Protein Binding and Functional Characterization of Plakophilin 2EVIDENCE FOR ITS DIVERSE ROLES IN DESMOSOMES AND �-CATENIN SIGNALING*

Received for publication, September 11, 2001, and in revised form, December 18, 2001Published, JBC Papers in Press, January 14, 2002, DOI 10.1074/jbc.M108765200

Xinyu Chen‡, Stefan Bonne§, Mechthild Hatzfeld¶, Frans van Roy§, and Kathleen J. Green‡�

From the ‡Departments of Pathology and Dermatology and the Robert H. Lurie Cancer Center, Northwestern UniversityMedical School, Chicago, Illinois 60611, the §Molecular Cell Biology Unit, Department of Molecular Biology,VIB-University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium, and the ¶Molecular Biology Groupof the Medical Faculty, University of Halle, 06097 Halle/Saale, Germany

Plakophilins are a subfamily of p120-related arm-re-peat proteins that can be found in both desmosomes andthe nucleus. Among the three known plakophilin mem-bers, plakophilin 1 has been linked to a genetic skindisorder and shown to play important roles in desmo-some assembly and organization. However, little isknown about the binding partners and functions of themost widely expressed member, plakophilin 2. To betterunderstand the cellular functions of plakophilin 2, wehave examined its protein interactions with other junc-tional molecules using co-immunoprecipitation andyeast two-hybrid assays. Here we show that plakophilin2 can interact directly with several desmosomal compo-nents, including desmoplakin, plakoglobin, desmoglein1 and 2, and desmocollin 1a and 2a. The head domain ofplakophilin 2 is critical for most of these interactionsand is sufficient to direct plakophilin 2 to cell borders.In addition, plakophilin 2 is less efficient than plakophi-lin 1 in localizing to the nucleus and enhancing therecruitment of excess desmoplakin to cell borders intransiently transfected COS cells. Furthermore, plako-philin 2 is able to associate with �-catenin through itshead domain, and the expression of plakophilin 2 inSW480 cells up-regulates the endogenous �-catenin/Tcell factor-signaling activity. This up-regulation by pla-kophilin 2 is abolished by ectopic expression of E-cad-herin, suggesting that these proteins compete for thesame pool of signaling active �-catenin. Our results dem-onstrate that plakophilin 2 interacts with a broader rep-ertoire of desmosomal components than plakophilin 1and provide new insight into the possible roles of plako-philin 2 in regulating the signaling activity of �-catenin.

Plakophilins (PKPs)1 1–3 belong to a subfamily of p120-

related armadillo proteins found in both the desmosomalplaque and the nucleus (1–5). Each is composed of a basicN-terminal head domain followed by a series of 10 imperfect42-amino acid repeats (arm repeats) and a short C-terminal tail(3). Two splice variants have been identified for PKP1 andPKP2, a shorter “a” form and a longer “b” form. Although theN-terminal head domains of PKPs exhibit relatively greatersequence diversity than the arm-repeat domains, a consensussequence termed HR2 is shared by all the PKP head domains(3, 4). PKP1 is concentrated in desmosomes of the suprabasallayers of stratified and complex epithelia (5). PKP3 can bedetected in desmosomes of most simple and almost all strati-fied epithelia with the exception of hepatocytes and hepatocel-lular carcinoma cells (3, 4). PKP2 has the broadest tissuedistribution in desmosomes of all simple, complex, and strati-fied epithelia as well as non-epithelial tissues such as myocar-dium and lymph node follicles, in which PKP1 was not detectedin desmosomes. PKP2 is concentrated in the basal layer of moststratified squamous epithelia, whereas PKP1 is mostly concen-trated in desmosomes of the upper layers (2–4, 6). On the otherhand, PKP3 is more uniformly expressed in the living epider-mal layers (3, 4). PKPs have been detected in both desmosomesand nuclei in desmosome-possessing cells and only in nuclei indesmosome-lacking cells, but the mechanisms responsible forthis dual location and their functions in these two differentenvironments are still poorly understood.

Members of the armadillo family, to which PKPs belong, playcritical structural and regulatory roles through their interac-tions with proteins in two related intercellular adhesive junc-tions, desmosomes and adherens junctions, which anchor in-termediate filament (IF) networks and actin filaments to sitesof cell-cell contact (7–10). The best characterized interactions ofthe desmosomal armadillo proteins are those in which plako-globin (Pg) participates. Pg associates directly with the cyto-plasmic domains of the desmosomal cadherins, transmem-brane glycoproteins of desmosomes that are further subdividedinto the desmoglein (Dsg) and desmocollin (Dsc) subfamilies.Three isoforms exist for each of these subfamilies, which areexpressed in a cell type- and differentiation-dependent manner(8, 11, 12). Plakoglobin links desmosomal cadherins to IFthrough its interactions with the plakin family member desmo-plakin (DP). Functionally, loss of Pg function through mutationor genetic ablation leads to heart and skin defects, supportingthe fact that this link between the cadherins and the IF-des-moplakin complex plays a key role in tissue integrity (13–15).

PKP1 also plays a critical role in tissue integrity, as patientsnull for PKP1 show histological evidence of aberrant desmo-somes and poorly anchored IF and suffer from ectodermaldysplasia accompanied by skin fragility (16). Whereas DP innormal epidermis is concentrated in desmosomes, this plaque

* This work is supported by a R. H. Lurie Baseball Charities CancerFellowship (to X. C.), an Institute for the Promotion of Innovation byScience and Technology-Flanders Fellowship (to S. B), DeutscheForschungsgemeinschaft Grant Hal791/3-4 (to M. H.), a Fund for Sci-entific Research-Flanders grant (to F. v.-R.), and National Institutes ofHealth Grants RO1 AR43380, PO1 DE12328 (project 4), and AR41836(to K. G.). The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

� To whom correspondence should be addressed: Dept. of Pathology,Northwestern University Medical School, 303 East Chicago Ave.,Chicago, IL 60611. Tel.: 312-503-5300; Fax: 312-503-8240; E-mail:kgreen@northwestern.edu.

1 The abbreviations used are: PKP, plakophilin; IF, intermediatefilament; Pg, plakoglobin; Dsg, desmoglein; Dsc, desmocollin; DP, des-moplakin; DPNTP, desmoplakin N-terminal polypeptide; IP, immuno-precipitation; nt, nucleotide(s); kb, kilobase; PBS, phosphate-bufferedsaline; X-�-gal, 5-bromo-4-chloro-3-indolyl �-D-galactopyranoside; TCF,T cell factor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 12, Issue of March 22, pp. 10512–10522, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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protein exhibits a largely diffuse, cytoplasmic distribution inPKP1 null patients. This observation raises the possibility thatPKP1 is required for the efficient association of DP with thejunctional plaque. Subsequent studies provided evidence sup-porting this hypothesis, showing that PKP1 can directly inter-act with the DP N terminus and enhance its recruitment to theplasma membrane. Furthermore, PKP1 collaborates with Pgand DP to promote the clustering of desmosomal plaque com-plexes at cell-cell borders and to enhance IF association withthe plaque (17–19). Together, these results have led to theproposal that PKP1 is important in strengthening the desmo-somal plaque by enhancing lateral protein-protein interactions.However, virtually nothing is known about the roles of otherPKPs in desmosomes and how their participation in desmo-somes affects the specific organization or functions of desmo-somes in different tissues and stages of differentiation.

