The Journal of Cell Biology, Volume 149, Number 1, April 3, 2000 209–222
The Rockefeller University Press, 0021-9525/2000/04/209/14 $5.00
The Function of Plakophilin 1 in Desmosome Assembly and Actin
Mechthild Hatzfeld, Christof Haffner, Katrin Schulze, and Ute Vinzens
Molecular Biology Group of the Medical Faculty, University of Halle, 06097 Halle/Saale, Germany
multigene family, is a protein with dual localization in
the nucleus and in desmosomes. To elucidate its role in
desmosome assembly and regulation, we have analyzed
its localization and binding partners in vivo. When over-
expressed in HaCaT keratinocytes, plakophilin 1 local-
ized to the nucleus and to desmosomes, and dramati-
cally enhanced the recruitment of desmosomal proteins
to the plasma membrane. This effect was mediated by
plakophilin 1’s head domain, which interacted with des-
moglein 1, desmoplakin, and keratins in the yeast two-
hybrid system. Overexpression of the armadillo repeat
domain induced a striking dominant negative pheno-
Plakophilin 1, a member of the armadillo type with the formation of filopodia and long cellular
protrusions, where plakophilin 1 colocalized with actin
filaments. This phenotype was strictly dependent on a
conserved motif in the center of the armadillo repeat
domain. Our results demonstrate that plakophilin 1
contains two functionally distinct domains: the head do-
main, which could play a role in organizing the desmo-
somal plaque in suprabasal cells, and the armadillo re-
peat domain, which might be involved in regulating the
dynamics of the actin cytoskeleton.
cell adhesion • cell motility
keratinocytes • desmoglein • armadillo •
Desmosomes are adhering junctions that anchor interme-
diate filaments to sites of cell–cell contact. Biochemically,
they are distinct, but are related to the adherens junctions
that anchor actin filaments. They contain two types of
transmembrane proteins of the cadherin superfamily, the
desmogleins (Dsgs) and desmocollins (Dscs). There are at
least three different desmogleins, and three different des-
mocollin genes (Dsg1-3 and Dsc1-3) that are differen-
tially expressed (Koch and Franke, 1994; Schmidt et al.,
1994). Whereas Dsg2 and Dsc2 are ubiquitously expressed
in all cells that possess desmosomes, the expression of
Dsgs1 and 3 and Dscs1 and 3 is restricted to stratified epi-
thelia (Schmidt et al., 1994; Garrod et al., 1996). Expres-
sion patterns of isoforms of the desmosomal cadherins
overlap, and individual desmosomes can contain more
than one isoform (North et al., 1996). Clustering of desmo-
somal cadherins and desmosome formation depends on
both Dsgs and Dscs (Chitaev and Troyanovsky, 1997).
The intracellular domains of the desmosomal cadherins
associate with a number of plaque proteins that establish
the link to the intermediate filament system (Troyanovsky
et al., 1993, 1994a, 1996; Mathur et al., 1994; Chitaev et al.,
1996; Kowalczyk et al., 1996; Witcher et al., 1996). Plako-
globin and desmoplakin are essential components of the
plaque. Plakoglobin associates with both types of desmo-
somal cadherins and binds to several Dsg and Dsc iso-
forms (Mathur et al., 1994; Troyanovsky et al., 1994a,b,
1996; Chitaev et al., 1996; Wahl et al., 1996; Witcher et al.,
1996). The binding of plakoglobin to E-cadherin is a pre-
requisite for desmosome formation, and its COOH termi-
nus is involved in regulating desmosome size (Ruiz et al.,
1996; Lewis et al., 1997; Palka and Green, 1997). More-
over, a signaling role in the Wnt pathway, which is similar
to that of
-catenin and the Drosophila
, has been reported (Karnovsky and Klymkowsky,
1995; Rubenstein et al., 1997). Direct interactions between
desmosomal cadherins and desmoplakin have been re-
ported only in vitro (Smith and Fuchs, 1998), and it ap-
pears that plakoglobin is necessary to link these proteins
in vivo (Kowalczyk et al., 1996, 1997). Desmoplakin binds
to intermediate filaments through its COOH-terminal do-
main and connects desmosomes to the cytoskeleton (Stap-
penbeck et al., 1993, 1994; Kouklis et al., 1994; Bornslae-
ger et al., 1996; Meng et al., 1997). Thus, intermediate
filaments seem to be linked to the plasma membrane
A ddress correspondence to M. Hatzfeld, Molecular Biology Group of the
Medical Faculty, University of Halle, Magdeburger Strasse 18, 06097
Halle/Saale, Germany. Tel.: 49-345-557-4422. Fax: 49-345-557-4421. E-mail:
Abbreviations used in this paper:
-terminal polypeptide; Dsc, desmocollin; Dsg, desmoglein;
arm, armadillo; DP-NTP, des-
The Journal of Cell Biology, Volume 149, 2000
through a linear sequence of interactions between ker-
atins, desmoplakin, plakoglobin, and the cytoplasmic tail
A dditional components of the desmosomal plaque are
plakophilins 1, 2, and 3, and p0071 (Hatzfeld et al., 1994;
Heid et al., 1994; Hatzfeld and Nachtsheim, 1996; Mertens
et al., 1996; Bonne et al., 1999; Schmidt et al., 1999). These
proteins contain a central domain that consists of a series
of 45 amino acid repeats (arm repeats), and are members
of the p120
family of armadillo (arm) -related proteins
(Reynolds et al., 1994; Hatzfeld and Nachtsheim, 1996;
Daniel and Reynolds, 1997). Plakophilin 1 is a major com-
ponent of desmosomes from stratified and complex epi-
thelia, and it is predominantly expressed in the suprabasal
layers (Kapprell et al., 1988). It binds to keratins in vitro
(Kapprell et al., 1988; Hatzfeld et al., 1994; Smith and
Fuchs, 1998), but the significance of this interaction in vivo
has not yet been established. More recently, plakophilin 1
has been described as a widespread nuclear protein that is
also expressed in nondesmosome-bearing cells, where it
accumulated in the nucleoplasm (Schmidt et al., 1997;
Klymkowsky, 1999). The function of plakophilin 1 in the
nucleus remains unknown so far. A n essential role in des-
mosome organization and stability has been suggested re-
cently on the basis of a genetic skin disease. Patients lack-
ing plakophilin 1 suffer from a skin fragility syndrome.
