Carboxyl Terminus of Plakophilin-1 Recruits It to Plasma Membrane, whereas Amino Terminus Recruits Desmoplakin and Promotes Desmosome Assembly*

Plakophilins are armadillo repeat-containing proteins, initially identified as desmosomal plaque proteins that have subsequently been shown to also localize to the nucleus. Loss of plakophilin-1 is the underlying cause of ectodermal dysplasia/skin fragility syndrome, and skin from these patients exhibits desmosomes that are reduced in size and number. Thus, it has been suggested that plakophilin-1 plays an important role in desmosome stability and/or assembly. In this study, we used a cell culture system (A431DE cells) that expresses all of the proteins necessary to assemble a desmosome, except plakophilin-1. Using this cell line, we sought to determine the role of plakophilin-1 in de novo desmosome assembly. When exogenous plakophilin-1 was expressed in these cells, desmosomes were assembled, as assessed by electron microscopy and immunofluorescence localization of desmoplakin, into punctate structures. Deletion mutagenesis experiments revealed that amino acids 686–726 in the carboxyl terminus of plakophilin-1 are required for its localization to the plasma membrane. In addition, we showed that amino acids 1–34 in the amino terminus were necessary for subsequent recruitment of desmoplakin to the membrane and desmosome assembly.

Desmosomes are prominent cell-cell adhesive junctions found in a variety of epithelial tissues. Desmosomes are disc-shaped junctions that link the intermediate filament cytoskeleton from one cell to the intermediate filament cytoskeleton of an adjacent cell. Thus, the primary role of the desmosome is to provide epithelial tissues a mechanism to resist mechanical stress (reviewed in Refs. 1 and 2). The transmembrane core of the desmosome is composed of the desmosomal cadherins, desmogleins, and desmocollins. A number of cytoplasmic desmosomal plaque proteins cooperate to link the desmosomal cadherins to the intermediate filament cytoskeleton. Desmoplakin and plakoglobin are desmosomal plaque proteins expressed in all desmosome-containing epithelial cells. Plakoglobin is an armadillo repeat-containing protein that interacts directly with the cytoplasmic domain of the desmosomal cadherins (3)(4)(5). Desmoplakin is a member of the plakin family of cytoskeletal linker proteins (2, 6) that contains a carboxyl-terminal intermediate filament binding domain (7) and an amino-terminal head domain required for desmosome association (8,9).
In addition to desmoplakin and plakoglobin, members of the plakophilin family (PKP1-3) are also present in the desmosomal plaque (10 -13). How-ever, the role of the plakophilins in desmosome assembly is not fully understood. Plakophilin-1 is expressed abundantly in the epidermis (14) and mutations in the PKP1 gene, resulting in loss of plakophilin-1 protein expression, and are the underlying cause of ectodermal dysplasia/skin fragility (15). Ultrastructural examination of skin from these patients revealed that desmosomes are reduced in number and in size compared with normal individuals. Therefore, plakophilin-1 is thought to play a role in desmosome stability (16,17). Recently, plakophilin-2 null mice have been generated and characterized. Interestingly, these mice display a lethal phenotype due to defects in heart morphogenesis (18). Based on these findings, plakophilins have been proposed to play an important role in the organization and stability of the desmosome.
The desmosome is a complex cell-cell junction both in composition and in the regulation of assembly. The composition of the desmosome in stratified epithelia varies in a differentiation-specific manner. Multiple desmoglein, desmocollin, and plakophilin genes are spatiotemporally expressed during keratinocyte differentiation (14,19,20). In addition, desmosome assembly is dependent upon the assembly of another cell-cell adhesive junction, the adherens junction. The adherens junction is structurally similar to a desmosome, except that the transmembrane protein is a classical cadherin, the plaque proteins are catenins, and the complex is associated with the actin cytoskeleton. It is well accepted that, in order for cells to assemble desmosomes, they must first adhere to one another via adherens junctions (21)(22)(23). The mechanisms underlying junctional cross-talk between adherens junctions and desmosomes are poorly understood.
