA- before separation by SDS-polyacrylamide gel electrophoresis (PAGE) AGGCAGCCAGC (3′). Following digestion with Bsu36I restriction on 7% polyacrylamide gels. Antibodies used for immunoprecipitation enzyme (NEB), the product was subcloned between flanking 5′ were ARVCF mAb 4B1 (Mariner et al., 1999), p120 mAb 15D2 (Wu hARVCF and 3′ hp120-1A cDNA sequence in pcDNA3.1. et al., 1998), E-cadherin mAb rr1, β-catenin pAb (Sigma C2206) and For p120-ARVCF, human p120-1A cDNA corresponding to amino hemagglutinin-antigen epitope tag mAb 12CA5. acids 1-477 was joined to cDNA corresponding to amino acids 470- Western blotting was carried out as described (Reynolds et al., 962 of human ARVCF, essentially as described for ARVCF-p120. 5′ 1994). Proteins were detected by enhanced chemiluminescence p120-1A cDNA was amplified from pcDNA3.1/hp120-1A plasmid (Amersham ECL reagent). ARVCF was detected with an ARVCF with the oligonucleotides (5′) GCCCTCTAGACT CGAGCGGCC (3′) mouse pAb generated against the carboxy-terminal 115 amino acids and (5′) GGATGACAGGTTCCACAGGGTTCCGGTAATAACTTC- of human ARVCF diluted 1:5000. Other antibodies used for western AGT (3′). 3′ human ARVCF cDNA sequence was amplified from blotting include p120 mAb pp120 (Transduction Laboratories pcDNA3.1/hARVCF plasmid with (5′) ACCGGAACCCTGTGGA- #P17920), 0.1 µg/ml; E-cadherin mAb (Transduction Laboratories ACCTGTCATCCTATGAGCCCCTG (3′) and (5′) CAACTAGAAG- #C20820), 0.1 µg/ml; β-catenin pAb (Sigma #C2206), 1:1000. GCACAGTCGAGGC (3′) (complementary to pcDNA3.1 polylinker For protein staining with Coomassie Blue, 7% polyacrylamide gels sequence). These products provided the template for a subsequent were agitated in Coomassie Blue stain (50% methanol, 10% acetic amplification reaction with the primers (5′) GCCCTCTAGACT- acid, 0.2% Coomassie Brilliant Blue R) overnight. Destaining was CGAGCGGCC (3′) and (5′) CAACTAGAAGGCACAGTCGAGGC performed in 30% methanol, 7% acetic acid for several hours, (3′). The HindIII digestion product was subcloned between flanking followed by further incubation in 10% methanol, 7% acetic acid. 5′ hp120-1A and 3′ hARVCF cDNA sequence in pcDNA3.1. The 5′ subcloning junctions and fusion regions between ARVCF Phosphatase treatment and p120 cDNAs were verified by sequencing. For phosphatase treatment, ARVCF was immunoprecipitated as described above. Immunoprecipitates were washed 3× with lysis buffer lacking protease and phosphatase inhibitors, and twice with RESULTS TBS to remove detergent. Washed immunoprecipitates were then incubated with 200 units λ phosphatase (New England Biolabs #753) Characterization of ARVCF expression for 30 minutes at 30¡C, in a 50 µl reaction mixture containing 50 mM Monoclonal antibodies (mAbs) were generated previously Tris-HCl, pH 7.5, 0.1 mM Na2EDTA, 5 mM EDTA, 0.01% Brij 35, against the carboxy-terminal 115 amino acids of ARVCF, a 2 mM MnCl2. The reaction was terminated by removing the region with relatively low similarity to p120 (Mariner et al., supernatant and boiling the immunoprecipitate in Laemmli sample 1999). Fig. 1 shows the region of ARVCF used as antigen to buffer for 3 minutes. generate ARVCF mAb 4B1, and illustrates the structural Immunofluorescence labeling similarity between ARVCF and p120. The monoclonal Cells were labeled by immunofluorescence as described