Desmosomes in their skin are small and poorly formed
with widening of keratinocyte intercellular spaces and per-
turbed desmosome/keratin filament interactions (McGrath
et al., 1997). Desmoplakin was found predominantly cyto-
plasmic in these patients, suggesting a role for plakophilin
1 in organizing suprabasal desmosomes. These findings
point to an essential role of plakophilin 1 in establishing
stable cell contacts, desmosomal plaque size, and organi-
zation. In a recent paper, a direct interaction between the
terminus and plakophilin 1 and a role of
plakophilin 1 in recruiting desmoplakin to the membrane
was described (Kowalczyk et al., 1999). It was proposed
that this interaction may be important for clustering of
desmosomal components through lateral interactions.
To learn more about the function of plakophilin 1, we
have analyzed its function in desmosome assembly in
more detail. Wild-type plakophilin 1 recruited endogenous
desmosomal proteins into the plaque when overexpressed
in keratinocytes. This function was mediated by its head
domain, and we show that this domain interacts with Dsg1,
desmoplakin, and keratins. In contrast, the arm repeat do-
main had a dominant negative phenotype: it promoted for-
mation of filopodia and long cellular protrusions, where it
colocalized with actin filaments. Deletion of a conserved
motif in the center of the arm domain abolished the ability
of plakophilin 1 to modulate cellular morphology and to
associate with actin. Our data suggest that plakophilin 1 is
involved in regulation of desmosome assembly as well as
dynamics of the actin cytoskeleton.
Materials and Methods
RNA Isolation and Plasmid Constructs
RNA was prepared according to the LiCl/urea extraction method (A uf-
fray and Rougeon, 1980), and cDNA s were synthesized by reverse tran-
scriptase–PCR, with expand reverse transcriptase and expand high fidelity
polymerase (Roche Diagnostics). Suitable restriction sites for cloning
were included in the primer sequences. PCR products were either directly
cloned into the expression vectors or first ligated into the PCRII vector
using the TOPO TA cloning kit (Invitrogen BV ). A ll PCR products were
Prokaryotic expression was performed in the pRSET A vector, which
includes an NH
-terminal His tag (Invitrogen Corp.). Expression in eu-
karyotic cells was performed with the following vectors: pCMV 5 (with
-terminal T7 tag; A ndersson et al., 1989), pCMV script (without tag;
Stratagene), pOPRSV (without tag; Stratagene), and pEGFP (with NH
terminal GFP tag; CLONTECH Laboratories). V ectors for the expression
of GA L4 fusion proteins in yeast were the pBD and pA D vectors (Strat-
agene) and the pA S2-1 and pGA D424 vectors (CLONTECH Laborato-
ries). Fig. 1 a gives an overview of the plakophilin 1 constructs used in this
study. The intracellular domains of human Dsg1, Dsg2, and Dsg3, and
Dsc1a, 1b, Dsc2a, 2b, and Dsc3a, 3b were amplified by reverse tran-
scriptase–PCR from HaCaT cell RNA and cloned into the pGA D424 vec-
tor. Dsg1 domains IA (amino acids 568–643), CS (amino acids 644–764),
and the Dsg-specific domain (Dsg; amino acids 765–1,049) were amplified
by PCR and cloned into the pGA D424 vector. Human keratins 8, 18, 6,
and 17 and their individual domains in pA D and pBD have been de-
scribed elsewhere (Schnabel et al., 1998). The complete coding sequence
-actin was amplified by PCR from HeLa cell RNA and cloned
into pGA D424.
In Vitro Mutagenesis
In vitro mutagenesis was performed to delete the 5–amino acid motif
ENCMC (amino acids 452–456) in the plakophilin 1 arm repeat domain.