To date, experiments to understand the role of plakophilin-1 in the desmosome have been performed primarily in cells that already assemble endogenous desmosomal structures, such as COS (24,25), HeLa, and HaCat cells (26). These experiments have relied on the transient expression of full-length plakophilin-1 or fragments of plakophilin-1 and assessing their incorporation into previously assembled desmosomes. We sought to develop a cell culture system designed to examine the role of plakophilin-1 in de novo assembly of desmosomes. To accomplish this, we generated a model cell system that is plakophilin-1-null and does not assemble desmosomes (A431DE cells). Upon stable expression of plakophilin-1 in A431DE cells, desmosomes are assembled. Using this system, we showed that two distinct amino acid sequences in the amino and carboxyl termini of plakophilin-1 are required for desmosome assembly and plasma membrane localization, respectively. (5,27,28) and are maintained in medium containing 2 g/ml puromycin (Sigma).
Retrovirus Production and Infection-Retrovirus production and infection were performed essentially as described by Ireton et al. (29). Briefly, phoenix cells (5 ϫ 10 5 /100-mm dish) were transfected with constructs prepared in the LZRS-MS-neo vector using calcium phosphate (Stratagene, La Jolla, CA). Forty-eight hours following transfection, the cells were switched to medium containing 2 g/ml puromycin (Sigma) to select for virus-producing cells. A population of puromycin-resistant cells was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum lacking puromycin and grown at 32°C for 24 h for virus production. Virusconditioned medium was collected and passed through a 0.45-m syringe filter and polybrene (Sigma) was added to 4 g/ml. For infection of target cells, 2 ϫ 10 5 cells were plated in 1 well of a 6-well dish 18 h prior to infection. Fresh virus-conditioned medium was incubated with target cells for 6 h at 32°C. Following infection, the medium was replaced with fresh medium, and the cells were returned to 37°C. Two days after infection, a selective medium containing 1 mg/ml G418 was added, and a population of cells expressing the desired protein product was isolated.
Detergent Extraction of Cells and Immunoblot Analysis-For total cell lysates, cells in T25 flasks were grown to confluence, rinsed three times with phosphate-buffered saline containing 2 mM sodium orthovanadate, and extracted in 1 ml of Empigen BB extraction buffer (10 mM Tris-HCl, pH 7.0, 0.1% Empigen BB (Calbiochem), 5 mM EDTA, 2 mM EGTA, 30 mM sodium fluoride, 40 mM ␤-glycerophosphate, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, and 2 mM phenylmethylsulfonyl fluoride). The cells were placed on ice, scraped, and triturated vigorously for 2 min. Insoluble material was pelleted by centrifugation at 14,000 ϫ g for 15 min at 4°C, and the supernatant was used immediately or stored at Ϫ70°C. The protein concentration was determined using DC protein assay (Bio-Rad) with bovine serum albumin as a standard. Proteins were electrophoretically transferred overnight to nitrocellulose membranes and blocked in 5% nonfat dry milk in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 45 min. Blocking solution was removed by washing for 15 min followed by washing twice for 5 min each time in TBST. For the immunoblots, hybridoma-conditioned medium was used at 1:100 dilution in TBST for 1 h. Membranes were washed 15 min followed by washing twice for 5 min each time in TBST. Membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody (Jackson Immunoresearch, West Grove, PA) at 1:10,000 for 1 h. Secondary antibody was removed by washing 15 min followed by washing four times for 5 min each time in TBST. Immunoreactive bands were detected using Super Signal Pico substrate (Pierce).