previously antibodies 4B1 (ARVCF) and 15D2 (p120) are highly specific (Reynolds et al., 1994). Briefly, cells were cultured on glass coverslips for their respective antigens (Mariner et al., 1999). for 24-48 hours. After two rinses in room temperature PBS, cells were Initial efforts to localize ARVCF by immunofluorescence fixed and permeabilized in ice-cold methanol for 7 minutes at −20¡C, were unsuccessful. Similarly, ARVCF was difficult to detect then washed twice in PBS. Coverslips were then blocked in blocking by immunoprecipitation and western blotting under conditions solution (PBS containing 3% nonfat milk) for 5 minutes, then routinely used to assay p120. Subsequent experiments (Fig. 2) incubated in primary antibody diluted in blocking buffer (mAb 4B1, µ revealed that ARVCF is nearly ubiquitously expressed, as has 8D11, 12F4, 1 g/ml) for 30 minutes at room temperature in a been demonstrated previously by northern analysis (Sirotkin et humidified chamber. Following three 5 minute PBS washes, coverslips were rinsed with blocking buffer and incubated with al., 1997), but is present in very low amounts at the protein secondary antibody diluted in blocking buffer. Secondary antibodies level. To obtain roughly equivalent signal intensities for used include Alexa488-coupled goat anti-mouse IgG (Molecular ARVCF and p120, the amount of cell lysate required for Probes), diluted 1:1200; fluorescein isothiocyanate (FITC)-coupled ARVCF immunoprecipitation reactions (Fig. 2A) was typically goat anti-IgG1 and tetramethyl rhodamine isothiocyanate (TRITC)- 10- to 20-fold more than that required for p120 (Fig. 2B). coupled goat anti-IgG2a (Southern Biotechnology Associates, Inc.), Levels were also compared in semiquantitative fashion by diluted 1:200. The chimera ARVCF-p120 was detected with mAb immunoprecipitating ARVCF and p120 from HCT 116 cell 12F4, and p120-ARVCF was detected with mAb 4B1. lysates with excess antibody, and analysis by Coomassie Blue Generation of bimolecular chimeric proteins staining (data not shown). HCT116 cells were chosen because they express higher levels of ARVCF than any of our other cell ARVCF-p120 and p120-ARVCF chimeric proteins were generated by molecular sewing and polymerase chain reaction (PCR) amplification lines. Immunoprecipitated p120, but not ARVCF, was readily with Pfu Turbo (Stratagene). detectable by this method, suggesting that even in HCT116 For ARVCF-p120, human ARVCF (hARVCF) cDNA, cells, ARVCF is expressed at significantly lower levels than corresponding to amino acids 1-469 of hARVCF, were joined to p120. In general, ARVCF levels were highest in epithelial cDNA corresponding to amino acids 478-962 of human p120 isoform cells (e.g. Fig. 2A, lanes 1-4), but decreased levels were also 1484 D. J. Mariner, J. Wang and A. B. Reynolds

human ARVCF mAb Ag 1234 56* 78910 AA 1 962

human p120ctn 1A 1234 5 6* 78910 AA 1 962

Fig. 1. Structural similarity of ARVCF and p120ctn isoform 1A, and location of ARVCF monoclonal antibody epitopes. Individual Armadillo repeats are numbered. An asterisk indicates the position of a putative nuclear localization signal, and the filled box depicts a coiled-coil motif. Monoclonal antibodies were generated against a 115-amino-acid carboxy-terminal ARVCF fragment (hatched), a region with low homology to p120.

