The Armadillo Family Protein p0071 Is a VE-cadherin- and Desmoplakin-binding Protein*

Cathárine C. CalkinsDagger , Bridgett L. HoepnerDagger , Christine M. LawDagger , Matthew R. NovakDagger , Shannon V. SetzerDagger , Mechthild Hatzfeld§, and Andrew P. KowalczykDagger

From the Dagger  Departments of Dermatology and Cell Biology and the Emory Skin Diseases Research Center, Emory University School of Medicine, Atlanta, Georgia 30322 and the § Department of Biochemistry and Pathobiology, Medical Faculty of University of Halle, 06097 Halle/Saale, Germany

Received for publication, June 8, 2002, and in revised form, October 31, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

p0071, a member of the armadillo protein family, localizes to both adherens junctions and desmosomes in epithelial cells and exhibits homology to the adherens junction protein p120 and the desmosomal protein plakophilin-1. p0071 is also present at dermal microvascular endothelial intercellular junctions and colocalizes with VE-cadherin, an endothelium-specific cadherin that associates with both actin and intermediate filament networks. To define the role of p0071 in junction assembly, p0071 was tested for interactions with other components of the endothelial junctional complex. In transient expression assays, p0071 colocalized with and formed complexes with both VE-cadherin and desmoplakin. Deletion analysis using the yeast two-hybrid system revealed that the armadillo repeat domain of p0071 bound directly to VE-cadherin. Site-directed mutagenesis experiments demonstrated that p0071 and p120 bound to the same region on the cytoplasmic tail of VE-cadherin and that overexpression of p0071 could displace p120 from intercellular junctions. In contrast to VE-cadherin, desmoplakin was found to associate with the non-armadillo head domain of p0071. Cotransfections and triple-label immunofluorescence analysis revealed that VE-cadherin colocalization with desmoplakin in transfected COS cells required p0071, suggesting that p0071 may couple VE-cadherin to desmoplakin. Based on previous findings that both VE-cadherin and desmoplakin play central roles in vasculogenesis, these new results suggest that p0071 may play an important role in endothelial junction assembly and in the morphogenic events associated with vascular remodeling.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vascular endothelial cells form a continuous cell layer along the wall of blood vessels and participate in a wide range of biological processes that regulate vascular function. One important function of the endothelial lining is to control the movement of solutes and fluid from the vascular space to the tissues (1, 2). The loss of cell adhesion between endothelial cells results in tissue edema, inflammation, and poor wound healing. In addition, these cell-cell contacts also function as plasma membrane attachment sites for cytoskeletal networks, such as actin and intermediate filaments, thereby influencing cell shape and tissue integrity (3-5). It is now apparent that intercellular junctions are macromolecular complexes that integrate informational cues derived from cell adhesion events with intracellular signaling pathways that regulate cell proliferation, apoptosis, and gene expression (6, 7).

The mechanisms by which adhesive interactions are established between vascular endothelial cells have been studied extensively (8). Endothelial cells express a unique cadherin, VE-cadherin (where VE is vascular endothelial; cadherin-5), which plays a central role in the establishment and maintenance of endothelial monolayer integrity and angiogenesis (9, 10). Significantly, endothelial cells assemble unique junctional complexes that couple VE-cadherin to both actin and intermediate filament networks (11), although the precise nature of these protein interactions remains only partially characterized. Like other classical cadherins such as E-cadherin, VE-cadherin associates with the cytoplasmic proteins beta -catenin and plakoglobin, members of the armadillo protein family (12). Both plakoglobin and beta -catenin bind directly to the cytoplasmic domain of VE-cadherin, thus providing a link to the actin cytoskeleton through their association with alpha -catenin, a vinculin homolog that plays a key role in linking the cadherin complex to the actin cytoskeleton. In addition, plakoglobin (but not beta -catenin) provides a link from VE-cadherin to the intermediate filament network by recruiting desmoplakin, an intermediate filament-binding protein, to intercellular junctions (11).

Desmoplakin, a member of the plakin family of cytoskeletal cross-linking proteins, is an abundant constituent of the desmosomal plaque and functions to couple intermediate filaments to membrane-associated adhesive junctions (13, 14). Previous studies demonstrated that the carboxyl terminus of desmoplakin interacts directly with intermediate filament networks (15-18), whereas the amino terminus of desmoplakin associates with desmosomes through interactions with plakoglobin (19, 20) and the plakophilins (21-23). In addition to playing fundamental roles in desmosome assembly and epidermal integrity (24), desmoplakin also plays important roles in adherens junction formation (24, 25). Interestingly, vascular and lymphatic endothelial cells, which do not assemble true desmosomes, also express desmoplakin (26, 27). In cultured human umbilical vascular endothelial cells and primary dermal microvascular endothelial cells, desmoplakin was shown to localize to intercellular junctions (11, 28). Recent studies also indicate that desmoplakin plays an important role in vasculogenesis (29), underscoring the need to determine how desmoplakin interacts with other components of endothelial intercellular junctions.