In addition to their function in intercellular junctions, somearmadillo family members also play roles in cellular activitiesoutside of the junctions. Both Pg (20) and p120 (21) have beenfound in the nucleus and implicated in various signaling path-ways (22–26); however, the best-studied example is �-catenin.This close relative of Pg connects the cytoplasmic tails of clas-sical cadherins with actin-binding proteins such as �-catenin toprovide the linkage between actin filaments and adherens junc-tions (27). Besides its important roles in cell-cell adhesion,�-catenin is a critical downstream effector of the Wnt-signalingpathway, which mediates a large variety of developmentalprocesses (28, 29). The cellular level of �-catenin is tightlycontrolled by a complex of proteins that facilitates its rapiddegradation, whereas activation of the Wnt-signaling pathwayantagonizes the degradation process and leads to the accumu-lation of free, cytoplasmic �-catenin. �-Catenin can then trans-locate into the nucleus and activate target gene transcription inassociation with TCF/Lef1 transcription factors (30). Since therecognition of its role in Wnt signaling, an increasing numberof proteins have been discovered to interact with �-catenin andregulate its signaling activity through various mechanisms(31). PKPs, like their armadillo family relatives, are also foundin the nucleus. So far evidence for participation of PKPs in�-catenin-dependent signaling has not been reported. A recentstudy showed that nuclear PKP2 is complexed with the RNApolymerase III holoenzyme and interacts directly with its larg-est subunit RPC155 in vitro, opening up additional possibilitiesfor the nuclear activity of PKP2 (32). However, because only asubset of PKP2 was observed in these complexes, other nuclearfunctions for this arm-repeat subfamily of proteins may exist.

PKP2 is the most ubiquitously expressed PKP family mem-ber, suggesting that it has broad cellular functions. In thisstudy, to elucidate the biological functions of PKP2, we havecharacterized its protein interactions with other junctionalmolecules. Here we show that PKP2 interacts directly with theobligate desmosome components Pg and DP as well as thedesmosomal cadherins Dsg1, Dsg2, Dsc1a, and Dsc2a and mayinteract indirectly with Dsg3. PKP2 is less efficient than PKP1in enhancing the recruitment of excess DP to cell borders,which is consistent with the weaker interaction observed be-tween PKP2 and DP and indicates distinct roles for these twoPKPs in desmosome assembly. In addition, PKP2 can associatewith non-cadherin-bound �-catenin and regulate its signalingactivity in TOPFLASH reporter assays. This novel associationwith a component of adherens junctions and the Wnt-signalingpathway raises the possibility that PKP2 might participate incross-talk between the two major types of intercellular junc-tions and signal transduction pathways connecting the cellsurface and the nucleus.

EXPERIMENTAL PROCEDURES

Generation of cDNA Constructs—To generate the probe used forlibrary screening, a partial PKP2 cDNA clone in pRSET-A vector (p673)was digested with BamHI and NcoI to isolate the insert nucleotides1009–2401 of the PKP2 a splicing variant (PKP2a) cDNA. Numberingof the PKP2a cDNA sequence is based on the mRNA sequence forPKP2a and 2b (GenBankTM accession number X97675) and the proteinsequence of PKP2a (GenBankTM accession number CAA66265). Thisfragment was used to screen a normal human keratinocyte �ZAPIIlibrary (provided by Dr. Masayuki Amagai). Clone (C1-1) containingPKP2a nucleotides 1–1849 was digested with KpnI and EcoRI to purifythe fragment containing PKP2a nucleotides 1–1318, which was ligatedinto the EcoRI and KpnI sites of pBS vector to generate plasmid p793(nt 1–1318). p793 was digested with XbaI, end-filled, and then digestedwith KpnI to cut out the insert, which was then ligated into a 3.4-kbfragment of p673, which had been digested with NcoI, end-filled, anddigested with KpnI. The resulting plasmid p792 contains PKP2a nucle-otides 1–2401 in pBS. To obtain the missing C terminus of PKP2a (nt2402–2514), PCR was performed using the total phage DNA isolatedfrom the above library with forward primer XC5� (5�-GGACCAATGC-CAACATC-3�) and reverse primer XC3� (5�-GTCGACGTCTTTAAGG-GAG-3�). The resulting PCR product, spanning nucleotides 2008–2511and containing an engineered SalI site at the 3� end, was directly clonedinto the pGEM-T vector (Promega) to generate clone XC1–4. XC1–4was double-digested with MscI and SphI to isolate the C terminus ofPKP2a, which was then cloned into the 4.9-kb fragment of p792 di-gested with MscI and SphI, thus creating the complete PKP2a cDNA inpBS (p829). Complete sequencing of the PKP2a cDNA revealed threenucleotide differences compared with the originally published PKP2asequence (2) (GenBankTM accession number X97675). The first differ-ence is a C to T transition at nt 1097 that changes a Pro to Leu. Becausethis difference came from a phage clone and was also found in multipleEST clones, it was considered as a polymorphic difference in the PKP2acDNA sequence. The other two differences are a G to T change at nt1886 and an A to G change at nt 2164, both of which were found inPCR-generated plasmid p673 and were not present in any EST clones inthe GenBankTM data base. So they were interpreted as errors intro-duced by the cloning process and corrected as described below.

A mammalian expression construct of PKP2a (p830) was constructedby ligating an EcoRI-SalI fragment from p829 into pFLAG-CMV-5vector. The two random mutations at PKP2a cDNA sequence 1886 andnt 2164 were corrected using the QuikChange site-directed mutagene-sis kit (Stratagene), and the resulting plasmid (p915) was fully se-quenced to ensure that there were no additional mutations. Constructp915 is the expression construct for PKP2a used in this study. TheN-terminal head domain of PKP2a (PKP2a-H) was generated by PCRusing primers PHN (5�-GATGAATTCCACGATGGCAGCCCCCG-3�)and PHC (5�-CATGTCGACGTCTGCATTCCCCAGC-3�) with p915 asthe template. The PCR product contains an EcoRI site and a Kozakconsensus sequence at the 5� end and a SalI site at the 3� end. Afterligation of the PCR product into pGEM-T vector and subsequent diges-tion with EcoRI and SalI, the PKP2a head domain (nt 1–1044) wasligated into pFLAG-CMV-5. The procedure of generating the remainingportion of PKP2a (PKP2a-A) including the arm-repeat domain and theC terminus into the pFLAG-CMV-5 vector was the same as aboveexcept for the primers used in the PCR reaction. The forward primerused was PAN (5�-GATGAATTCCACGATGGAGATGACTCTGGAG-3�),and the reverse primer was PAC (5�-CATGTCGACGTCTTTAAGG-GAGTGGTAGGC-3�). To generate the full-length PKP1a with a FLAG-epitope tag at its C terminus, a full-length a form of PKP1 in pCMVscript vector p724 (19) was used as the template for PCR using primersPKP1-nt-1745 (5�-CCTGCAATCTGGCAACTCTG-3�) and PKP1.FLAG(5�-CCTACTTGTCATCGTCGTCCTTGTAATCGAATCGGGAGGTGA-AG-3�). The PCR product that contains a FLAG epitope tag at its Cterminus was subsequently blunt end-ligated into pSK vector digestedwith EcoRV. This intermediate construct was diagnostically digested todetermine the orientation of the PCR insert and was double-digestedwith BstEII and SalI to isolate the PCR insert, which was then ligatedinto p724 digested with BstEII and SalI to generate full-length FLAG-tagged PKP1a in pCMV script vector.

The HR2 domain of PKP2a contains amino acids 29–60. To generatethe expression construct of PKP2a with internal deletion of its HR2domain (PKP2a �HR2), a SOEing (splicing by overlap extension) pro-cedure was utilized. PCR was performed with p915 as the templateusing primer A (5�-GTATCATATGCCAAGTC-3�) and primer B (5�-GGCGAGGGTCTGCTGGGAGCTGTCCAGTTG-3�) to obtain the PCRproduct AB. A second PCR was performed with p915 as the template

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using primer C (5�-CAGCAGACCCTCGCC-3�) and primer D (5�-CTG-CAGAAGCTTGAGG-3�) to obtain the PCR product CD. Then the finalPCR was performed with a mixture of product AB and CD as thetemplate using primer A and primer D to produce the PCR product AD.Product AD was digested with NdeI and HindIII and cloned into the5.6-kb fragment from digesting p915 with NdeI and HindIII, thuscreating PKP2a �HR2. It was then used as the template to generate thePKP2a head domain with HR2 deletion (PKP2a-H �HR2) following thesame procedure described above for generating PKP2a-H. To generatefull-length DP with a C-terminal Myc tag, a plasmid (p350) pCMV.DP.myc, which has a stop codon at nucleotide position 1754 due tomutation, was digested with DraIII and SalI. The 7.5-kb fragment wasligated with the 5.1-kb fragment from DraIII and SalI digestion ofplasmid p612, pSK.DP.�C. This ligation generates full-length DP in thepCMV vector with a C-terminal Myc tag (p613).