Fig. 3. Post-translational modification and isoform distribution of ARVCF. mAb 4B1 immunoprecipitates of ARVCF from various cell lines were split equally, incubated in the absence (lanes 1-5) or presence (lanes 6-10) of lambda (λ) phosphatase, and western blotted with ARVCF antibodies. Lanes contain immunoprecipitates from untransfected MDCK cells (lanes 1,6), neo vector alone transfected MDCK cells (lanes 2,7), MDCK cells stably transfected with human ARVCF (lanes 3,8), human HCT116 colon cancer cells (lanes 4,9), and human kidney 293 cells (lanes 5,10). Because exogenous ARVCF is difficult to detect under conditions used for p120 immunoprecipitation (lanes 1,2 and 6,7), tenfold more total protein was present in lysates used for immunoprecipitation from HCT116 (lanes 4,9) and 293 (lanes 5,10) than was present in MDCK lysates (lanes 1-3, 6-8). HCT116 (lanes 4,9) and 293 (lanes 5,10) Fig. 2. Comparison of ARVCF and p120 expression in different cell cells contained several ARVCF isoforms, the largest of which types. ARVCF (mAb 4B1) and p120 (mAb 15D2) were comigrated exactly with the ectopic human ARVCF expressed in immunoprecipitated from NP-40 cell lysates of the indicated human MDCK cells (lanes 3,8). Phosphatase treatment resulted in cell lines, and western blotted with ARVCF pAb (A) or mAb pp120 sharpening and increased mobility of the bands in many cell types (B). Total protein was normalized before immunoprecipitation to (e.g. compare lane 4 with lane 9), consistent with removal of control for relative abundance. However, fivefold more lysate was used phosphate. for ARVCF immunoprecipitates than for the p120 immunoprecipitates. Note that the exposure time for the ARVCF western blot was 15 minutes, while that for the p120 blot was 30 seconds. stable MDCK cell lines ectopically expressing human ARVCF. Immunoprecipitated ARVCF from these cell lines was easily detected by SDS-PAGE and semiquantitative Coomassie Blue detected in fibroblasts (e.g. lane 8) and a T-lymphocyte cell line staining (data not shown) at levels approximating those of (lane 9). ARVCF was expressed as multiple isoforms, endogenous p120. The exogenous ARVCF from the MDCK suggesting extensive alternative splicing similar to that ARVCF overexpressor cell lines reacted strongly with ARVCF characterized previously for p120 (Keirsebilck et al., 1998; Mo monoclonal antibodies and comigrated with the largest form of and Reynolds, 1996). endogenous human ARVCF from the human cell lines HCT116 To further examine and exclude the possibility that the and 293 (Fig. 3, compare lane 3 to lanes 4 and 5, and lane 8 to apparently low levels of ARVCF relative to p120 were due to lanes 9 and 10). Upon treatment of ARVCF immunoprecipitates differences in antibody affinity, we coated ELISA plates with with lambda phosphatase (lanes 6-10), a broad spectrum protein recombinant fragments of p120 and ARVCF at equivalent phosphatase acting on phospho-serine, -threonine and -tyrosine, molar concentrations, and compared the signals generated by both the endogenous and ectopic ARVCF bands collapsed into our various p120 and ARVCF monoclonal antibodies (data not sharp, tightly migrating bands (compare lanes 1-5 with 6-10), shown). The ARVCF antibodies behaved similarly to well- indicating that ARVCF is extensively phosphorylated in vivo, characterized high-affinity p120 monoclonal antibodies, as is p120 (Thoreson et al., 2000). indicating that differences in antibody affinities are unlikely to account for the low levels of ARVCF detected above. Localization of ARVCF to adherens junctions To circumvent problems with abundance, we utilized several Only very faint junctional staining of ARVCF could be ARVCF in the nucleus and adherens junction 1485

cell lines stably expressing vector alone (lanes 1-5) or ARVCF (lanes 6-10). Interactions in the absence of ARVCF overexpression were not detected due to the low levels of ARVCF (lanes 1-5). In the ARVCF overexpressing cell lines, ectopic ARVCF, p120 and β-catenin each coprecipitated with E-cadherin (Fig. 5B,C,D, lane 7) and vice versa (Fig. 5A, lanes 8-10), demonstrating association with one another. However, ARVCF was not present in p120 immunoprecipitates (Fig. 5B, lane 9) and vice versa (Fig. 5C, lane 8), while both proteins coimmunoprecipitated β-catenin (Fig. 5D, lanes 8 and 9). These data suggest that ARVCF and p120 are mutually exclusive for one another in E-cadherin complexes, consistant with the hypothesis that ARVCF interacts with E- cadherin directly and competes with p120 for binding to the juxtamembrane