An exciting development over the last several years is the realization that intercellular junctions contain a newly identified subset of armadillo family proteins termed the p120/plakophilin subfamily (30, 31). In general, p120 assembles into actin-associated adherens junctions, whereas the plakophilins assemble into intermediate filament-based desmosomes. Interestingly, one member of the subfamily (termed p0071) assembles into both adherens junctions and desmosomes (32). This dual targeting of p0071 to both types of junctions raises the possibility that p0071 plays a role in the molecular cross-talk that occurs between different intercellular junctions, thus influencing a range of cellular events that are regulated by either the actin or intermediate filament cytoskeleton. To understand how p0071 might perform specific roles in junction assembly, we initiated an investigation into the subcellular localization of p0071 in dermal microvascular endothelial cells. In addition, we tested p0071 for interactions with other intercellular junction proteins using transient transfection of COS-7 and 293 cells, co-immunoprecipitation, and yeast two-hybrid analysis. The data indicate that p0071 associates with both adherens junction and desmosomal proteins and that p0071 is a cadherin- and desmoplakin-binding protein.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cDNA Constructs

p0071-- A cDNA construct encoding full-length human p0071 was generated as described previously (32) and subcloned into the NotI site of the pKS vector (Stratagene, La Jolla, CA). Expression in eukaryotic cells was performed with the FLAG-tagged expression system pCMV-Tag4 or pCMV-Tag2 vector, containing a carboxyl- or an amino-terminal FLAG epitope, respectively (Stratagene). p0071-(1-1016)-FLAG was constructed using the 3.0-kb NotI/SalI fragment from the pKS vector containing the p0071 insert and subcloned into pCMV-Tag4. Similarly, p0071-(1-553)-FLAG was generated from a 1.8-kb NotI/PstI fragment and subcloned into pCMV-Tag4. p0071-(554-1016)-FLAG was generated from a 1.8-kb PstI/SalI fragment and subcloned into the pCMV-Tag2 vector. To generate full-length FLAG-tagged p0071 (p0071-(1-1192)-FLAG), a point mutation was introduced to eliminate the stop codon using the primer pair 5'-GAC TCA TGG GTG GCG GAT CAA GCT TCC CAA CAG AGG and 5'-CCT CTG TTG GGA AGC TTG ATC CGC CAC CCA TGA GTC and the QuikChangeTM site-directed mutagenesis kit (Stratagene). The resulting 3.5-kb BamHI/HindIII fragment was subcloned into the pCMV-Tag4 vector. The p0071-(1-508)-FLAG construct was generated by PCR using a p0071 5'-primer (5'-CGC GGA TCC AGA GGA ATG CCA GCT CCT GAG CAG GCC), which generates a BamHI site (underlined) at the 5'-end of p0071-(1-508)-FLAG, and the p0071-(1-508)-FLAG 3'-primer (5'-TTT AAC GTC GAC CTA CTT ATC GTC GTC ATC CTT GTA ATC GGT GCC ATC ATC AGC CGG CAC), which generates the FLAG tag (italicized) and a 3'-SalI site (underlined). p0071-(509-992)-FLAG was also generated using a p0071 5'-primer (5'-CGC GGA TCC AGA GGA ATG ACA AGA TCC CCA TCA ATA GAC), which generates a BamHI site (underlined) at the 5'-end of the construct, and the p0071-(509-992)-FLAG 3'-primer (5'-CCG CTC GAG CTA CTT ATC GTC GTC ATC CTT GTA ATC CCA TAA TGT ATT CAA GAC CTG GGC), which generates the FLAG tag (italicized) and a 3'-XhoI site (underlined). p0071-(992-1192)-FLAG was generated by PCR using a p0071 5'-primer (5'-CGC GGA TCC AGA GGA ATG CAA TAT CGG GAC CTC CGG AGC), which generates a BamHI site (underlined) at the 5'-end of p0071-(992-1192)-FLAG, and the p0071-(992-1192)-FLAG 3' primer (5'-CCG CTC GAG CTA CTT ATC GTC GTC ATC CTT GTA ATC CAC CCA TGA GTC TGG GGA CCC), which generates a FLAG tag (italicized) and a 3'-XhoI site (underlined). The PCR products were subcloned into donor vectors of the Creator system (Clontech, Palo Alto, CA), and PCR-generated regions were sequenced to verify that errors were not introduced into the coding sequence. Expression of all constructs was further verified by Western blotting and immunofluorescence. A diagram illustrating the different domains of the recombinant proteins used in this work is shown in Fig. 1.


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Fig. 1.   Schematic diagram of the cDNA constructs used in this study. A, p0071 constructs encoding full-length (FL) p0071 or various domains of the protein were constructed and tested in mammalian expression assays and in the yeast two-hybrid system. PDZ-BD, PDZ domain-binding domain. B, shown are full-length VE-cadherin and its cytoplasmic domain. Site-directed mutagenesis was used to alter the region encoding amino acids 652-654 to E652A/M653A/D654A (652-654EMD-AAA). JMD, juxtamembrane domain; CBD, catenin-binding domain. C, constructs encoding full-length human desmoplakin with a carboxyl-terminal Myc epitope tag (DP.myc) or the mutant lacking the amino-terminal domain (DPDelta N) were used for mammalian expression assays. DPNTP (DP-NTP) is the 584-amino acid region of the desmoplakin amino-terminal polypeptide and was used in yeast two-hybrid experiments. aa, amino acids. Refer to "Experimental Procedures" for additional information.