The following constructs were described previously: full-length hu-man Pg cDNA in a mammalian expression vector LK44 under thecontrol of the human �-actin promoter (33); the Pg�N and Pg�C ex-pression constructs under the control of the �-actin promoter (34); anexpression construct encoding a polypeptide comprising the first 584amino acids of DP, called DPNTP (35). Full-length �-catenin in mam-malian expression vector pCAN was provided by Dr. Paul Polakis.

Mammalian expression constructs of full-length human Dsg1 with aC-terminal Myc tag and full-length human Dsg2 with a C-terminal Myctag were described previously (33, 36). The full-length human Dsg3with C-terminal Myc tag in the expression vector was a gift fromDr. John Stanley.

Cell Culture and Transfections—COS-7, HEK293, A431, and HaCaTcells were cultured in Dulbecco’s minimal essential medium with pen-icillin/streptomycin and 10% fetal bovine serum. The SCC9 oral squa-mous cell carcinoma cells were grown in Dulbecco’s minimal essentialmedium/F-12 medium with 10% fetal bovine serum and penicillin/streptomycin. The SW480 human colon carcinoma cell line was culturedin Leibovitz’s L-15 medium supplemented with penicillin/streptomycinand 10% fetal bovine serum. For transient transfection of COS cells andHEK293 cells, calcium phosphate transfection was performed as previ-ously described (37), and experiments were performed 42–46 h later.SCC9 and SW480 cells were transfected using FuGENE 6 reagent(Roche Applied Science) according to the manufacturer’s protocol andassayed 24 h later.

Antibodies—A rabbit polyclonal antibody poly-Myc 2026 directedagainst the c-Myc epitope and a rabbit polyclonal antibody 1880 againstDsg3 were kindly provided by Dr. John Stanley. A rabbit polyclonalantibody against E-cadherin was a gift from Drs. Randy Marsh andRobert Brackenbury. A mouse monoclonal antibody PC28 directedagainst the DP central rod domain was a gift from Dr. Pamela Cowin.A monoclonal anti-c-Myc antibody 9E10 and rabbit polyclonal antibod-ies against �-catenin (C2206) and �-catenin (C2081) were purchasedfrom Sigma. A monoclonal anti-�-catenin antibody C19220 was ob-tained from Transduction Laboratories. A rabbit polyclonal antibodyOct-A probe against the FLAG epitope was purchased from Santa CruzBiotechnology, Inc. The following antibodies were described previously:rabbit polyclonal antibodies NW161 (35) and NW6 (38), directed againstdesmoplakin; a chicken polyclonal antibody 1407 (39), directed againstplakoglobin; a mouse monoclonal antibody 6D8, directed against Dsg2(35, 40).

Co-immunoprecipitation, Immunoblot, and Sequential Detergent Ex-traction—For co-immunoprecipitations, 100 or 60 mm dishes of cellswere washed in PBS, extracted in 1 ml or 500 �l of cold co-immunopre-cipitation buffer containing 1% Triton X-100, 145 mM NaCl, 10 mM

Tris-HCl, pH 7.4, 5 mM EDTA, 2 mM EGTA, and 1 mM phenylmethyl-sulfonyl fluoride. Lysates were then processed for immunoprecipitationas described previously (41) using specific antibodies. For immunopre-cipitation of FLAG-tagged proteins, anti-FLAG� M2-agarose affinitygel (Sigma) was used. Immunocomplexes were separated on 7.5% poly-acrylamide gels and electrophoretically transferred to nitrocellulose,which was blocked with 5% dry milk in PBS containing 0.1% Tween 20and probed with appropriate primary and secondary antibodies(Kirkegaard and Perry Laboratories) diluted in 5% dry milk in PBS.Sequential detergent extraction was performed as described (34) exceptthat the amount of buffer used at each step was adjusted so that thefinal volume of each pool was 400 �l.

Immunofluorescence—Cells were plated on glass coverslips the daybefore transfection in 6-well tissue culture dishes. 24 h after transfec-tion using FuGENE 6 reagent, cells were washed in PBS, fixed inmethanol for 2 min at �20 °C, and incubated with appropriate antibod-ies diluted in complete PBS. Cells were incubated with primary anti-bodies for 30 min at 37 °C, washed, and incubated with secondary

antibodies for 30 min at 37 °C. The following antibodies were used atindicated dilutions: PC28 at 1:200, C2206 at 1:50, C19220 at 1:50,Oct-A-probe at 1:50, Alexa Fluor�-conjugated goat anti-mouse or goatanti-rabbit secondary antibodies (Molecular Probes, Eugene, OR) wereused at 1:300. Images were obtained on a Leitz DMR microscope usinga Hamamatsu Orcal digital camera and Openlab imaging software(Improvision).

Yeast Two-hybrid Constructs and Assays—The plakoglobin constructlacking the N-terminal domain (Pg�N) in pACTII vector was describedpreviously (p515) (42). The cDNA sequence encoding Pg�N was re-moved from p515 by digestion with NcoI and XhoI and subcloned intothe same sites of pAS-CYH2 to create Pg�N in pAS-CYH2. Plasmidp525, which contains cDNA sequence of DPNTP in pBluescript (42),was digested with BamHI and SalI, and DPNTP was subcloned into theBamHI and XhoI sites of pACTII vector (p900). The PKP1a head do-main in DNA binding domain vector pBD-GAL4 (Stratagene) was de-scribed previously (19). The intracellular domains of Dsg1, Dsg2, Dsg3,Dsc1a, and Dsc2a in pGAD424 vector were described previously (18).These inserts were removed from the pGAD424 vector by digestion withEcoRI and SalI and cloned into the EcoRI and XhoI sites of pGADT7vectors. The mouse E-cadherin cytoplasmic domain comprises aminoacids 741–884 and was generated by PCR with primers containingEcoRI and SalI sites and cloned into the EcoRI and XhoI sites ofpGADT7. Full-length human �-catenin was cloned into the SmaI andBamHI sites of pGAD424. Both the human p120 catenin isoform 3ACand the cytoplasmic domain of protocadherin � 15 were cloned into theEcoRI and SalI sites of pGBKT7. Full-length PKP2a, its head domain,and the headless portion of PKP2a were amplified by PCR and clonedinto the SmaI site of pAS-CYH2 vector and confirmed by DNAsequencing.

To assay interactions between proteins, yeast strain Y189 or AH109was co-transformed with two testing plasmids, and dual transformantswere selected by growth on plates lacking both tryptophan and leucine.Transformations were performed according to methods in the Match-makerTM two-hybrid product protocol (CLONTECH Laboratories Inc.).Briefly, competent yeast cells were transformed with 1–5 �g of eachplasmid DNA using a standard lithium acetate/polyethylene glycolmethod. For the Y189 strain, positive interactions were quantified bymeasuring the �-galactosidase activity using 4-mythylumbelliferyl �-D-galactopyranoside as a substrate following the methods described be-fore (43). For AH109 strain, dual transformants were tested for theirability to grow on SD-Leu-Trp-His-Ade plates and their ability to turn bluein the presence of X-�-gal. Materials for base media and agar werepurchased from Difco, and materials for the defined media were pur-chased from CLONTECH Laboratories Inc.

TOPFLASH Luciferase Assay—TOPFLASH and FOPFLASH lucifer-ase reporter gene constructs were generously provided by Dr. BertVogelstein. TOPFLASH reporter gene construct contains optimizedTCF-binding sites upstream of a luciferase reporter gene, whereas theFOPFLASH contains mutated sites that do not bind TCF (44). SW480cells were seeded at 2.75 � 105 cells/well in 6-well dishes 24 h beforetransfection. Each well was transfected with 0.25 �g of either TOP-FLASH or FOPFLASH, 0.05 �g of pRL-TK (Promega) as an internalcontrol for transfection efficiency, and 1 �g of each indicated test con-struct. The total amount of DNA in each transfection was kept constantby the addition of an empty expression vector plasmid (pCMV-FLAG).Transfections were performed in triplicate using FuGENE 6 reagent(Roche Applied Science), and luciferase assays were performed 24 hlater following the protocol provided for the Dual-Luciferase™ reporterassay system (Promega). Luciferase activity was corrected for transfec-tion efficiency by using the control pRL-TK activity. Each experimentwas performed at least three times independently.