Immunofluorescence Microscopy-Cells grown on glass coverslips were washed briefly in Hepes-buffered Hanks' balanced salts solution (HHBSS) and fixed using 1% paraformaldehyde in 1ϫ HHBSS. Cells were rinsed briefly in phosphate-buffered saline (PBS) and blocked in PBS containing 10% heat-inactivated goat serum (Sigma) and 0.2% Triton X-100 (Fisher). Cells were incubated in primary antibody for 1 h, and primary antibody was removed by washing once in blocking solution and three times in PBS. Fluorescein isothiocyanate-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) were diluted (1:100) in blocking buffer and added to cells for 1 h. Excess secondary antibody was removed by washing once in blocking solution and three times in PBS. Coverslips were briefly washed in distilled water and mounted using vectashield mounting medium with 4Ј,6diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Images were collected on a Zeiss Axiovert 200M microscope equipped with an ORCA-ER (Hamamatsu) digital camera. Images were collected and processed using OpenLab software from Improvision, Inc. (Boston, MA).
Calcium Switch Experiment-2 ϫ 10 5 cells were grown on glass coverslips in a 6-well dish for 24 h, and the medium was replaced with calcium-free Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% dialyzed fetal bovine serum (Hyclone Laboratories). 18 h later, the medium was replaced with Dulbecco's modified Eagle's medium (1.8 mM calcium) supplemented with 5% fetal bovine serum, and cells were processed for immunofluorescence for the indicated times in calcium-containing medium.
Electron Microscopy-Electron microscopy was performed at The University of Nebraska Medical Center electron microscopy core facility. Cells were grown on Thermanox plastic coverslips, fixed using 1% glutaraldehyde, and processed for electron microscopy as described in Ref. 33.
Yeast Two-hybrid Assays-Protein-protein interactions were tested using the Matchmaker 3 system from Clontech (Palo Alto, CA). Plakophilin-1 bait constructs were subcloned into pGBKT7 to create Gal4 DNA binding domain fusion proteins, and desmoplakin was subcloned into pGADT7 to create a prey plasmid encoding desmoplakin amino acids 1-584 fused to the Gal4 activation domain. Two-hybrid assays were performed according to the manufacturer's protocol.

RESULTS
Generation of A431DE Cells Expressing Plakophilin-1-Because our goal was to investigate the role of plakophilin-1 in desmosome assembly, it was necessary to generate a model cell system that expressed all of the necessary desmosomal components (except for plakophilin-1) and did not assemble desmosomes. Thus, we turned to the plakophilin-1-negative A431D cell line previously developed in our laboratory (27). A431D cells are a derivative of the A431 cervical squamous cell carcinoma cell line, which has lost expression of the classical E-and P-cadherins. As a result, A431D cells lack the ability to form adherens junctions and do not aggregate. Interestingly, A431D cells retain expression of the desmosomal cadherins (desmoglein 2 and desmocollin 2), plakoglobin, a small amount of plakophilin-2, and desmoplakin; however, they do not assemble desmosomes (27). A431D, similar to the parental A431 cells, do not express endogenous plakophilin-1.
It is widely believed that cells must interact with one another via the classical cadherin containing adherens junction to assemble a desmosome (22,23). Therefore, we generated stable clones of A431D cells transfected with a 2ϫ Myc epitope-tagged E-cadherin cDNA construct (A431DE cells). The A431DE clones were then screened by immunofluorescence for desmoplakin expression, because it was noted that the parental A431D cell population was heterogeneous for desmoplakin expression (data not shown). Multiple clones of A431DE cells were identified that expressed relatively high levels of desmoplakin. These cells were then retrovirally infected with a cDNA encoding full-length plakophilin-1, and stable populations of plakophilin-1-expressing A431DE cells (A431DE/pkp1) were isolated.