VE-cadherin-- Full-length cDNA encoding human VE-cadherin, subcloned into the pECE vector using the SV40 promoter, was generated as described previously (9, 33). The cDNA clone encoding the cytoplasmic domain of human VE-cadherin was generated by PCR as described previously (34). The cytoplasmic domain was recovered from the TA vector (Invitrogen) by restriction digestion with EcoRI/XhoI and subcloned into the Creator system donor vector. To generate the triple-alanine mutation in the VE-cadherin juxtamembrane domain (E652A/M653A/D654A), we used the QuikChange site-directed mutagenesis kit and the primer pair 5'-GAG GGC GGC GGC GCG GCG GCC ACC ACC AGC TAC G and 5'-CGT AGC TGG TGG TGG CCG CCG CGC CGC CGC CCT C.

Desmoplakin-- cDNAs encoding full-length desmoplakin with a carboxyl-terminal Myc epitope tag, the first 584 amino acids of desmoplakin (DPNTP, for desmoplakin N-terminal polypeptide) (35), and a truncated desmoplakin polypeptide lacking the amino-terminal domain (DPDelta N) (15, 16) were generously provided by Dr. K. J. Green (Fig. 1).

Yeast Two-Hybrid Assays

Yeast two-hybrid vectors encoding the Gal4 DNA-binding domain (pLP-GBK) or transcription activation domain (pLP-GAD) were purchased from Clontech. The p0071 constructs described above were subcloned from the Creator system donor vector into the Creator system acceptor vector pLP-GBK. The VE-cadherin cytoplasmic domain was subcloned into the Creator system donor vector, followed by recombination subcloning into the acceptor vector pLP-GAD. All constructs were then verified by sequencing. To assay for interactions between proteins, 5-10 µg of plasmid DNA was transformed into the yeast strain AH109 (Clontech) using LiAc, and double transformants were selected by growth in the absence of leucine and tryptophan. Expression of the HIS and ADE reporter genes was analyzed by monitoring colony growth on plates lacking histidine, adenine, leucine, and tryptophan.

Cell Lines and Transfections

The monkey kidney cell line COS-7 and the human embryonic kidney cell line 293 were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and penicillin/streptomycin/amphotericin B (Invitrogen). Primary cultures of human dermal microvascular endothelial cells were purchased from the Emory Skin Diseases Research Center (Core B) and cultured in MDCB131 (Invitrogen) supplemented with 10% fetal bovine serum, 100 µg/ml cAMP (Sigma), 1 µg/ml hydrocortisone (Sigma), 10 ng/ml epidermal growth factor (Intergen Co., Purchase, NY), and penicillin/streptomycin/amphotericin B (36). For transient transfection experiments, a subclone of COS-7 cells (COS-7-20) was transfected by calcium phosphate precipitation. The cells were fixed and processed for immunofluorescence analysis after 24-48 h.

Antibodies and Immunofluorescence Microscopy

The distribution of p0071 in microvascular endothelial cells and transiently transfected COS-7 cells was analyzed by immunofluorescence. Cells grown on coverslips were rinsed in phosphate-buffered saline and fixed in methanol at -20 °C for 4 min. Alternatively, cells were fixed in 3.7% paraformaldehyde in phosphate-buffered saline, followed by permeabilization in 0.5% Triton X-100 in phosphate-buffered saline. VE-cadherin was monitored using mouse anti-cadherin-5 monoclonal antibody (Transduction Laboratories, Lexington, KY). Desmoplakin was detected using rabbit polyclonal antibody NW6, directed against the desmoplakin carboxyl-terminal domain (37), or the Myc epitope tag (Bethyl Laboratories, Inc., Montgomery, TX). Endogenous p0071 was analyzed with a rabbit polyclonal antibody (32), whereas FLAG-tagged p0071 was detected by an anti-FLAG tag monoclonal antibody (Stratagene). Appropriate species cross-absorbed secondary antibodies conjugated to rhodamine, fluorescein, or various Alexa Fluors (Molecular Probes, Inc., Eugene, OR) were used for dual-label immunofluorescence. Control experiments were carried out routinely to verify that fluorescence was not due to secondary antibody cross-reactivity. A Leica DMR-E fluorescence microscope equipped with narrow band-pass filters and a Hamamatsu Orca camera was used. Images were captured and processed using Open Lab software (Improvision, Inc., Lexington, MA).

Immunoprecipitation and Western Blot Analysis

Immunoprecipitation was carried out as described previously (19, 21). Briefly, cells were scraped into Tris-buffered saline containing 0.5% Triton X-100, vortexed, and subjected to centrifugation at 14,000 × g. Antibody M2 (directed against the FLAG tag) conjugated to agarose beads (Sigma) was incubated with the cell lysate for 1 h at 4 °C. Immune complexes were captured by centrifugation, and the beads were washed four times in Tris-buffered saline containing 0.5% Triton X-100 for 10 min with gentle rotation at 4 °C. Immune complexes were released by incubation in reducing SDS-PAGE sample buffer at 95 °C and then separated on 7.5% acrylamide gels and transferred to nitrocellulose according to standard protocols. Membranes were washed and incubated with horseradish peroxidase-coupled secondary antibodies, and bound antibodies were visualized by chemiluminescence reagent (Amersham Biosciences).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