RESULTS

The Head Domain of PKP2a Is Sufficient for Its Cell BorderLocalization—Because PKP1a associates with desmosomalcomponents and is targeted to desmosomes through its headdomain (18), we tested whether the PKP2 head domain issimilarly responsible for its localization to cell borders. PKP2a,PKP2a-H, or PKP2a-A (Fig. 1) were transiently transfectedinto the SCC9 keratinocytes, and their subcellular distributionwas examined with respect to the endogenous desmosomalmarker DP. Ectopic full-length PKP2a was distributed primar-ily in a continuous fashion along cell borders (Fig. 2a), whichoverlapped, but was not completely coincident, with the punc-tate discontinuous pattern exhibited by endogenous DP (Fig.

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2b). This distribution pattern of PKP2a suggests that overex-pressed PKP2a might be associated with non-desmosomal com-ponents at the plasma membrane.

The head domain of PKP2a (PKP2a-H) colocalized more ex-tensively with endogenous DP and appeared more punctatecompared with full-length PKP2a (Fig. 2c). In addition, thePKP2a head domain was detected in the cytoplasm as discretedots. In contrast to full-length ectopic PKP2a, which was rarelyseen concentrated in the nucleus, in about 50% of the trans-fected cells the PKP2a head domain was detected in the nu-cleus. The PKP2a head domain was also localized to both cellborders and the nucleus in Madin-Darby canine kidney, HeLa,and COS cells (data not shown), reminiscent of the intracellu-lar localization reported for the PKP1a head domain (17, 18). Incontrast, PKP2a-A exhibited a largely diffuse distributionthroughout the cytoplasm without any specific pattern or cellborder localization. Cells overexpressing PKP2a-A were alsofrequently found to form many filopodia-like membrane pro-trusions and to have altered morphology. These results suggestthat the PKP2a head domain, which also has the ability toaccumulate in the nucleus, is sufficient for the membrane tar-geting of PKP2a.

PKP2a Co-immunoprecipitates with DP and the N-terminal584 Amino Acids Polypeptide DPNTP—It was shown previ-ously that PKP1a interacts directly with the N-terminal do-main of DP and enhances the recruitment of ectopic and en-dogenous DP to cell-cell borders (18, 19). To compare the abilityof PKP2a and PKP1a to interact with DP, full-length DP orDPNTP was transiently expressed in HEK293 cells with orwithout one of the PKP constructs, followed by immunoprecipi-tation using M2-agarose to precipitate FLAG-tagged PKP1a orPKP2a and the associated proteins. As shown in Fig. 3, likePKP1a, PKP2a co-immunoprecipitated DP and DPNTP. Nei-ther DP nor DPNTP was nonspecifically detected in the immu-nocomplexes with M2-agarose when expressed alone. Althoughsimilar amounts of PKP1a and PKP2a were immunoprecipi-tated by M2-agarose, substantially more full-length DP orDPNTP was present in the immunocomplex with PKP1a, sug-gesting that DP may interact more robustly with PKP1a thanwith PKP2a. This possible difference in the strength of theinteractions between DP and two plakophilins was further

supported by yeast two-hybrid results (Fig. 6).PKP2a Co-immunoprecipitates Pg and the Arm-repeat Domain

of Pg Is Sufficient for This Association—In a previous study, itwas reported that PKP1a was unable to interact with Pg in an invitro overlay assay (45). Here, the ability of PKP1 and PKP2 tointeract with Pg was compared by co-immunoprecipitation fromtransiently transfected COS cells. In agreement with previousfindings, we detected little or no Pg association with PKP1a (Fig.4A). In contrast, when Pg was co-expressed with FLAG-taggedPKP2a in COS cells followed by immunoprecipitation using M2-agarose, Pg was efficiently co-precipitated together with PKP2a(Fig. 4B). Deletion of either the N-terminal head or C-terminaltail of Pg did not adversely affect its association with PKP2a,indicating that the Pg arm-repeat domain primarily mediates theinteraction. A similar amount of PKP2a co-immunoprecipitatedconsiderably more Pg�C than either Pg�N or full-length Pg.Next, the ability of the PKP2a head domain (PKP2a-H) andPKP2a arm-repeats with the C-terminal tail (PKP2a-A) to co-immunoprecipitate Pg was tested (Fig. 4C). PKP2a-H co-

FIG. 1. Schematic diagram of plakophilin constructs used inthis study. The top two constructs are full-length PKP1a and PKP2a.Deletion constructs of PKP2a were generated including the PKP2ahead domain (PKP2a-H), the PKP2a headless fragment containing thearm-repeat domain and C-terminal tail (PKP2a-A). The HR2 domain ofPKP2a (amino acids 29–60) was internally deleted to generate PKP2awith deletion of the HR2 domain (PKP2a �HR2) and PKP2a headdomain with deletion of HR2 domain (PKP2a-H �HR2). The numbersindicate amino acid position, and the black box represents the HR2domain. PKPs have 10 arm-repeats, shown as open boxes. All theconstructs were C-terminally tagged with a FLAG epitope (star).

FIG. 2. The head domain of PKP2a is sufficient for its borderlocalization. SCC9 cells, which assemble numerous desmosomes, weretransiently transfected with PKP2a (a and b), the deletion constructsPKP2a-H (c and d), or PKP2a-A (e and f). 24 h after transfection, cellswere processed for double-label immunofluorescence using the polyclonalantibody Oct-A-probe to detect the FLAG epitope of ectopic polypeptides(a, c, and e) and the monoclonal antibody PC28 to detect endogenous DP(b, d, and f). Both PKP2a and its head domain are distributed in thecytoplasm and along cell borders, where they partially colocalize withendogenous DP (a–d). PKP2a-A is diffuse throughout the cytoplasm with-out specific border localization (e). PKP2a head domain is often found toconcentrate in the nucleus (c). Scale bar, 10 �m.

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precipitated less Pg compared with full-length PKP2a, andPKP2a-A was even less efficient in doing so, considering themuch higher amount of PKP2a-A present in the cell lysates andthe immunocomplex.

PKP2a Co-immunoprecipitates Dsg1 and Dsg2 but Not Dsg3When Co-expressed in COS Cells, and the HR2 Domain ofPKP2a Is Not Necessary for Its Complex Formation with OtherDesmosomal Components—To investigate possible interactionsbetween PKP2a and the three desmoglein isoforms Dsg1, -2,and -3, co-immunoprecipitation experiments were performedusing lysates from transiently transfected COS cells. PKP2aspecifically co-immunoprecipitated both Dsg1 and Dsg2 but notDsg3 (Fig. 5A). The PKP2a head domain, but not its arm-repeatdomain, mediated the interaction between PKP2a and Dsg2(data not shown). The parallel experiment could not be success-fully performed with Dsg1 due to quenched expression of theDsg1 construct when co-transfected with PKP2a-H. BecauseCOS cells express endogenous Pg, which might be able tointeract with both PKP2a and Dsg1 or Dsg2 in the same com-plex, we attempted to determine if the association of Dsg2 withPKP2a was mediated by a limiting amount of free endogenousPg. If this were the case, it should be possible to elevate theamount of Dsg2 associated with PKP2a by co-expression ofectopic Pg. However, the presence of ectopic Pg did not influ-ence the level of Dsg2 co-precipitated with PKP2a (Fig. 5B).

The conserved HR2 domains in the head domains of PKPshave been postulated to be important for certain common func-tions (3, 4). However, deletion of HR2 from PKP2a did notaffect its ability to co-immunoprecipitate other desmosomalcomponents (Fig. 5C). In addition, both PKP2a �HR2 and

PKP2a-H �HR2 exhibited the same distribution pattern astheir respective wild-type counterparts PKP2a and PKP2a-Hwhen expressed in SCC9 cells (data not shown). These resultssuggest that the HR2 domain of PKP2a is not critical for eitherits association with other desmosomal components or its properlocalization in cells.