Immunofluorescence microscopy was performed to verify the expression and proper subcellular localization of E-cadherin, plakophilin-1, desmoplakin, and desmoglein 2 in these cells. As expected, parental A431D cells did not express endogenous E-cadherin or plakophilin-1 (Fig. 1, A and D). When we expressed E-cadherin in A431D cells (A431DE cells), it was localized to the plasma membrane as expected (Fig. 1B). In addition, we also observed intracellular E-cadherin staining in A431DE cells. We suspect that this intracellular E-cadherin is a newly synthesized protein that is in the endoplasmic reticulum and Golgi complex and is in the process of being transported to the plasma membrane (data not shown). To support this hypothesis, we also observed a small amount of unprocessed E-cadherin that migrated slightly slower than expected in immunoblots (see Fig. 3, arrow). Plakophilin-1 expression was not restored upon E-cadherin expression (Fig. 1 E). When we expressed plakophilin-1 in A431DE cells (A431DE/pkp1 cells), it was homogeneously expressed and was localized in the nucleus and cell borders as previously reported (26,35,36) (Fig. 1F). Although plakophilin-1 was readily detectable at sites of cell-cell contact (Fig. 1F, arrows), the majority was localized to the nucleus. This subcellular localization is similar to the subcellular localization of endogenous plakophilin-1 in HaCat cells (26) or primary keratinocytes (data not shown). The intense nuclear signal of plakophilin-1 in infected cells frequently obscured the plakophilin-1 signal at the cell-cell border. Therefore, images were captured that allowed visualization of the plasma membrane-associated protein, and as a consequence, the nuclear signal was often over exposed. The nuclear function of plakophilin-1 is unknown and was not further pursued in this study. Rather, we focused on the role of plakophilin-1 in the desmosome.
Expression of Plakophilin-1 in A431DE Cells Promotes Desmosome Assembly-We used immunofluorescence microscopy to examine the assembly of desmosomes in A431DE/pkp1 cells. In parental A431D and A431DE cells, both desmoplakin (Fig. 1, G and H) and desmoglein 2 (Fig.  1, J and K) were diffusely localized in the cytosol (desmoplakin) or on the cell surface (desmoglein) and did not assemble into characteristic punctate structures that are indicative of desmosomes. However, A431DE/ pkp1 cells recruited desmoplakin (Fig. 1I) and desmoglein 2 (Fig. 1L) to areas of cell-cell contact. The staining pattern obtained with these desmosomal components was punctate and resembled the staining pattern expected in cells that assemble desmosomes. To rule out the possibility that desmosome assembly was unique to a single clone of A431DE cells, multiple clones of A431DE cells were retrovirally infected to overexpress plakophilin-1. Each clone effectively assembled desmosomes (data not shown). In addition to immunofluorescence microscopy, we used electron microscopy to analyze desmosomes in A431DE/pkp1 cells.
The desmosomal structures in these cells were indistinguishable from desmosomes in A431 cells, which make numerous endogenous desmosomes (Fig. 2) (37). Thus, it is clear that forced expression of plakophilin-1 in A431DE cells allowed these cells to assemble functional, fully formed desmosomes.
Immunoblot analysis of whole cell lysates prepared from A431D, A431DE, and A431DE/pkp1 cells revealed that expression of the desmosomal components plakophilin-2, plakoglobin, desmoglein 2, and desmoplakin was not changed by forced expression of E-cadherin or plakophilin-1 (Fig. 3). The expression of desmocollin 2 was slightly increased following E-cadherin expression and further increased in response to plakophilin-1 expression, which may be due to stabilization of desmocollin 2 upon desmosome assembly. Thus, the A431D cells provide a unique system to investigate the role of plakophilin-1 in desmosome assembly.
To more closely examine the sequence of desmosome assembly in A431DE/pkp-1 cells, we performed a calcium switch experiment.  . Equal protein from each lysate was resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using antibodies specific for E-cadherin, plakophilin-1, plakophilin-2, desmoplakin, plakoglobin, desmoglein 2, and desmocollin 2. The arrow in the anti-E-cadherin (E-cad) panel denotes a pool of unprocessed E-cadherin likely present in the endoplasmic reticulum and Golgi apparatus.