p0071 Is Localized at Intercellular Junctions in Primary Cultures of Dermal Microvascular Endothelial Cells-- The precise localization of p0071 in different cell types and the mechanisms by which p0071 incorporates into junctions have been only partially characterized. p0071 has been shown previously to localize at cell-cell contacts in epithelial compartments of various tissues (32, 38) and was found to be expressed and assembled into intercellular junctions of a variety of cultured epithelial cell lines, including HeLa, A431, and HaCaT (32). Using previously characterized antibodies specific for p0071 (32), the distribution of p0071 was examined and compared with that of VE-cadherin in cultured primary human microvascular endothelial cells. In confluent monolayers of primary microvascular endothelial cells, p0071 localized at intercellular junctions (Fig. 2A) and colocalized with VE-cadherin (Fig. 2, C and D). These results are consistent with previous studies indicating that p0071 is broadly expressed and assembled into intercellular junctions in a variety of cell types and tissues (32).


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Fig. 2.   p0071 localization in dermal microvascular endothelial cells. Primary cultures of dermal microvascular endothelial cells were processed for immunofluorescence using a rabbit polyclonal antibody (Ab) directed against p0071 (32) and a monoclonal antibody directed against VE-cadherin (Cad-5). p0071 was detected predominantly at intercellular junctions (A). No staining was observed in the absence of the anti-p0071 primary antibody (B). Dual-label immunofluorescence demonstrated colocalization of p0071 (C) with VE-cadherin (D).

p0071 Binds VE-cadherin-- The localization of p0071 at cell-cell borders suggested that this protein may interact with other components within the intercellular junctions of microvascular endothelial cells, such as VE-cadherin. To begin the analysis of p0071 binding partners, a FLAG-tagged p0071 construct (p0071-(1-1016)-FLAG) was generated. When expressed in COS-7 cells, the p0071-(1-1016)-FLAG protein localized to both the cytoplasm and intercellular junctions (Fig. 3B). To determine whether p0071 interacts with VE-cadherin, p0071-(1-1016)-FLAG was cotransfected with VE-cadherin (Fig. 3, C and D). p0071-(1-1016)-FLAG (Fig. 3C) and VE-cadherin (Fig. 3D) exhibited extensive colocalization, primarily at cell-cell borders. Moreover, in cells coexpressing p0071-(1-1016)-FLAG and VE-cadherin, p0071 recruitment to intercellular junctions was dramatically increased. The central armadillo domain of p0071, but not the non-armadillo head domain, colocalized with VE-cadherin when cotransfected into COS-7 cells (data not shown), suggesting that the armadillo domain of p0071 binds to VE-cadherin. To test for direct interactions between p0071 and VE-cadherin, full-length p0071 and the cytoplasmic domain of VE-cadherin were examined for direct binding using the yeast two-hybrid system. Direct interactions between proteins were assayed by growth in the absence of histidine and adenine (Fig. 3E). In agreement with the COS-7 cell transient transfection assays, growth in the absence of histidine and adenine was consistently observed when yeast were cotransformed with plasmids encoding p0071 in the DNA-binding domain vector and the VE-cadherin cytoplasmic domain in the transcription activation domain vector (Fig. 3E). Similar results were obtained using beta -galactosidase as a reporter for direct interactions (data not shown), indicating that p0071 and VE-cadherin are direct binding partners. To further define the domain of p0071 that binds to VE-cadherin, deletion mutants of p0071 (p0071-(1-508), p0071-(509-992), and p0071-(324-1192)) were generated and analyzed for the ability to bind to the VE-cadherin cytoplasmic domain (Fig. 3E). The results indicate that the central armadillo domain of p0071 (amino acids 509-992) is necessary and sufficient for binding to the cytoplasmic domain of VE-cadherin. These observations are consistent with the hypothesis that, like the related proteins ARVCF (armadillo repeat gene deleted in velo cardio facial syndrome), delta -catenin, and p120, p0071 is a cadherin-binding protein.


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Fig. 3.   p0071 colocalizes with VE-cadherin in COS-7 cell transient transfections and binds directly to VE-cadherin in the yeast two-hybrid system. A cDNA construct encoding p0071-(1-1016)-FLAG was expressed in COS-7 cells and detected using an antibody (Ab) directed against the FLAG epitope tag. FLAG staining was not observed in untransfected COS-7 cells (A). In transfected (T) cells, the p0071-(1-1016)-FLAG polypeptide was observed at intercellular junctions and in other regions of the cytoplasm and plasma membrane (B). In cells cotransfected with VE-cadherin (VE-cad), both cadherin (C) and the p0071-(1-1016)-FLAG polypeptide (D) accumulated at intercellular junctions and exhibited extensive colocalization. Yeast two-hybrid experiments were carried out to test for direct interactions using growth in the absence of histidine and adenine as a reporter for direct interactions between VE-cadherin and multiple p0071 polypeptides (E). p0071 polypeptides were expressed as fusions with the Gal4 DNA-binding domain and tested for interactions either with the empty activation domain (AD) vector or with VE-cadherin in the Gal4 transactivation domain vector (VE-cadcyto). Constructs encoding the p0071 central armadillo domain consistently interacted with the VE-cadherin cytoplasmic tail. FL, full-length.