Association of Ectopic PKP2a with Endogenous DesmosomalComponents—To determine whether PKP2a exhibits the samerepertoire of interactions with endogenous desmosomal pro-teins, we analyzed endogenous desmosomal proteins that co-precipitated with FLAG-tagged PKP2a in SCC9 epithelial cells.When transiently expressed FLAG-tagged PKP2a was immu-

FIG. 3. PKP2a co-immunoprecipitates both full-length DP andits N-terminal polypeptide DPNTP. DP or DPNTP was transientlyexpressed in HEK293 cells either alone or together with FLAG-taggedPKPs, which were then immunoprecipitated by M2-agarose, whichbinds the FLAG epitope of ectopic PKPs. The cell lysates and immuno-complexes were analyzed by Western blot to detect DP and DPNTPusing the anti-DP antibody NW161 and to detect ectopic PKPs using theanti-FLAG antibody Oct-A-probe. Both PKP1a and PKP2a specificallyco-precipitate DP and DPNTP, but more DP and DPNTP are found to beco-immunoprecipitated by PKP1a than by PKP2a. The variable levels ofDP and DPNTP observed in cell lysates are likely due to promotercompetition that results from transfection of multiple plasmids.

FIG. 4. PKP2a co-immunoprecipitates Pg and its terminal de-letion mutants. A, PKP1a is unable to co-immunoprecipitate Pg effi-ciently. Pg was expressed either alone or together with FLAG-taggedPKP1a followed by immunoprecipitation with M2-agarose and Westernblot analysis with 1407 and Oct-A-probe. B, terminal domains of Pg arenot essential for its association with PKP2a. Pg and its deletion mu-tants were transiently expressed in COS cells either alone or togetherwith FLAG-tagged PKP2a followed by immunoprecipitation with M2-agarose and Western blot analysis. An anti-Pg polyclonal antibody 1407was used to detect Pg, and anti-FLAG antibody Oct-A-probe was used todetect FLAG-tagged PKP2a. PKP2a co-immunoprecipitates Pg and itsdeletion mutants lacking either the N-terminal or C-terminal domain.C, deletion of the head or arm-repeat domain of PKP2a abrogates itsassociation with Pg. Pg was transfected either alone or together withdifferent FLAG-tagged PKP2a constructs followed by immunoprecipi-tation with M2-agarose and Western blot analysis. The ability of PKP2ato co-precipitate Pg is severely compromised by the deletion of PKP2aarm-repeat domain plus C-terminal tail and almost completely abol-ished by the deletion of PKP2a head domain.

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noprecipitated from the 1% Triton X-100-soluble cell lysates ofSCC9 cells, endogenous Pg was consistently detected in theimmunocomplex (Fig. 5D). DP, which is mostly insoluble, wasvariably detected in the complex with PKP2a (not shown),suggesting a weak and/or transient association. Surprisingly,whereas Dsg3 was present at low levels in the immunocomplexwith ectopic PKP2a, we could not detect endogenous Dsg2co-precipitated by ectopic PKP2a from SCC9 cells.

Yeast Two-hybrid Analysis of Protein Interactions betweenPKP2a and Other Desmosome Components—To extend the re-sults obtained using co-immunoprecipitation assays, the abilityof PKP2a to interact directly with DPNTP, Pg�N, and thedesmosomal cadherin tails was tested using the yeast two-hybrid system. The relative strength of interactions betweenDPNTP and PKP1a or PKP2a was compared using yeast strainY187 because it is more suitable for quantitative �-galactosid-ase assays. Consistent with previous reports (19), DPNTP in-teracted strongly with the head domain of PKP1a (Fig. 6).DPNTP directly interacted with full-length PKP2a and its headdomain at a much weaker level but not with the arm-repeatdomain of PKP2a. PKP2a and its head domain, but notPKP2a-A, interacted directly with Pg�N at a similar level.

Yeast two-hybrid assays employing growth and blue/whiteselection demonstrated that PKP2a directly interacted withthe cytoplasmic domains of Dsg1, Dsc1a, and Dsc2a but notwith Dsg2 or Dsg3 (Table I). The PKP2a head domain inter-acted with both Dsg1 and Dsg2 directly. However, no interac-tions were detected between PKP2a-A and any of the cytoplas-mic domains, nor was an interaction detected between PKP2aand E-cadherin. As a positive control, the E-cadherin cytoplas-

mic domain interacted with p120 catenin in the same assay(data not shown).

PKP1a and PKP2a Exhibit Different Localization Patternsand Abilities to Enhance the Border Recruitment of Excess DPWhen Overexpressed in COS Cells—The protein interactionstudies described above suggest that PKP1a and PKP2a haveoverlapping but distinct repertoires of desmosomal bindingpartners and that they may contribute differently to desmo-some assembly. It has been suggested that a critical function ofPKP1 is its ability to enhance DP recruitment to desmosomes(19). Therefore we compared the abilities of PKP1a and PKP2ato recruit DP to cell-cell borders in transiently transfected COScells. As reported previously (17), PKP1a localized to both thenucleus and cell borders in COS cells when it was expressedalone (Fig. 7A, a�), and its expression resulted in increasedborder staining for endogenous DP compared with adjacent,non-transfected cells (Fig. 7A, a, arrow). Staining for endoge-nous DP in the population of non-transfected cells is weak toundetectable (Fig. 7A, a and b, arrowhead). In contrast to theintense nuclear accumulation of PKP1a observed in more than95% of transfected COS cells, less than 10% of cells transfectedwith PKP2a showed weak nuclear staining. Like PKP1a,PKP2a enhanced the recruitment of endogenous DP to cellborders (Fig. 7A, b and b�).

Interestingly, the difference between PKP1a and PKP2a intheir ability to enhance DP recruitment was revealed whenboth PKP and DP were co-transfected into COS cells. Whenexpressed alone, ectopic DP predominantly colocalized with theIF network in the cytoplasm and sometimes localized to cellborders in transfected COS cells as described before (Fig. 7B, a,

FIG. 5. PKP2a co-immunoprecipi-tates Dsg1, Dsg2 but not Dsg3 fromco-transfected COS cells, and endog-enous Pg and Dsg3 from singly trans-fected SCC9 cells. A, each of the threeDsg isoforms was transiently expressed inCOS cells either alone or together withFLAG-tagged PKP2a followed by immu-noprecipitation with M2-agarose andWestern blot analysis of the immunocom-plex and cell lysates. Dsg1 was detectedwith the poly-Myc antibody, Dsg2 was de-tected with the anti-Dsg2 antibody 6D8,and the anti-Dsg3 antibody 1880 wasused to detect Dsg3. B, co-expression of Pgdoes not enhance the association betweenPKP2a and Dsg2. Dsg2, Pg, and FLAG-tagged PKP2a were transiently expressedin COS cells in combinations as indicatedfollowed by immunoprecipitation withM2-agarose and Western blot analysis.The poly-Myc antibody was used to detectthe Myc-tagged ectopic Pg. C, the HR2domain of PKP2a is not required for itsassociation with other desmosomal com-ponents. Different desmosomal molecules(DM) were transiently expressed inHEK293 cells either alone or togetherwith PKP2a or PKP2a�HR2, both ofwhich are FLAG-tagged and were immu-noprecipitated with M2-agarose. Deletionof the HR2 domain in PKP2a does notaffect its co-immunoprecipitation withother desmosomal components. D, SCC9cells were transiently transfected with ei-ther empty vector or FLAG-tagged PKP2aconstruct, and the Triton-soluble cell ly-sates were subjected to co-immunopre-cipitation using M2-agarose to isolateectopic PKP2a and its associated endog-enous proteins. Pg, Dsg3 but not Dsg2were detected in the complexes with ec-topic PKP2a.

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inset) (17). When PKP2a was co-expressed with DP, bothPKP2a and DP colocalized extensively along intermediate fil-aments (Fig. 7B, a and a�) and could also be detected along cellborders in certain cases (Fig. 7B, b and b�). In comparison,co-expression of PKP1a and DP resulted in a dramatic recruit-ment of DP to cell-cell borders together with PKP1a and aconcomitant disappearance of DP from IF networks in co-trans-fected cells (Fig. 7B, c and c�). Co-expression with DP did notaffect the accumulation of PKP1a in the nucleus (Fig. 7B, c�).These results showed that even though both PKPs can enhancethe recruitment of endogenous DP to cell borders, PKP1a ismuch more efficient than PKP2a in promoting the border lo-calization of excess DP.