A431DE/pkp-1 cells were grown in low calcium-containing medium overnight, and calcium was added to the cultures for the indicated times (Fig. 4). Plakophilin-1 and desmoplakin immunostaining of cells grown in low calcium was diffuse and remained diffuse after 5 min of calcium addition (Fig. 4, A and B). As soon as 10 min following the calcium addition, plakophilin-1 began to appear at sites of cell-cell contact (Fig.  4C), whereas desmoplakin remained diffuse (Fig. 4D). Thirty minutes following the addition of calcium to the cultures, plakophilin-1 was readily apparent at cell-cell borders, and desmoplakin was also present at cell borders (Fig. 4, E and F). These data suggest that plakophilin-1 is localized to the plasma membrane and desmoplakin is subsequently recruited.
Expression of Plakophilin-1 Deletion Mutants in A431DE Cells-To understand the mechanism whereby plakophilin-1 mediates desmosome formation, we wanted to determine 1) whether specific amino acid sequences in plakophilin-1 are involved in targeting it to the plasma membrane in the absence of pre-existing desmosomes and 2) whether specific amino acids in plakophilin-1 are necessary to promote the assembly of other desmosomal components into a desmosome. To accomplish this, we constructed a series of plakophilin-1 deletion mutants, expressed these mutants in A431DE cells, and examined the ability of these constructs to localize to the plasma membrane and/or promote the assembly of desmosomes. Desmosome assembly was assessed by redistribution of desmoplakin from a diffuse cytoplasmic localization into punctate structures. Fig. 5A shows a diagram of the plakophilin-1 mutants constructed for these studies. Plakophilin-1 comprises an amino-terminal "head domain," nine armadillo repeats (Fig. 5A, shaded boxes) and a short carboxyl-terminal tail domain. The epitope for the anti-plakophilin-1 monoclonal antibody (14B11), which was used to detect plakophilin-1 in cells, is also indicated. Pkp-1 C40 and Pkp-1 C10 are plakophilin-1 constructs in which the carboxyl-terminal 40 or 10 amino acids, respectively, have been deleted. Pkp-1 N34 and Pkp-1 N55 are plakophilin-1 mutants in which 34 or 55 amino acids have been deleted from the amino terminus of plakophilin-1. Plakophilins contain a conserved sequence in the amino-terminal head domain termed the HR2 domain (Fig. 5C) (38). The Pkp-1 N55 construct deletes the entire HR2 domain, whereas the Pkp-1 N34 construct deletes the amino-terminal portion of the HR2 domain, including the amino acid sequence LALPS, which is conserved among all three plakophilins. The Pkp-1 ⌬LALPS construct is a plakophilin-1 mutant lacking only the conserved LALPS sequence. The plakophilin-1 deletion constructs were expressed in A431DE cells by retroviral infection. To assess the level of protein expression, whole cell lysates were prepared, and equal protein content was separated by SDS-PAGE. Immunoblot analysis revealed that the constructs were expressed at nearly identical levels (Fig. 5B). A431DE/pkp-1 cells were grown on glass coverslips overnight in low calcium-containing medium. The medium was replaced with calciumcontaining medium (1.8 mM) for the indicated times, and cells were processed for immunofluorescence using anti-plakophilin-1 antibodies (A, C, and E) or anti-desmoplakin antibodies (B, D, and F). Note the accumulation of plakophilin-1 (arrows) at the cell borders after 10 min in high calcium-containing medium. Punctate desmoplakin was readily detectable after 30 min in high calcium-containing medium.