p0071 Binds to the Juxtamembrane Domain of VE-cadherin-- Recent studies have identified a core region within the juxtamembrane domain of E-cadherin responsible for binding to the armadillo protein p120 (39). The juxtamembrane domain sequence is highly conserved among type I and II classical cadherins (31). To test whether p0071 and p120 compete for binding sites at cell-cell junctions, full-length FLAG-tagged p0071 was transiently expressed in COS-7 cells. The cells were then processed for immunofluorescence microscopy using antibodies directed against the FLAG epitope tag to detect exogenously expressed p0071 along with antibodies directed against endogenous p120. In untransfected cells, p120 was detected in a continuous pattern along cell-cell contacts (Fig. 4, A and B). In contrast, p120 staining was dramatically reduced at the borders of adjacent cells expressing the FLAG-tagged p0071 protein (Fig. 4, C and D), suggesting that p0071 occupies membrane-binding sites in a mutually exclusive manner with p120. To determine whether p0071 and p120 bind to the same region of VE-cadherin, a triple-alanine substitution at residues 652-654 of the VE-cadherin juxtamembrane domain was generated and tested for the ability to bind to plakoglobin, p0071, or p120 in the yeast two-hybrid system. Protein-protein interactions were monitored by growth in the absence of histidine and adenine. Although plakoglobin bound to the mutated VE-cadherin cytoplasmic domain, neither p0071 (Fig. 4E) nor p120 (data not shown) interacted with the mutated cytoplasmic tail of VE-cadherin. Similar results were obtained when full-length VE-cadherin with the E652A/M653A/D654A mutation was expressed in COS-7 cells; the VE-cadherin mutant failed to colocalize with p0071 as observed by dual-label immunofluorescence (data not shown). These data indicate that p0071 binds to the juxtamembrane domain of VE-cadherin and that it may compete with p120 for binding to this highly conserved region of cadherins.


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Fig. 4.   p0071 displaces p120 from intercellular junctions and binds directly to the p120-binding site on the VE-cadherin cytoplasmic domain. Full-length FLAG-tagged p0071 was expressed in COS-7 cells, and the distribution of endogenous p120 was monitored. After 48 h, cells were fixed and processed for immunofluorescence to detect FLAG-tagged p0071 (A and C) and endogenous p120 (B and D). Note that in adjacent cells that were both transfected with p0071-FLAG (C), p120 staining was absent (D, arrows). The putative p120-binding site within the VE-cadherin cytoplasmic domain was mutated (E652A/M653A/D654A (VE-cad652-654EMD-AAA)) and tested for interactions with plakoglobin or p0071 in the yeast two-hybrid system (E). The mutated VE-cadherin tail interacted with plakoglobin, which binds to the distal catenin-binding domain of VE-cadherin, but failed to interact with p0071. Ab, antibody.

p0071 Forms Complexes with the Intermediate Filament-binding Protein Desmoplakin-- Vascular endothelial cells assemble unique intercellular junctions that are thought to couple VE-cadherin not only to actin microfilaments, but also to the vimentin intermediate filament cytoskeleton. Several studies have demonstrated that the intermediate filament-binding protein desmoplakin is expressed in endothelial cells and assembled into endothelial intercellular junctions (26, 27). Furthermore, desmoplakin and VE-cadherin have been shown to co-assemble into intercellular junctions of cultured endothelial cells (11, 28), and recent studies indicate that desmoplakin plays a central role in blood vessel formation during development (29). Based on these previous reports and on the observation that p0071 localizes to both adherens junctions and desmosomes (32), p0071 was tested for interactions with desmoplakin. p0071-(1-1016)-FLAG and full-length Myc-tagged desmoplakin were coexpressed in COS-7 cells (Fig. 5). In transfected cells, dual-label immunofluorescence demonstrated that p0071 colocalized with full-length desmoplakin (Fig. 5, A and B). The two proteins were often detected in a filamentous staining pattern and in perinuclear aggregates, resulting from the alignment of desmoplakin along intermediate filament networks when expressed in cultured cells (15, 16). Furthermore, exogenous p0071 targeted to both desmosomes and adherens junctions in transiently transfected A431 cells (data not shown). In contrast to plakophilin-1 (40), p0071 did not appear to enhance the recruitment of full-length desmoplakin to intercellular junctions in transfected COS-7 cells (Fig. 5, A and B).


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Fig. 5.   p0071 colocalizes with full-length desmoplakin, but not with the desmoplakin mutant lacking the amino-terminal domain. p0071-(1-1016)-FLAG was cotransfected in COS-7 cells with full-length Myc-tagged desmoplakin (DP.myc; A and B) or the desmoplakin mutant lacking the amino-terminal domain (DPDelta N) (C and D). p0071-(1-1016)-FLAG (B) exhibited extensive colocalization with full-length desmoplakin (A), but failed to colocalize with DPDelta N (C and D). Note that the filamentous pattern of both desmoplakin polypeptides results from the binding of the desmoplakin carboxyl-terminal domain to endogenous intermediate filament networks (15). Ab, antibody.