domain. Localization of ARVCF to the nucleus To assess the distribution of ARVCF in other cell types, we immunolocalized the protein after transient transfection of ARVCF cDNA into a variety of cell lines. Interestingly, under conditions of transient overexpression, ARVCF localized clearly to both cell-cell junctions and nuclei in multiple cell types including MDCK (data not shown). The nuclear localization was particularly striking in the human fibroblast cell line Va-2, where both junctional and nuclear localization was observed. To further examine ARVCF localization in this cell line, we generated stable cell lines by transfection and subcloning. ARVCF could not be detected in Fig. 4. Overexpressed ARVCF colocalizes with p120. Untransfected control MDCK the absence of overexpression (Fig. 6A), but cells (A,B), or stable MDCK cell lines overexpressing ectopic ARVCF (C,D), were localized prominently to the nucleus (Fig. 6C, methanol-fixed and immunolabeled with ARVCF mAb 4B1 (A,C) or p120 mAb 8D11 (B,D). ARVCF was barely detectable in untransfected control cells by arrows) as well as to points of cell-cell contact immunofluorescence microscopy, but colocalized precisely with p120 when (Fig. 6C, arrowhead) in multiple clonal Va-2 overexpressed. 63× magnification. cell lines overexpressing ARVCF. Endogenous p120, on the other hand, localized to the adherens junctions in these cells and was not detected by either monoclonal or polyclonal antibodies in the apparent in nuclei (Fig. 6B,D). The intensity of the nuclear absence of ARVCF overexpression (Fig. 4A), consistent with ARVCF staining varied widely, with some nuclei staining the low level of ARVCF detected in parental MDCK cells by brightly and others, not at all. Cell density did not affect the immunoprecipitation and western blotting. Immunofluorescent variable levels of nuclear staining, nor was the degree of localization in ARVCF-overexpressing MDCK cells, however, nuclear localization strictly dosage-dependent, since low- as revealed bright staining of ARVCF at cell contacts and precise well as high-level expressing cells exhibited nuclear ARVCF. colocalization with p120 (Fig. 4, compare C,D) and E-cadherin Transiently overexpressed ARVCF typically concentrated to (data not shown) in adherens junctions. Punctate staining at the high levels in the nuclei of transfected Va-2 cells, though it membranes, which is typical of desmosomal components, was occasionally appeared to be absent from nuclei. Ectopically not detected by ARVCF monoclonal antibodies. These data expressed p120 (isoform 1A), however, was detectable in the suggest that, like p120, ARVCF associates with cadherin nuclear compartment to a lesser extent, with lower frequency complexes. of nuclear translocation and decreased concentration despite high level expression. This tendency of overexpressed p120- ARVCF and p120 bind E-cadherin in mutually 1A to remain at junctions and in the cytoplasm occurred even exclusive complexes in the absence of exon B, which contains a functional nuclear To determine whether ARVCF associates directly with E- exclusion signal (van Hengel et al., 1999). Likewise, cadherin complexes, we assayed for protein-protein cotransfection of ARVCF and p120 in Va-2 cells frequently associations by immunoprecipitation and western blotting of showed accumulation of ARVCF but not p120 in the nucleus various components of the cadherin complex (Fig. 5) in MDCK of the same cell (Fig. 6, compare ARVCF in E to p120 in F), 1486 D. J. Mariner, J. Wang and A. B. Reynolds

Fig. 5. ARVCF and p120 coprecipitate with E-cadherin in mutually exclusive complexes. Adherens junction proteins indicated across the top of the blot were immunoprecipitated from 0.5% NP-40 detergent cell lysates of a control neo-resistant MDCK cell line (lanes 1-5) or a similar MDCK cell line stably overexpressing human ARVCF (lanes 6-10). The irrelevant epitope tag mAb 12CA5 (lanes 1,6) was used to control for nonspecific interactions. Coimmunoprecipitating proteins were detected by western blotting with an E-cadherin mAb (A), ARVCF pAb (B), p120 mAb pp120 (C) and anti-β-catenin pAb (D). Only 1/10 of the p120 immunoprecipitate was loaded for the p120 direct western blot (C, lanes 4,9). ARVCF was barely detectable in untransfected cells (B, lane 3), but was easily detected after transfection (B, lane 8). Note in the transfected cell line that ARVCF immunoprecipitates do not contain p120 (C, lane 8), and p120 immunoprecipitates do not contain ARVCF (B, lane 9), suggesting a mutually exclusive interaction with E-cadherin.