The Carboxyl Terminus of Plakophilin-1 Is Required for Its Localization at the Plasma Membrane-As expected, expression of wild-type plakophilin-1 in A431DE cells resulted in plakophilin-1 distribution to the plasma membrane and the nucleus (Fig. 6B). Desmoplakin staining in these cells revealed a punctate staining pattern, indicating that desmosomes were assembled at cell-cell borders (Fig. 6A). Expression of plakophilin-1, lacking 10 amino acids from the carboxyl terminus (Pkp-1 C10), also resulted in the mutant protein localizing to the plasma membrane and punctate desmoplakin staining at cell-cell borders (Fig.  6, C and D). Interestingly, the plakophilin-1 construct lacking 40 amino acids from the carboxyl terminus (Pkp-1 C40) was not localized to the plasma membrane but rather was exclusively localized to the nucleus (Fig. 6F). In addition, desmoplakin staining remained cytosolic, similar to the staining pattern observed in A431DE cells. These data suggest that amino acids present in the carboxyl terminus of plakophilin-1 are required for plakophilin-1 localization to the plasma membrane and that plakophilin-1 localization at the plasma membrane is required for subsequent desmoplakin recruitment.
The Amino-terminal Head Domain of Plakophilin-1 Is Required for Desmosome Assembly-Previous studies have indicated that the head domain of plakophilin-1 is responsible for targeting it to the desmosomal plaque in COS and HeLa cells, presumably via interactions with desmoplakin (24,26). Within the amino-terminal head domain, plakophilins share a region of high homology called the HR2 domain, which can be further divided into two smaller segments separated by a linker of poorly conserved sequence (12). Presently, the function of the HR2 domain is unknown. We constructed aminoterminal deletions of plakophilin-1 missing the first half of the HR2 domain (Pkp-1 N34) or the entire HR2 domain (Pkp-1 N55) (Fig. 4). Expression of either of these mutant plakophilin-1 constructs in A431DE cells resulted in plakophilin-1 localization at the plasma membrane and nucleus in a staining pattern very similar to fulllength plakophilin-1 (Fig. 7, compare D and F to B). However, desmoplakin was not recruited to cell borders in these cells but rather remained diffuse in the cytosol (Fig. 7, compare C and E to A). These data suggest that amino-terminal sequences in plakophilin-1 recruit desmoplakin to the plasma membrane.
To more closely examine the amino-terminal sequences that mediate desmoplakin recruitment, we constructed a plakophilin-1 mutant in which the conserved LALPS sequence in the amino-terminal half of the HR2 domain was deleted. The ⌬LALPS plakophilin-1 was localized to the nucleus and to cell-cell borders (Fig. 7, compare H to B). However, desmoplakin staining in these cells remained cytosolic (Fig. 7G). These data suggest that the LALPS sequence within the HR2 domain plays an important role in recruiting desmoplakin to sites of desmosome assembly.
To more closely examine desmosome assembly, we examined the staining pattern of desmocollin 2 and plakophilin-2 in A431/pkp-1, The black boxes represent the HR2 domain, and the shaded boxes represent the armadillo repeat domain. The monoclonal antibody 14B11 recognizes an epitope present between amino acids 56 and 103 of human plakophilin-1. B, whole cell lysates were prepared from A431DE cells expressing each plakophilin-1 mutant, and equal protein was separated by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose was immunoblotted using the anti-plakophilin-1 antibody 14B11. Molecular mass markers at the left represent 97 and 68 kDa. C, alignment of plakophilin-1, -2, and -3 amino-terminal head sequences. The HR2 sequence is underlined.
Plakophilin-2 staining in these cultures was unchanged regardless of plakophilin-1 expression. Endogenous plakophilin-2 was localized at sites of cell-cell contact; however, the plakophilin-2 was unable to assemble desmoplakin in A431DE/pkp N34 (Fig. 8E) cells and A431/ pkp-1 C40 cells (Fig. 8F). These experiments suggest that plakophilin-1 and plakophilin-2 function differently in desmosome assembly in A431DE cells. No change in plakoglobin localization was observed in these cultures. Plakoglobin was localized to sites of cell-cell contact where it co-localized with the exogenous E-cadherin. In addition, plakoglobin co-localized with the intracellular E-cadherin seen in Fig. 1  (data not shown).