To determine the domain of desmoplakin required for colocalization with p0071, the Myc-tagged desmoplakin mutant lacking the amino-terminal domain (DPDelta N-Myc) was coexpressed with p0071-(1-1016) in COS-7 cells (Fig. 5, C and D). As with full-length desmoplakin, DPDelta N aligned along intermediate filament networks in a filamentous pattern (Fig. 5D). However, p0071 (Fig. 5C) failed to colocalize with DPDelta N (Fig. 5D), suggesting that the desmoplakin amino-terminal domain is required for interactions with p0071. To further investigate the ability of these proteins to interact, co-immunoprecipitation studies were performed (Fig. 6). Protein complexes were captured from transiently transfected 293 cells using anti-FLAG tag antibody M2-agarose beads. Full-length desmoplakin and DPDelta N were detected by Western blot analysis using the Myc epitope tag. Full-length desmoplakin consistently co-immunoprecipitated with p0071, whereas the amino-terminally truncated desmoplakin failed to co-precipitate with p0071 (Fig. 6B). Parallel Western blots verified that p0071 (Fig. 6C) was captured by the immunoprecipitation procedure and that both full-length desmoplakin and DPDelta N were expressed in each transfection (Fig. 6A). Together with the colocalization data shown in Fig. 5, these experiments suggest that p0071 interacts with the amino-terminal region of desmoplakin.


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Fig. 6.   p0071 co-immunoprecipitates with desmoplakin. p0071-(1-1016)-FLAG was cotransfected in 293 cells with either full-length Myc-tagged desmoplakin (DP.myc) or the desmoplakin mutant lacking the amino-terminal domain (DPDelta N). Both full-length desmoplakin (FL-DP) and DPDelta N were detected in whole cell lysates of transfected 293 cells using an antibody directed against the c-Myc epitope tag (A). When p0071-(1-1016)-FLAG was captured from 293 cell lysates using antibody M2-agarose beads, full-length desmoplakin (but not DPDelta N) was detected in the complex with p0071-(1-1016)-FLAG (B). Expression of the p0071 protein was demonstrated by Western blotting using a rabbit polyclonal antibody directed against p0071 (C). IP, immunoprecipitate.

To determine whether p0071 binds directly to the amino-terminal domain of desmoplakin, interactions between p0071 and the desmoplakin amino-terminal polypeptide DPNTP (35) were tested using the yeast two-hybrid system. Growth in the absence of histidine and adenine was used as a reporter for protein interactions (Fig. 7A). As reported previously (19), DPNTP interacted directly with plakoglobin, but failed to interact with the VE-cadherin cytoplasmic domain. We were also unable to detect interactions between DPNTP and p120 (data not shown). However, direct interactions between multiple domains of p0071 and DPNTP were consistently observed by yeast two-hybrid analysis (Fig. 7A). To further investigate p0071 domains that interact with desmoplakin, the p0071 head or arm domain was coexpressed with full-length desmoplakin in COS-7 cells (Fig. 7, B-E). As expected, full-length desmoplakin aligned along intermediate filament networks (Fig. 7, B and D). The amino-terminal head domain of p0071 consistently colocalized with desmoplakin in a filamentous pattern (Fig. 7, B and C). In contrast, the armadillo domain of p0071 was unable to associate with desmoplakin when transiently expressed in COS-7 cells (Fig. 7, D and E). Fig. 7F summarizes colocalization results using COS-7 cells transfected with various domains of p0071 and full-length desmoplakin. The results of extensive cotransfection analysis in COS-7 cells suggest that the non-armadillo head domain of p0071 associates with the amino-terminal domain of desmoplakin. However, as indicated by the yeast two-hybrid analysis, it is likely that other domains of p0071 outside of the head domain also contribute to the interaction with the amino-terminal domain of desmoplakin.


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Fig. 7.   The p0071 head domain interacts with desmoplakin. cDNA constructs encoding various domains of p0071 were tested for interactions with the desmoplakin amino-terminal domain using the yeast two-hybrid system. Growth in the absence of histidine and adenine was used as a reporter for protein interactions (A). Each construct was tested for self-activation, and none of the p0071 constructs activated transcription as determined by growth in the absence of histidine and adenine (A and data not shown). In addition to the two-hybrid analysis, the head (B and C) and the central (D and E) armadillo domains of p0071 were tested for colocalization with desmoplakin in COS-7 cell transient transfections. Desmoplakin was often detected along intermediate filaments and colocalized extensively with the head domain of desmoplakin (B and C), but failed to colocalize with the arm domain of p0071 (D and E). These results, along with the results of additional colocalization experiments, are summarized in F. FL, full-length; VE-cadcyto, VE-cadherin cytoplasmic domain; Ab, antibody; ND, not determined; PDZ-BD, PDZ domain-binding domain.

p0071 Couples VE-cadherin to Desmoplakin-- Desmoplakin and VE-cadherin have been shown to colocalize in human dermal microvascular endothelial cells. However, the association is not through a direct linkage, but rather through a complex with plakoglobin (11). To examine if p0071 could perform a role similar to that of plakoglobin and link VE-cadherin to desmoplakin, triple-label immunofluorescence studies of transiently transfected COS-7 cells were undertaken. Full-length Myc-tagged desmoplakin and VE-cadherin were coexpressed in COS-7 cells in the absence (Fig. 8, A-D) or presence (Fig. 8, E-H) of p0071-(1-1192)-FLAG. In the absence of p0071, VE-cadherin failed to colocalize with desmoplakin (Fig. 8, A-D). In contrast, VE-cadherin exhibited striking colocalization with desmoplakin in the presence of p0071 (Fig. 8, E-H). These data suggest that p0071 plays a role in linking the endothelial cadherin to the vimentin intermediate filament network, although other proteins are likely to participate in this linkage.