indicating that the nuclear localization of these proteins is regulated by distinct mechanisms. ARVCF lacks the branching activity of p120 Transient transfection of p120 into most fibroblast cell lines induces an unusual dosage-dependent branching phenotype that is particularly striking in NIH 3T3 cells (Reynolds et al., 1996). To determine whether ARVCF also displays this activity, human ARVCF and human p120 were transiently expressed at high levels in NIH 3T3 cells from a plasmid containing a CMV promoter. Whereas the branching morphology of cells overexpressing p120 was striking (Fig. 7B), high levels of ARVCF had little or no effect on cell

Fig. 6. Overexpressed ARVCF localizes to both nuclei and cell-cell contacts in Va-2 fibroblasts. Wild-type Va-2 cells (A,B) were stably transfected with human ARVCF (C,D) or transiently cotransfected with both ARVCF and murine p120 (E,F). Cells were fixed with methanol and immunostained with ARVCF mAb 4B1 (A,C,E), or p120 mAbs 12F4 (B,D) and 8D11 (F). ARVCF was undetectable in Va-2 fibroblasts in the absence of overexpression (A), but was detected in cell-cell contacts (arrowhead) and in the nucleus (arrows) upon stable ARVCF expression (C). Endogenous p120, however, was not readily detectable in nuclei of wild-type (B) or stable ARVCF transfectants (D). In transient cotransfections of ARVCF and p120, ARVCF was frequently nuclear (E) in cells that simultaneously excluded p120 (F). 63× magnification. ARVCF in the nucleus and adherens junction 1487

Molecular mapping of the sequences responsible for the branching phenotype and nuclear localization To begin to define the sequences mediating the different phenotypic and nuclear properties of these proteins, molecular chimeras (ARVCF-p120 and p120-ARVCF) were generated by fusing human ARVCF with human p120 (isoform 1A) between Arm repeats 3 and 4. The proteins are highly conserved in this region and, in fact, are identical over a stretch of nine amino acids in the location of the joint. Thus, we anticipated that the overall structure of the chimeric proteins would not be changed significantly, and indeed, both chimeric proteins retained the Fig. 7. Transient ARVCF overexpression in NIH 3T3 cells does not ability to colocalize with E-cadherin at adherens junctions induce a branching phenotype. Human ARVCF (A) or human p120 (B) was transiently overexpressed in NIH 3T3 cells from pcDNA3.1, (data not shown), a function mediated by the central Armadillo a plasmid containing a CMV promoter. No obvious morphological repeat domain (Daniel and Reynolds, 1995). The p120 type 1A phenotype was apparent upon high-level expression of ARVCF (A), isoform was selected for these experiments because it most while a striking, dosage-dependent branching phenotype resulted closely resembles the cloned human ARVCF cDNA and is from p120 overexpression (B). Overexpressed ARVCF, but not p120, naturally abundant in fibroblasts (Mo and Reynolds, 1996; frequently formed bright, spherical cytoplasmic aggregates (A, Reynolds et al., 1994). arrowheads). To clarify the region of p120 responsible for its branching activity, NIH 3T3 cells were transiently transfected with the ARVCF-p120 and p120-ARVCF chimeras (shown morphology (Fig. 7A). In several cell types, ectopic ARVCF schematically in Fig. 8A), which were then localized by formed bright, spherical molecular aggregates in the immunofluorescence (Fig. 8B). ARVCF-p120 transfectants cytoplasm (Fig. 7A, arrowheads), which may be artifacts of exhibited a branching phenotype similar, albeit slightly less overexpression, but were nonetheless absent from p120 severe, to that induced by full-length p120 (Fig. 8Bi), transfectants. Interestingly, high level coexpression of ARVCF confirming previous data which demonstrated that the with p120 had no discernable effects on the branching Armadillo repeat domain of p120 mediates branching phenotype induced by p120 (data not shown). Thus, ARVCF (Reynolds et al., 1996). Additionally, the ARVCF-p120 lacks p120’s branching activity, and was not able to