Desmoplakin Interacts Directly with Amino Acids 1-34 of Plakophilin-1-Previous studies have demonstrated that the amino terminus of desmoplakin directly interacts with the amino-terminal head domain of plakophilin-1 (24,26). To more closely examine the interaction of plakophilin-1 with desmoplakin, we utilized in vitro binding assays and the yeast two-hybrid system. GST fusion proteins containing various plakophilin-1 domains were captured on glutathione-agarose, and the immobilized fusion proteins were then incubated with A431 cell lysate containing desmoplakin. GST fusion proteins that contain the entire plakophilin-1 head domain (GST head) were capable of interacting with desmoplakin from A431 cell lysates (Fig. 9A, lane 2). The GST fusion protein containing the plakophilin-1 head domain lacking the LALPS sequence (GST ⌬LALPS) weakly interacted with desmoplakin (Fig. 9A, lane 3). The GST fusion protein containing the plakophilin-1 head domain lacking the amino-terminal 34 amino acids (GST N34) did not associate with desmoplakin. As expected, the GST fusion proteins containing either the arm repeat domain of plakophilin-1 (GST arm repeats) or GST alone did not associate with desmoplakin (Fig. 9A, lanes  1, 4, and 5).
To further confirm the interaction between plakophilin-1 and desmoplakin, we utilized the yeast two-hybrid system (Fig. 9B). Plakophilin-1 bait constructs were co-transformed into yeast with a prey construct encoding amino acids 1-584 of desmoplakin fused to the Gal4 activation domain (24). As a control, the bait constructs were co-transformed with the empty vector to confirm that the plakophilin-1 bait  constructs were unable to activate reporter genes in the absence of an interaction. A positive interaction was assessed by the ability of the yeast to grow on SD medium lacking tryptophan, leucine, and histidine (Fig.  9B, ϪLeu/ϪTrp/ϪHis). As expected, the amino terminus of plakophilin-1 interacted with desmoplakin, whereas the arm repeat domain did not. A plakophilin-1 bait construct lacking the LALPS sequence interacts with desmoplakin, whereas a bait construct lacking the aminoterminal 34 amino acids was unable to interact with desmoplakin.
Plakophilin-1 mutants lacking the LALPS sequence were unable to recruit desmoplakin in A431DE cells; however, in vitro binding assays and yeast two-hybrid assays suggest that other amino acid sequences in the plakophilin-1 head domain mediate association with desmoplakin, and the LALPS sequence may regulate the strength of plakophilin-1/ desmoplakin interaction. These data indicate that plakophilin-1 amino acids 1-34 interact directly with the amino terminus of desmoplakin, and the interaction is mediated in part by the LALPS sequence present in plakophilin-1, -2, and -3.

DISCUSSION
Desmosomes are the major adhesive junctions in squamous epithelial tissues. However, the mechanisms that regulate the assembly of this junction are poorly understood. The plakophilin family of desmosomal plaque proteins has recently been shown to play important roles in assembly and maintenance of the desmosome (15,18,39). In this study, we sought to examine the role of plakophilin-1 in desmosome assembly. To accomplish this goal, we developed a plakophilin-1-negative cell culture system that would allow us to assemble desmosomes upon forced expression of plakophilin-1. A431D cells fail to express classical cadherins and were therefore defective in cell adhesion. Expression of E-cadherin restored cell-cell adhesion through the assembly of adherens junctions. However, desmosome assembly was not restored following the expression of E-cadherin alone. Expression of plakophilin-1, together with E-cadherin, allowed these cells to assemble desmosomes, suggesting that loss of plakophilin expression was one reason these cells were unable to assemble desmosomes. A separate study showed that plakophilin-2 coordinated desmosome assembly in HT1080 cells when these cells were also transfected with other necessary proteins such as plakoglobin (40). However, these authors did not investigate the functional domains of plakophilin-2 required for plakophilin-2 to localize to the plasma membrane or assemble junctions.