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Fig. 8.   VE-cadherin colocalizes with desmoplakin in the presence of p0071 in transfected COS-7 cells. Full-length Myc-tagged desmoplakin and full-length human VE-cadherin were transiently coexpressed in the absence (A-D) or presence (E-H) of p0071 in COS-7 cells. In the absence of p0071, desmoplakin (A) was detected in a filamentous pattern, and VE-cadherin (C) was diffuse at the cell surface, with some VE-cadherin accumulating at cell-cell contacts. In contrast, in the presence of p0071, desmoplakin (E), p0071-FLAG (F), and VE-cadherin (G) exhibited extensive colocalization (H).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies demonstrated that the amino-terminal domain of desmoplakin interacts with the armadillo family protein plakoglobin, which in turn links desmoplakin to the endothelial cadherin, VE-cadherin (11). The results of the present study indicate that the armadillo protein p0071 is also assembled into intercellular junctions of dermal microvascular endothelial cells and that VE-cadherin recruits p0071 to cell-cell borders. In addition, expression of p0071 resulted in the colocalization of VE-cadherin and desmoplakin in transient expression assays. Using the yeast two-hybrid system, p0071 was shown to be a direct binding partner for both VE-cadherin and the amino-terminal domain of desmoplakin. These findings suggest that, in endothelial intercellular junctions, p0071 functions as a protein that links VE-cadherin to the intermediate filament cytoskeleton.

p0071 is most closely related to members of the p120 subfamily of armadillo proteins. This group of proteins also includes the plakophilins, a family of desmosomal and nuclear proteins that are directly involved in the recruitment of desmoplakin to desmosomes in epithelial cells (21-23). Although amino acid sequence analysis has revealed similarities between p0071 and plakophilin-1-3 (32, 41), there are distinct differences in the tissue distributions of these proteins. The plakophilins are primarily expressed in epithelial tissues that assemble desmosomes, although cells lacking desmosomes have also been described to contain plakophilin-1-3 (41-43). In contrast, p0071 exhibits a broad tissue distribution and is unique among the p120/plakophilin family members in that p0071 is the only member of this subfamily described to date that assembles into both adherens junctions and desmosomes. However, the distribution of the various plakophilins in endothelial cells is not well characterized, and the manner in which vascular endothelial cells utilize p120/plakophilin family members to assemble adhesive junctions suited to the regulation of vascular permeability is poorly understood.

Although p0071 interacts with desmoplakin in a manner similar to the plakophilins, amino acid sequence analysis indicated that p0071 is more closely related to the p120 family members delta -catenin and ARVCF than to plakophilin-1-3 (41). Interestingly, p0071 exhibited strong interactions with the classical cadherins VE-cadherin (Fig. 3) and E-cadherin (data not shown).1 Furthermore, p0071 interacted with the VE-cadherin juxtamembrane domain in a region that is highly conserved in the classical cadherins. Mutational analysis of VE-cadherin indicated that a triple-amino acid substitution of the juxtamembrane domain abrogated both p0071 (Fig. 4) and p120 (data not shown) binding. When expressed transiently in COS-7 cells, p0071 also displaced p120 from intercellular junctions. The fact that a triple-alanine substitution abrogated binding to both p120 and p0071, combined with the observation that p0071 displaced p120 from cell-cell junctions, strongly suggests that p0071 may bind to the p120-binding site on classical cadherins and compete for p120 binding to cadherins. This prediction is similar to models proposed for other members of the p120 family (45, 46). A major challenge will be to discern how cells use this complex repertoire of armadillo proteins to modulate the functional state of adhesive contacts. In the case of endothelial cells, one possibility is that, by increasing p0071 expression, the cadherin-associated pool of p120 would be decreased, thereby shifting VE-cadherin association from actin-based to intermediate filament-based. Recent studies by Shasby et al. (47) indicate that, under quiescent conditions, VE-cadherin is tightly associated with vimentin and that this interaction is dramatically reduced upon exposure of endothelial cells to histamine. It will be interesting to determine what signaling pathways regulate p0071 expression and how this correlates with endothelial barrier function and, more broadly, with the balance between endothelial cell adhesion and cell migration.