act in a transfected cells lacked the cytoplasmic speckle-like dominant negative manner to suppress it. aggregates typically detected in ARVCF transfected cells, indicating that the carboxy-terminal end of ARVCF was responsible for the aggregation of ARVCF into cytoplasmic ARVCF-p120 A aggregates. Conversely, cells expressing the p120-ARVCF 1 234 5 6 78910 chimera (Fig. 8Bii) did not exhibit the branching phenotype AA 1 962 and displayed well-defined aggregates (arrowheads). To map the region responsible for the nucleophilic properties p120-ARVCF of ARVCF, the chimeras were transiently transfected into Va- 1234 5 6 78910 2 fibroblasts (Fig. 8C). ARVCF-p120 localized prominently to AA 1 962 the nuclei of most transfected cells (Fig. 8Ci), though it was hp120 hARVCF not as prominantly concentrated in the nucleus as full-length

Fig. 8. Molecular mapping of branching and nuclear localization activities. (A) Structure of ARVCF/p120 chimeric proteins. ARVCF sequences are depicted in gray, and p120 sequences in white. ARVCF and p120 human cDNAs were joined at the junction between Armadillo repeats 3 and 4 (as indicated), generating ARVCF-p120 and p120-ARVCF. The asterisk indicates the position of a predicted nuclear localization signal. The black box designates a coiled-coil motif. (B) Branching activity of the chimeric proteins in NIH 3T3 cells. Transient transfection and immunofluorescence detection of ARVCF-p120 and p120-ARVCF in NIH 3T3 cells revealed branching activity in cells expressing ARVCF-p120 (Bi), but not p120-ARVCF (Bii), and cytoplasmic aggregates in cells expressing p120-ARVCF (Bii, arrowheads) but not ARVCF-p120 (Bi). 40× magnification. (C) Localization of chimeric proteins in Va-2 cells. Transient transfection and immunofluorescence detection of ARVCF-p120 (Ci) and p120-ARVCF (Cii) in Va-2 fibroblasts reveal that nuclear localization segregates with the amino-terminal end of ARVCF. Arrowheads indicate nuclei. 63× magnification. 1488 D. J. Mariner, J. Wang and A. B. Reynolds

ARVCF. Conversely, p120-ARVCF was absent from the of ARVCF are similarly generated. In addition, like p120, nucleus of the majority of transfected cells (Fig. 8Cii), much ARVCF isoforms generally did not migrate on electrophoretic like full-length p120-1A. These data suggest that, in gels as sharply focussed bands, a situation readily rectified fibroblasts, the amino-terminal region of ARVCF is primarily by treatment with phosphatase. Thus, ARVCF appears to responsible for its localization with respect to the nucleus, be extensively modified by phosphorylation, especially in despite the presence of a classical bipartite nuclear localization epithelial cells. We have shown previously that p120 signal located in the carboxy-terminal region between phosphorylation is dependent on its recruitment to the Armadillo repeats 6 and 7. membrane by E-cadherin (Thoreson et al., 2000). By analogy to p120, the diffuse ARVCF bands likely reflect its presence at membranes. DISCUSSION Overexpression of ARVCF and p120 in a variety of cell lines revealed a clear propensity of ARVCF to localize to the nucleus The recent identification of novel proteins with high homology in certain cell types, and ARVCF was considerably more to p120 raises the possibility of competition and/or redundancy efficient than p120 (isoform 1A) in this process. Though the among members of the growing p120 family. Moreover, as nuclear accumulation of ARVCF was particularly striking in more of these proteins are definitively demonstrated to localize human Va-2 fibroblasts, ARVCF was less intensely nuclear and to the nucleus, a paradigm is emerging that suggests a general often excluded from the nucleus in NIH 3T3 cells, indicating role for p120 family proteins in junctional Ð nuclear signaling, that nuclear localization is mediated by cell type-specific perhaps relaying important information regarding the status of events that are not necessarily fibroblast-specific. In addition, cell-cell contacts. Here, we find that yet another p120 family in clonal