Parental A431 cells express plakophilin-2 and plakophilin-3 and lack plakophilin-1. The adhesion-defective A431D cells continue to express plakophilin-2, although at reduced levels compared with A431 cells, but do not express detectable plakophilin-3. Expression of E-cadherin in A431D cells did not restore plakophilin-3 expression nor did it increase expression of plakophilin-2. The endogenous plakophilin-2 in A431DE cells is either not competent to assemble desmosomes or is not expressed at levels sufficient to allow desmosome assembly. Interest-FIGURE 8. Plakophilin-1 does not assemble desmocollin without desmoplakin recruitment. A431/pkp-1 (A and D), A431/pkp-1 N34 (B and E ), and A431/pkp-1 C40 cells were grown on glass coverslips and processed for immunofluorescence microscopy. Cells were stained using antidesmocollin-2 (A-C ) and anti-plakophilin-2 (D-F ). Full-length plakophilin-1 expression resulted in punctate desmocollin localization, whereas plakophilin-1 mutants that were unable to recruit desmoplakin were also unable to organize desmocollin. ingly, endogenous plakophilin-2 was localized to cell-cell contacts and co-localized with the adherens junction component, ␤-catenin, in A431DE cells in the absence of desmosomes (data not shown). Whether or not plakophilin-2 plays any role in the adherens junction is not known. Expression of plakophilin-3 in A431DE cells also induced assembly of desmosomes in a manner similar to plakophilin-1 (data not shown). These data suggest that plakophilin-1 and -3 are functionally distinct from plakophilin-2. Future experiments will be aimed at understanding the functional differences among the individual plakophilins.
Previous studies examining the subcellular localization of plakophilin-1 have generated conflicting results. Investigators using transient expression of plakophilin-1, and fragments thereof, in COS, HeLa, and HaCat cells concluded that the amino-terminal head domain targeted it to the plasma membrane, presumably through interactions with desmoplakin, desmoglein, and/or plakoglobin (24,26). Data presented here and results from the Klymkowsky laboratory indicate that the carboxyl terminus of plakophilin-1 is necessary for plasma membrane localization (35). Interacting partners for the carboxyl terminus of plakophilin-1 that target it to the plasma membrane are unknown. However, plakophilin-1 localization to the plasma membrane is clearly independent of desmoplakin. In desmoplakin-null epidermis, plakophilin-1 was found to localize to desmosomal plaques (41). In addition, our data indicate plakophilin-1 is required for recruitment of desmoplakin to cell borders.
Earlier studies aimed at understanding the role of plakophilin-1 in desmosomes have used cell lines that constitutively assemble fully functional desmosomes (e.g. COS, HeLa, and HaCat cells). In these studies, the authors investigated the ability of plakophilin-1 fragments to be recruited into fully assembled desmosomes at the plasma membrane. Our system has the advantage of allowing us to distinguish desmosome assembly from recruitment to the plasma membrane. Amino-terminal plakophilin-1 mutants were able to localize to the plasma membrane; however, desmoplakin was not recruited to punctate structures. The plakophilin-1 amino terminus appears to be critical for desmoplakin recruitment in this cell culture system, and the LALPS sequence in the HR2 domain likely contributes to the interaction of plakophilin-1 with desmoplakin. Interestingly, in A431DE cells, plakophilin-1 amino-terminal mutants can localize to the plasma membrane, even though they do not recruit desmoplakin. This further suggests that plakophilin localization at the plasma membrane precedes both desmoplakin membrane association and intermediate filament attachment to the desmosomal plaque. In vivo studies have suggested that plakophilins may be important in recruiting desmoplakin to desmosomes. For example, plakophilin-2-null mice fail to assemble fully functional cell junctions in the heart, and these mice die because of defects in heart morphogenesis (18). Ultrastructural examination of these mice revealed that the desmosomal plaque was assembled; however, desmoplakin was not associated with the junctions and thus the junction was not connected to the cytoskeleton. Our studies are the first to define the molecular mechanism whereby plakophilin-1 mediates the connection of desmoplakin to the desmosome, thus promoting the important linkage of the desmosomal plaque to the intermediate filament cytoskeleton.