An important finding in this study is that p0071 appears to physically link VE-cadherin to desmoplakin. As shown in Fig. 8 and as described previously (11), VE-cadherin did not colocalize with desmoplakin when coexpressed transiently in COS-7 cells. However, in the presence of full-length p0071, VE-cadherin and desmoplakin were found to colocalize extensively. Both yeast two-hybrid and COS-7 cell transient transfection experiments demonstrated that the central armadillo domain of p0071 bound directly to VE-cadherin. Multiple domains of p0071 were found to bind to desmoplakin in yeast two-hybrid experiments, but only the head domain of p0071 colocalized with full-length desmoplakin in COS-7 cells (Fig. 7). These results suggest that desmoplakin may bind to multiple sites on p0071, but that the head domain is the primary binding domain in the context of a cadherin complex. Consistent with this interpretation, neither the head nor arm domain of p0071 alone was sufficient to drive colocalization between VE-cadherin and desmoplakin in triple-transfection experiments in COS-7 cells (data not shown). Based on the ability of these two domains of p0071 to interact differentially with desmoplakin and VE-cadherin, these observations suggest that p0071 may actually link VE-cadherin to desmoplakin, with the p0071 head domain binding to desmoplakin and the p0071 arm domain interacting with VE-cadherin. In this manner, p0071 appears unique among the other members of the p120/plakophilin family. For example, p120 does not appear to play any direct role in linking classical cadherins to the actin cytoskeleton. Likewise, plakophilin-1 binds strongly to desmoplakin, but a direct role for plakophilin-1 in linking the desmosomal cadherins to desmoplakin has not been demonstrated. It is likely that plakophilin-1 plays a more important role in lateral associations among desmosomal components (21, 22). Recently, plakophilin-2 was found to bind to a number of intercellular junction proteins, including desmoplakin and several members of the desmosomal cadherin family (23). The detailed manner in which the plakophilins contribute to junction assembly remains unclear, and understanding how these proteins contribute to junction assembly is an important area of investigation.

The suggestion that p0071 functions as a linker between VE-cadherin and desmoplakin predicts that this armadillo family protein may have some overlapping function with plakoglobin. Like p0071, plakoglobin is widely expressed and assembles into both adherens junctions and desmosomes (48, 49). Unlike beta -catenin, which associates predominantly with adherens junctions, plakoglobin is thought to facilitate cross-talk between adherens junctions and desmosomes (50). The hypothesis that p0071 may be able to substitute for some of these functions of plakoglobin is supported by knockout experiments in which various components of the endothelial adhesion complex were ablated. VE-cadherin-null embryos were found to undergo normal vasculogenesis, but then failed to properly remodel the vascular system and ultimately died at embryonic day 9.5 (51, 52). Conventional approaches to knock out the desmoplakin gene result in early embryonic lethality (53), well before initiation of vasculogenesis, thus precluding the ability to assess the role of desmoplakin in vascular development. However, an important role for desmoplakin in blood vessel morphogenesis was revealed more recently using a tetraploid aggregation approach to rescue the early embryonic lethality of the desmoplakin-null mutation (29). Like VE-cadherin, desmoplakin was found to be required for blood vessel remodeling, and embryos lacking desmoplakin exhibited markedly fewer capillaries. In contrast, plakoglobin-null animals exhibit heart and skin defects, but a vascular phenotype has not been described for the plakoglobin-knockout mice (54, 55). Because plakoglobin plays a role in linking desmoplakin to VE-cadherin, it is likely that, in the plakoglobin-null animals, other armadillo family proteins compensate for plakoglobin in linking VE-cadherin and desmoplakin. We have been unable to demonstrate a role for beta -catenin in this linkage. However, based on the ability of p0071 to bind to both VE-cadherin and desmoplakin and to mediate VE-cadherin and desmoplakin colocalization, it is possible that p0071 may compensate for some plakoglobin functions in blood vessels in the plakoglobin-null animals.

In addition to the junctional binding partners for p0071 described here and by Hatzfeld et al.,1 p0071 has also been shown to interact with presenilin (56) and several PDZ (PSD-90/Dlg/ZO-1) motif-containing proteins (57, 58). The carboxyl-terminal domain of p0071 (along with ARVCF and delta -catenin) terminates with a PDZ domain-binding motif (30, 31) and interacts with the PDZ motif-containing protein PAPIN (59) and with the LAP protein Erbin (57, 58). LAP family proteins such as Erbin and Scribble play important roles in the development of cell polarity during morphogenic events in Caenorhabditis elegans and Drosophila (44). The ability of p0071 to interact with cadherins and PDZ motif-containing proteins strongly suggests that p0071 and the other PDZ domain-binding proteins ARVCF and delta -catenin function not only as structural components of intercellular junctions, but also as scaffolds for the recruitment of signaling molecules involved in cell polarity and cell migration. It will be of significant interest to determine how p0071 may integrate VE-cadherin-mediated adhesion with the function of signaling proteins involved in cell polarity, particularly in the context of vascular morphogenesis and the loss of endothelial barrier function associated with inflammation.

    ACKNOWLEDGEMENTS

We thank Drs. K. J. Green, A. B. Reynolds, and E. Dejana for antibodies and cDNA reagents that made this work possible.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01 AR48266-01, R03 AR47147, K01 AR002039, HP30 AR042687, and T32 AR007587; the Dermatology Foundation; and Deutsche Forschungsgemeinschaft Grant Hal 791/3-3.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Dermatology, Emory University School of Medicine, Atlanta, GA 30322. Tel.: 404-727-8517; Fax: 404-727-5878; E-mail: akowalc@emory.edu.

Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M205693200

1 M. Hatzfeld, K. J. Green, and H. Sauter, J. Cell Science, in press.

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