populations of Va-2 cells, ARVCF was cell junction- member, ARVCF, interacts with E-cadherin in a manner that is specific in some cells and mostly nuclear in others. This result mutually exlusive with p120’s direct interaction with the E- was independent of cell density, raising the possibility that cadherin juxtamembrane domain. In addition, ARVCF is ARVCF localization is affected by the cell cycle, as has been strikingly nucleophilic in particular cell types that, for the most reported previously for the Drosophila Armadillo repeat part, exclude p120 or at least prohibit its accumulation to high protein Pendulin (Kussel and Frasch, 1995). In addition, levels in the nucleus. Interestingly, ARVCF completely lacked transiently overexpressed ARVCF, but not p120, tended to the branching activity associated with p120. Thus, sequence form aggregates in the cytoplasm. This phenomenon was differences appear to eliminate an interaction or activity that in exacerbated by high level expression and was dependent on p120 leads to the dramatic induction of highly branched sequences downstream of Arm repeat 3. cellular processes. Therefore, while several similarities Additional characteristics further distinguish ARVCF from between p120 and ARVCF are evident, the data suggest that p120. ARVCF completely lacked the strong branching activity these proteins are unlikely to have significant functional induced in fibroblasts by high-level p120 expression. This redundancy. effect is not induced by other Armadillo repeat proteins (e.g. In support of this conclusion is the finding that ARVCF is β-catenin) and requires an intact Armadillo repeat domain expressed at very low levels in most cells. To generate an (Reynolds et al., 1996). Apparently, the 56% identity to p120 ARVCF signal on western blots that was comparable to that within the Arm domain is not sufficient to preserve the activity. for p120, it was necessary to immunoprecipitate ARVCF from The simplest explanation is that p120, but not ARVCF, at least tenfold more cell lysate than required for p120. In fact, associates with an activity that somehow mediates the there was not enough ARVCF in any of the untransfected cell branching phenotype. It is also noteworthy that stable lines examined to suggest either an important role as a stuctural overexpression of ARVCF in both MDCK and Va-2 cells was protein in junctions, or that displacement of p120 by ARVCF easily achieved, whereas we have been unable to stably could be quantitatively significant. Rather, we propose a overexpress p120 in these and most other cell lines. Thus, specialized role for ARVCF in modulating cadherin function, constitutive expression of p120, but not ARVCF, is apparently for which high-level expression is not necessary. However, it incompatible with most cells for reasons that are as yet is also possible that ARVCF is expressed at high levels under unknown but might relate to these different activities. certain circumstances, perhaps during development, which Surprisingly, sequences necessary for the strong nuclear could increase its presence in the cadherin complex relative to presence of ARVCF were located in its amino-terminal end. other p120 family members. For example, δ-catenin/NPRAP/ All p120 family members contain conserved stretches of basic neurojungin, a close relative of p120 and ARVCF, is expressed amino acids located between repeats 6 and 7, which resemble at highest levels during brain development and is decreased in prototypical nuclear localization signals, or in the case of adult neural tissues (Lu et al., 1999; Paffenholz and Franke, ARVCF, a classic bipartite nuclear localization signal. The 1997). The possibility of competition among p120 family motif is the obvious candidate for the sequence that regulates members for cadherin binding is an important concept, the the ability of these proteins to translocate to nuclei. Bipartite consequences of which remain poorly understood. nuclear targeting motifs are found in more than 50% of nuclear Analysis of ARVCF expression