PKP2a Co-immunoprecipitates the Adherens Junction Com-ponent �-Catenin, Which Forms Mutually Exclusive Complexeswith either E-cadherin or PKP2a—In contrast to plakoglobin,which can be found in both desmosomes and adherens junc-tions, �-catenin is found exclusively in adherens junctionsthrough its direct interaction with classical cadherins such asE-cadherin. Because PKP2a interacts with the arm-repeat do-

main of plakoglobin, which is highly homologous to that of�-catenin, we tested whether �-catenin also binds to PKP2a. Inco-immunoprecipitation experiments performed from tran-siently co-transfected COS cells, �-catenin specifically co-pre-cipitated with both full-length PKP2a and its head domain butonly very weakly with PKP2a-A, suggesting that the headdomain of PKP2a is mainly responsible for its interaction with�-catenin (Fig. 8A). Full-length PKP2a did not co-precipitatetwo other adherens junction components �-catenin and E-cad-herin in co-transfected COS cells, showing the specificity of itsassociation with �-catenin in this assay (data not shown). Full-length PKP2a interacted with �-catenin in yeast two-hybridassays (Table I), suggesting that they directly bind to eachother. The negative control interaction between �-catenin andprotocadherin � 15 was negative (data not shown).

To determine if �-catenin forms distinct complexes withPKP2a and E-cadherin, COS cells were co-transfected withconstructs expressing E-cadherin, �-catenin, and PKP2a. Afterimmunoprecipitation of either �-catenin or PKP2a, the immu-nocomplex was analyzed for the presence of E-cadherin. Asshown in Fig. 8B, E-cadherin efficiently associated with �-cate-nin when �-catenin was immunoprecipitated with an anti-�-catenin antibody; however, E-cadherin was not detected in thecomplex with PKP2a and �-catenin when PKP2a was immu-noprecipitated by M2-agarose beads. This observation suggeststhat the pool of �-catenin associated with PKP2a is unable tointeract with E-cadherin and that �-catenin forms mutuallyexclusive complexes with E-cadherin and PKP2a.

To examine the complex formation between PKP2a and otheradherens junction components in a more physiological environ-ment in keratinocytes, SCC9 cells transfected with only FLAG-tagged PKP2a were used for M2-agarose immunoprecipitationexperiments. Both endogenous E-cadherin and �-catenin, butnot �-catenin, co-immunoprecipitated with PKP2a (Fig. 8C).The ability of ectopic PKP2a to associate with endogenous�-catenin is consistent with the protein interaction data. Thepresence of endogenous E-cadherin in a complex with ectopicPKP2a raises the possibility that in certain cell types, PKP2amight associate indirectly with E-cadherin through other pro-tein partners such as Pg. Indeed, when Pg was overexpressedin COS cells with ectopic E-cadherin and PKP2a, a complexcontaining all three proteins could be detected (not shown),which is in contrast to the co-immunoprecipitation experimentdescribed above for �-catenin (Fig. 8B).

Because endogenous PKP2a and �-catenin are localized todifferent junctional complexes and do not usually colocalizewith each other at cell borders (Ref. 2; data not shown), wesought to determine whether overexpressed PKP2a could colo-calize with endogenous �-catenin at the membrane. BothPKP2a and its head domain colocalized with endogenous�-catenin at cell borders in transiently transfected SCC9 cells(Fig. 9). The expression of PKP2a or PKP2a-H did not alter thedistribution of endogenous �-catenin or affect its accumulationalong cell borders. These data are consistent with PKP2a hav-ing the ability to associate with �-catenin in cells.

The Effects of PKP2a on �-Catenin/TCF Signaling—Free,cytoplasmic �-catenin that is not bound to cadherins can par-ticipate in the Wnt-signaling pathway through its interactionwith TCF/Lef1 transcription factors (28). PKP2a co-precipi-tates with a non-cadherin-bound pool of �-catenin, raising thepossibility that PKP2a might be involved in regulating �-cate-nin/TCF signaling. To better understand the physiological con-sequence of the PKP2a/�-catenin interaction, we investigatedthe effect of PKP2a on �-catenin/TCF-regulated transcriptionin human colon carcinoma line SW480 cells, which have beenshown to have constitutive �-catenin/TCF transcription activ-

FIG. 6. PKP2a interacts directly with DPNTP and Pg�N inyeast two-hybrid assays. Yeast strain Y187 cells were co-trans-formed with DPNTP or Pg�N in pACTII vector and various PKP con-structs as indicated. Co-transformants were selected on plates lackingtryptophan and leucine (�Trp/�Leu), and individual clones were usedfor quantitative �-galactosidase assay. Each bar represents the averageof triplicate measurements for an independent clone. Interaction be-tween p53 and large T antigen (LgT) is shown as a positive control. Themeasurements for the interactions between PKP2a-A and eitherDPNTP or Pg �N are negligible. DPNTP interacts most strongly withPKP1a head domain and at a much weaker level with both PKP2aand its head domain. Pg�N directly interacts with PKP2a and itshead domain but not with PKP2a-A. No significant measurementswere obtained from clones co-transformed with PKP constructs andpACTII empty vector (data not shown). MUG, 4-mythylumbelliferyl�-D-galactopyranoside.

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ity (44, 46). TOPFLASH luciferase assays were performed tomeasure the activation of �-catenin/TCF signaling. 24 h aftertransfection, TOPFLASH reporter was strongly activated byendogenous �-catenin/TCF signaling in SW480 cells comparedwith negligible FOPFLASH activity (0.012 � 0.002) (Fig. 10).Overexpression of PKP2a significantly elevated the activationof the TOPFLASH reporter by � 33% compared with vector

FIG. 7. Subcellular localization of PKP1a and PKP2a in COScells and their abilities to enhance the cell border recruitmentof DP. A, localization of overexpressed PKP1a and PKP2a and theireffects on the distribution of endogenous DP in COS cells. COS cellswere transiently transfected with either FLAG-tagged PKP1a (a anda�) or FLAG-tagged PKP2a (b and b�). Double-label immunofluores-cence was carried out to detect the localization of ectopic PKPs usingthe anti-FLAG antibody Oct-A-probe (a� and b�) and the localizationof endogenous DP using the anti-DP antibody PC28 (a and b). BothPKP1a and PKP2a are localized to cell borders, where they colocalizewith and enhance the staining of endogenous DP. PKP1a is also foundintensely in the nucleus (a�). The arrows (a and b) indicate enhancedborder staining of endogenous DP. The arrowheads (a and b) point tocell borders between untransfected cells. B, COS cells were co-transfected with DP together with FLAG-tagged PKP2a (a, b, a�, andb�) or FLAG-tagged PKP1a (c and c�) followed by double-label immu-nofluorescence using antibodies against the FLAG epitope (a�, b�, andc�) and DP (a–c). Ectopic PKP2a colocalizes extensively with overex-pressed DP along intermediate filaments and cell borders (a, b, a�,and b�). Overexpression of DP alone in COS cells results in itscolocalization mainly along intermediate filaments (a, inset). EctopicPKP1a is localized predominantly to the nucleus and along cell bor-ders, where DP is strongly recruited. The IF pattern of distribution ofoverexpressed DP is dramatically reduced when PKP1a is co-expressed (c and c�). Scale bars, 10 �m.

FIG. 8. PKP2a associates with �-catenin that is not bound toE-cadherin. A, the head domain of PKP2a is required for its associa-tion with �-catenin (cat). �-Catenin was transiently transfected intoCOS cells either alone or with different FLAG-tagged PKP2a constructsas indicated, which were then immunoprecipitated with M2-agarose.Western blots of cell lysates and immunocomplexes were performedusing the polyclonal anti-�-catenin antibody C2206 and anti-FLAGantibody Oct-A-probe. PKP2a and its head domain efficiently co-immu-noprecipitate �-catenin, whereas the headless fragment PKP2a-A al-most completely loses this ability. B, PKP2a forms complexes with�-catenin that is not bound to E-cadherin. �-Catenin, E-cadherin, andFLAG-tagged PKP2a were co-expressed in COS cells. Specific antibod-ies against �-catenin (C19220) and the FLAG epitope (M2) were used toimmunoprecipitate �-catenin and PKP2a, respectively. �-Catenin co-immunoprecipitates both PKP2a and E-cadherin, but E-cadherin is notdetected in the immunocomplex associated with PKP2a, indicatingdistinct pools of �-catenin in association with either PKP2a or E-cadherin but not both of them. The upper two bands recognized by theanti-E-cadherin antibody represent the precursor and mature form, andthe lower band is likely a breakdown product lacking part of the cyto-plasmic �-catenin binding domain. C, SCC9 cells were transientlytransfected with either empty vector or FLAG-tagged PKP2a constructand the Triton-soluble cell lysates were subjected to co-immunoprecipi-tation using M2-agarose to isolate ectopic PKP2a and its associatedendogenous proteins. E-cadherin and �-catenin but not �-catenin weredetected in the complexes with ectopic PKP2a.

TABLE IYeast two-hybrid analysis of protein-protein interactions

AH109 yeast cells were co-transformed with PKP2a or its subdomains in pAS-CYH2 and with the cytoplasmic domains (cyto) of desmosomalcadherins in pGADT7, full-length �-catenin in pGAD424 (�-cat), or the cytoplasmic domain of E-cadherin in pGADT7 (E-cad cyto). Positiveinteractions were determined through analyzing the abilities of co-transformants to grow and turn blue on selection plates lacking adenine,histidine, leucine and tryptophan and containing X-�-gal. Three separate clones from each co-transformation were assessed, and the same resultswere obtained for all three clones in all the assays. Clones that could grow as blue colonies on selection plates were considered as positive and aredesignated with plus symbols, and clones that did not show growth on the selection plates were considered as negative and are designated withminus symbols. White colonies were not observed. ND, not determined.

Dsg1 cyto Dsg2 cyto Dsg3 cyto Dsc1a cyto Dsc2a cyto �-Cat E-cad cyto

PKP2a � � � � � � �PKP2a-H � � � ND ND ND �PKP2a-A � � � ND ND ND �

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control, and the FOPFLASH activity was not significantly af-fected (0.011 � 0.001). The level of �-catenin in the cell lysateswas not affected by expression of PKP2a. Double-label immu-nofluorescence showed that overexpressed PKP2a was largelydiffuse in the cytoplasm and excluded from the nucleus, and nochanges in the subcellular localization of �-catenin were de-tected (data not shown). E-cadherin expression dramaticallyreduced the TOPFLASH activity as has been reported previ-ously (47, 48). This inhibition of �-catenin/TCF signaling byE-cadherin requires its �-catenin binding site and was shownto involve the binding and sequestering of �-catenin from thesignaling pool by E-cadherin (48). Co-expression of E-cadherinwith PKP2a in SW480 cells resulted in a similar level of inhi-bition of TOPFLASH activity as by E-cadherin alone. Thisinability of PKP2a to up-regulate TOPFLASH activity in thepresence of E-cadherin suggests that PKP2a may exert itseffect on �-catenin/TCF signaling through interaction with thesame pool of signaling active �-catenin that E-cadherinsequesters.

DISCUSSION

PKPs represent a family of arm-repeat proteins present inboth desmosomes and the nucleus. Commonly found in strati-fied and complex epithelia, PKPs exhibit cell type- and differ-entiation-dependent patterns of desmosomal localization, sug-gesting distinct roles in the regulation and function ofdesmosomes. Their constitutive nuclear localization independ-ent of their presence in desmosomes points to possible novelfunctions in the nucleus. The identification of human patientsbearing PKP1 gene mutations has provided compelling evi-dence for its critical involvement in organizing and stabilizingdesmosomes and further catalyzed the study on its role indesmosomes. However, little is known about the function of itsmost widely expressed relative, PKP2. In the present study, weinvestigated the ability of PKP2a to interact with other junc-tional molecules and conducted functional analyses on its rolein desmosome formation and regulation of �-catenin-signalingactivity.

PKP1a has been shown to interact directly with DP, Dsg1,and keratin (18, 45, 49) through its head domain (18, 19). Weshowed here by co-immunoprecipitation from co-transfectedcells that PKP2a associates with DP, Pg, Dsg1, and Dsg2. Yeasttwo-hybrid analysis demonstrated furthermore that interac-tions with DP, Pg, Dsg1, and possibly Dsg2 are direct andfurther uncovered Dsc1a and 2a as PKP2a interaction part-

ners. Pg co-precipitated with PKP2a and the deletion of eitherthe N-terminal head or C-terminal tail of Pg failed to abrogatethis interaction, suggesting that it is primarily mediated by thearm-repeat domain of Pg. Intriguingly, the association betweenPg and PKP2a was further enhanced by deletion of the PgC-terminal end domain. Previous work showed that Pg�C ex-

FIG. 10. Effect of PKP2a on �-catenin/TCF signaling in SW480cells. SW480 cells were transiently transfected with TOPFLASH lucif-erase reporter gene and a control luciferase reporter gene pRL-TK tocontrol for transfection efficiency. The results are expressed as relativeactivity, which is derived from dividing the TOPFLASH luciferaseactivity by the activity of pRL-TK. The results are the means and S.D.of triplicate values obtained in one representative experiment of three.Negligible activity was observed using FOPFLASH reporter gene,which contains mutated TCF-binding sites (data not shown). PKP2aexpression significantly elevated the TOPFLASH activity by �33% (p �0.011), whereas E-cadherin expression inhibited its activity and pre-vented its up-regulation by PKP2a. Mean relative activities were notsignificantly different between cells expressing E-cadherin and cellsexpressing both E-cadherin and PKP2a (p � 0.405 0.05). Cell lysatesprepared for the reporter gene assay were quantitated, and the sameamount of total protein was analyzed by SDS-PAGE and Western blotusing the anti-�-catenin (cat) antibody C2206. The total level of �-cate-nin is not altered by the expression of either PKP2a or E-cadherin. Theasterisk designates values significantly different from control by Stu-dent t test at p 0.05.

FIG. 9. PKP2a and its head domaincolocalize with �-catenin (cat) at thecell membrane. SCC9 cells were tran-siently transfected with FLAG-taggedPKP2a (a, b, and c) or its head domainPKP2a-H (d, e, and f), and double-labelimmunofluorescence was carried out todetect endogenous �-catenin (b and e) andFLAG-tagged ectopic proteins (a and d).Composite pictures are shown to demon-strate the colocalization of �-catenin withPKP2a and PKP2a-H along the cell bor-ders, as indicated in yellow (c and f). Someof the overlapped signals do not appearas yellow after the overlay because theintensity of the individual signals isunequal.

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pression in A431 cells caused formation of longer desmosomesor groups of tandemly linked desmosomes that were still at-tached to keratin intermediate filaments (34). It was proposedthat deletion of the Pg C terminus might enhance interactionswith other desmosomal components and lead to generation oflonger desmosomes. Together with our data, it thus seemspossible that enhanced interaction between Pg�C and PKP2acould contribute to the effect on desmosome morphology previ-ously attributed to Pg�C expression.

PKP2a co-immunoprecipitated Dsg1 and Dsg2, but not Dsg3in co-transfected COS cells, and its interaction with Dsg1 wasconfirmed by yeast two-hybrid assays as being directly medi-ated by the PKP2a head domain. It is unclear why we detecteda direct interaction between the Dsg2 cytoplasmic domain andthe PKP2a head domain but not full-length PKP2a in yeasttwo-hybrid assays, but it seems likely that a direct binding sitefor Dsg2 is contained in the head domain of PKP2a in light ofthe abilities of both PKP2a and PKP2a-H to co-precipitate Dsg2in transfected cells. The fact that increasing the level of cellularPg does not enhance the association between PKP2a and Dsg2(Fig. 5B) is consistent with the hypothesis that Pg does notinteract with PKP2a and Dsg2 simultaneously. The bindingsites in Pg for Dsg1 and Dsc1a reside in its arm-repeat domain(40, 50, 51), as does the binding site for PKP2a as we show here.Thus, it is possible that desmosomal cadherins and PKP2acompete with each other for binding to Pg. How the observeddirect interactions discussed here translate into the formationof the physiological complexes in cells may depend on otherfactors, including the availability of specific protein partners inspecific cell types. For instance, although direct interactionswith Dsg3 were not observed in co-transfection or yeast two-hybrid assays, Dsg3 was present in an endogenous complexwith ectopic PKP2, whereas Dsg2 was not (Fig. 5D). Togetherthese data suggest that Pg or other unknown proteins maymediate an indirect association between PKP2a and Dsg3 butnot Dsg2. The absence of an identifiable complex between ec-topic PKP2a and endogenous Dsg2 by co-immunoprecipitationmay also indicate that in SCC9 cells, which express both Dsg3and Dsg2, Dsg3 may compete more effectively for PKP2 due toits relative abundance compared with Dsg2 in the detergent-soluble pool or other factors favoring this interaction.