by western blotting revealed proteins and less than 5% of non-nuclear proteins (Dingwall multiple bands in all cell types examined. Alternative splicing and Laskey, 1991). Nonetheless, our data strongly suggest that of p120 results in the use of as many as four different start strong nuclear localization of ARVCF is dependent on codons and the alternative use of three exons in the carboxy- sequences amino-terminal to repeat 4. The data are consistent terminal half of the protein (Mo and Reynolds, 1996; van with reports that the cognate motif in δ-catenin and Hengel et al., 1999). Given the nearly identical intron-exon plakophilins is not required for nuclear translocation structure of these proteins, it is likely that the multiple isoforms (Klymkowsky, 1999; Paffenholz and Franke, 1997). Moreover, ARVCF in the nucleus and adherens junction 1489 in preliminary experiments, we find that deletion of the basic Driscoll, D., Salvin, J., Sellinger, B., Budarf, M., McDonald-McGinn, D., motif in p120 has no effect on its ability to translocate to the Zackai, E. and Emanuel, B. (1993). Prevalence of 22q11 microdeletions nucleus, even under conditions where nuclear translocation of in DiGeorge and velocardiofacial syndromes: Implications for genetic counselling and prenatal diagnosis. J. Med. Genet. 30, 813-817. p120 is efficient (not shown). Therefore, the basic motif is Driscoll, D., Spinner, N., Budarf, M., McDonald-McGinn, D., Zackai, E., likely to have a different function that has yet to be elucidated. Goldberg, R., Shprintzen, R., Saal, H., Zonana, J., Jones, M. et al. In summary, ARVCF and p120 associate with E-cadherin in (1992). Deletions and microdeletions of 22q11.2 in velo-cardio-facial a mutually exclusive fashion, suggesting competition for syndrome. Am. J. Med. Genet. 44, 261-268. interaction with the juxtamembrane domain. Thus, it is Fagotto, F., Gluck, U. and Gumbiner, B. (1998). Nuclear localization signal- independent and importin/karopherin-independent nuclear import of β- theoretically possible that high level ARVCF expression could catenin. Curr. Biol. 8, 181-190. compete for cadherin occupancy, thereby substituting its own Frixen, U. H., Behrens, J., Sachs, M., Eberle, G., Voss, B., Warda, A., functions or acting as a dominant negative inhibitor of p120 Lochner, D. and Birchmeier, W. (1991). E-cadherin-mediated cell-cell function. However, the low level expression of ARVCF relative adhesion prevents invasiveness of human carcinoma cells. J. Cell Biol. 113, 173-185. to p120 suggests that replacement of p120 is not Hatzfeld, M., Kristjansson, G. I., Plessmann, U. and Weber, K. (1994). physiologically relevant, at least in most adult cell types. Band 6 protein, a major constituent of desmosomes from stratified epithelia, Instead, ARVCF is likely to have a different and probably is a novel member of the armadillo multigene family. J. Cell Sci. 107, 2259- additive role in cell-cell junctions that is not directly 2270. competitive with p120. In addition, ARVCF appears to be more Hatzfeld, M. and Nachtsheim, C. (1996). Cloning and characterization of a new armadillo family member, p0071, associated with the junctional plaque: nucleophilic than p120 in many cell types. Thus, ARVCF may evidence for a subfamily of closely related proteins. J. Cell Sci. 109, 2767- have increased importance in the nucleus, or shuttle between 2778. membranes and the nucleus, thereby conveying information Hazan, R. B., Kang, L., Roe, S., Borgen, P. I. and Rimm, D. L. (1997). related to cell-cell contact. One possibility is that docking on Vinculin is associated with the E-cadherin adhesion complex. J. Biol. Chem. E-cadherin, or other resident cadherins, may be a regulatory 272, 32448-32453. Heid, H. W., Schmidt, A., Zimbelmann, R., Schafer, S., Winter- mechanism restricting access of ARVCF to the nucleus. The Simanowski, S., Stumpp, S., Keith, M., Figge, U., Schnolzer, M. and inability of ARVCF to induce cellular branching, and the Franke, W. W. (1994). 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