The presence of PKP2 in desmosomes is widespread as it isfound in all simple epithelia and in complex epithelia where itis localized throughout the layers and concentrated in desmo-somes of the basal layer. These localization data are consistentwith our observations, and together they suggest that PKP2acan associate with all Dsg isoforms, either directly or indi-rectly. Thus PKP2 may be involved in desmosome function ona much broader scale than PKP1, and it is intriguing to con-sider that it may serve different functions when it is associatedwith different desmosomal cadherins.

In COS cells, ectopic PKP1a efficiently enhances the recruit-ment of DPNTP to cell borders, and this ability is conferred bythe PKP1a head domain (19). Because DPNTP contains onlythe first 584 amino acids of DP and does not bind to IF, wecompared the ability of PKP1a and PKP2a to enhance the cellborder recruitment of full-length DP that is sequestered alongthe IF network. Co-expression of PKP1a with DP in COS cellsresulted in a pattern of DP distribution vastly different fromthat when DP was expressed alone. Instead of exhibiting ex-tensive co-alignment with the IF network and occasional bor-der localization, DP was mostly concentrated along the borders,colocalizing with PKP1a. In contrast, the majority of DP re-mained sequestered along IF in cells co-expressing PKP2a andDP. The fact that both PKPs enhanced the border localizationof endogenous DP to a similar level (Fig. 7A, a and b) could be

due to a limiting amount of endogenous DP available for re-cruitment and that to reveal the higher capacity of PKP1a torecruit DP requires that excess DP be expressed.

The different abilities of PKP1 and PKP2 to enhance cellborder recruitment of ectopic DP in COS cells might be ex-plained by several mechanisms. Biochemical evidence sug-gested that DP interacts with PKP2a less robustly than withPKP1a, and an interaction with a certain strength could berequired to directly recruit DP to cell borders by PKPs. Sec-ondly, the two PKPs might enhance DP recruitment indirectlythrough their differential ability to recruit other desmosomalcomponents or as yet unidentified proteins. Furthermore,PKP2a could differ from PKP1a in its propensity to incorporateinto desmosomes, and PKP1a may preferentially assemble intodesmosomes in COS cells. Because PKP2 has been foundmostly concentrated in desmosomes in the basal layer of vari-ous stratified epithelial tissues (2), perhaps the participation ofPKP2 in desmosomes of the basal layer contributes to theformation of smaller (52) and possibly more dynamic desmo-somes that are suitable for the amplification and generation ofnew cells from the basal layer. This may partly be achievedthrough the reduced capacity of PKP2 to recruit and stabilizeother desmosomal components such as DP.

Another striking difference between PKP1a and PKP2a istheir ability to accumulate in the nucleus in cells overexpress-ing these proteins. PKP1a and its head domain efficiently con-centrated in the nuclei when expressed in a variety of cell lines(17–19). Here we show that PKP2a is mostly excluded from thenucleus in transiently overexpressing cells. The PKP2a headdomain can localize to the nucleus more efficiently than full-length PKP2a, indicating that the arm-repeat domain ofPKP2a either contains nuclear export signals or has the abilityto retain PKP2a in the cytoplasm through interactions withother proteins.

Besides interacting with other desmosome plaque proteins,PKP2a is also capable of associating with �-catenin mainlythrough the PKP2a head domain. Co-expression of E-cadherin,�-catenin, and PKP2a led to the formation of mutually exclu-sive complexes that contained �-catenin with either E-cadherinor PKP2a, suggesting that PKP2a is unlikely to interact with�-catenin in the adherens junction. However, ectopic PKP2aformed complexes with endogenous E-cadherin and �-cateninbut not with �-catenin in SCC9 cells. This result suggests thatother proteins such as Pg could mediate the association be-tween E-cadherin and PKP2a. Because this complex lacks�-catenin, it is unlikely to be found in mature adherens junc-tions, but it perhaps represents a junction intermediate in-volved in sorting and segregation of components during theearly stages of junction formation or during the dynamic reg-ulation of junctional structures. Even though we were unableto detect colocalization of endogenous PKP2a and �-catenin inSCC9 cells, we did observe extensive colocalization of ectopicPKP2a and its head domain with endogenous �-catenin alongthe cell borders. It has been shown previously that there is apool of �-catenin localized in the lateral membrane of Madin-Darby canine kidney cells that does not contain E-cadherin(53), so possibly ectopic PKP2a could interact with this pool of�-catenin at the membrane but not the E-cadherin bound poolof �-catenin.

Despite a lack of apparent effect on the total level of �-cate-nin, PKP2a overexpression resulted in a modest, but reproduc-ible increase in the level of endogenous �-catenin/TCF signal-ing in SW480 cells. Because we could only achieve about 15%transfection efficiency in SW480 cells, some moderate effect on�-catenin protein level by expression of PKP2a may not havebeen detectable in the total cell population. PKP2a expression

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did not seem to change the subcellular localization of �-cateninin transfected cells, and experiments using COS cells andHEK293 cells, which could be transfected at efficiencies greaterthan 50%, did not reveal any significant effect by PKP2a ex-pression on either the total level of �-catenin or its partitioninginto different detergent soluble pools (data not shown). Theseresults suggest that PKP2a modulates �-catenin signalingthrough mechanisms other than regulating its stability, ashave been suggested for several regulators of �-catenin/TCFsignaling (54–56). Expression of ectopic E-cadherin inhibitedendogenous �-catenin/TCF signaling and suppressed PKP2a-dependent increases in �-catenin/TCF signaling in SW480cells. A recent paper presented evidence for the existence of asmall pool of transcriptionally active �-catenin among themuch larger pool of cytosolic but transcriptionally inactive�-catenin in SW480 cells (47). Our result is consistent with themodel in which E-cadherin sequesters the transcriptionallyactive pool of �-catenin and prevents its binding to PKP2a.

The data presented in this study show that PKP2 and PKP1exhibit overlapping but distinct properties in their repertoiresof binding partners and functional relationships with DP.These results, coupled with the observed partially overlappingyet different expression profiles, suggest specific participationof individual or combinations of PKPs in creating functionallydistinguishable desmosomes that are tailored for the needs ofdifferent tissues or differentiation stages. The ability to inter-act with �-catenin and modulate its signaling activity indicatesthat PKP2a might be involved in other cellular processes suchas the cross-talk between desmosomes and adherens junctionsand the transduction of signals from the cell surface to thenucleus.

Acknowledgments—We thank all those who have generously contrib-uted antibodies, plasmids, and other reagents, including M. Amagai,P. Cowin, R. Marsh, R. Brackenbury, J. Stanley, P. McCrea, P. Polakis,J. Nelson, and B. Vogelstein. Thanks also to Yejia Zhang for assistancein the initial cloning of PKP2, Claudia Horn for technical assistance inthe cloning of yeast two-hybrid constructs containing the cytoplasmicdomains of desmosomal cadherins, and Spiro Getsios, Lisa Godsel, andAndrew Kowalczyk for helpful discussion and critical reading of themanuscript.

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Xinyu Chen, Stefan Bonné, Mechthild Hatzfeld, Frans van Roy and Kathleen J. Green-CATENIN SIGNALINGβITS DIVERSE ROLES IN DESMOSOMES AND

Protein Binding and Functional Characterization of Plakophilin 2: EVIDENCE FOR

doi: 10.1074/jbc.M108765200 originally published online January 14, 20022002, 277:10512-10522.J. Biol. Chem. 

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