The reaction was performed with the QuickChange site-directed mu-
tagenesis kit (Stratagene). The deletion was verified by sequence analysis.
Plasmids were transformed into the yeast strain Y RG2 (Stratagene) by
electroporation. Double transformants were grown on plates lacking leu-
cine and tryptophane. Expression of the His reporter gene was analyzed
on plates lacking histidine in addition to leucine and tryptophane. lacZ re-
porter gene expression was analyzed in the colony lift filter assay and
quantitated using the ONPG (o-nitrophenyl-
strate as described in the yeast protocols handbook (CLONTECH Labo-
Recombinant Protein Purification and
The plakophilin 1 head and arm repeat domains in pRSET were ex-
pressed in BL21 DE3 bacteria and purified under denaturing conditions
on Ni-NTA resin (Qiagen). The purified protein fragments were used for
immunization of rabbits.
Cell Lines, Wound Healing Assay, and Transfections
HeLa and HaCaT (Boukamp et al., 1988) cells were routinely cultured in
DME supplemented with 10% FBS. Normal human epidermal kerati-
nocytes cells and keratinocyte medium were obtained from Promocell.
Wound healing assays were performed with HaCaT cells grown to conflu-
ency, and a wound was inserted by scraping. Cells were analyzed 24 h after
wounding. For transient transfection experiments, cells were plated 12–16 h
before transfection. Cells were transfected either by the calcium phos-
phate precipitation method (5prime
liposomal transfection reagent (Roche Biochemicals). Cells were fixed
and processed for immunofluorescence analysis after 20–44 h.
3prime, Inc.) or using the DOSPER
Antibodies and Immunofluorescence Microscopy
Cells grown on coverslips were rinsed in PBS and fixed in methanol at
C for 10 min, followed either by acetone treatment for 1 min or by
treatment in 0.2% Triton X -100 in PBS for 20 min. A lternatively, cells
were fixed in 3.7% formaldehyde in PBS freshly prepared from paraform-
aldehyde and permeabilized in 0.2% Triton in PBS. Cells were washed in
PBS and incubated with 1% BSA in PBS before antibody application.
Plakophilin 1 and its fragments were detected by the polyclonal rabbit
sera against the head and repeat domains. A lternatively, plakophilin 1
Hatzfeld et al.
Function of Plakophilin 1
head and repeat domains, which were expressed in the pCMV 5 vector,
were detected with a T7 mA b (Novagen, Inc.). For double labeling the
following antibodies were used: antidesmoplakin 1 and 2, DP1&2 2.15 or
DP1&2 mix (2.15
U114 were obtained from Progen. A ntiplakoglobin and anti–Pan-cadherin
were from Sigma Chemical Co.; the keratin antibody RCK107 was from Dr.
F. Ramaekers (University Hospital, Rotterdam, The Netherlands).
Secondary antibodies were donkey anti–rabbit or anti–mouse coupled
to Cy2 or Cy3 (Jackson ImmunoResearch Laboratories, Inc., through Di-
anova) or A lexa 488 goat anti–mouse or goat anti–rabbit IgG (Molecular
Probes, Inc.). A ctin filaments were visualized by incubation with FITC- or
TRITC-labeled phalloidin (Sigma Chemical Co.).
Microscopy was carried out with a Nikon Eclipse E600 microscope with
narrow band filters.
2 DG3.10, anti-Dsc3 Dsc3
Laser Scanning Microscopy
Cells processed for immunofluorescence microscopy were analyzed using
a Zeiss LSM 510 laser scanning microscope equipped with a helium-neon
and an argon laser and a Plan-A pochromat 63
wavelengths were 488 nm for A lexa 488 and 543 nm for Cy3. The used de-
tection filters were BP505-530 for A lexa 488 and LP560 for Cy3. Fluores-
cence was recorded using the multitracking procedure to get complete
separation of the fluorescence signals.
Western Blot Analysis
Total protein extracts were prepared by adding SDS sample buffer heated
C to the cell culture dishes. Y east protein extracts were prepared
according to the SDS-urea method in the presence of the complete pro-
tease inhibitor cocktail tablets (Roche Diagnostics), as described in the
Y east Protocols Handbook. Samples were separated on 8 or 10% acryl-
amide gels and transferred to nitrocellulose. Filters were blocked in 5%
nonfat dry milk in TBS with 0.05% Tween 20. Primary antibodies were
applied for 2 h at room temperature or overnight at 4
washed and incubated with alkaline phosphatase–coupled secondary anti-
bodies, and bound antibodies were visualized either with the CDP-Star
chemiluminescence reagent (Tropix) or with NBT/BCIP (Boehringer In-
gelheim Bioproducts). In some experiments, the ECL detection system
(A mersham Pharmacia Biotech) was used.
C. Filters were
Plakophilin 1 Constructs and Antibodies
To address the function of plakophilin 1 in desmosome as-
sembly and structure, we studied targeting of its domains
in epithelial cells and analyzed its direct binding partners
in the yeast two-hybrid system. Fig. 1 a summarizes the
plakophilin 1 constructs tested in transfection assays and
in the yeast two-hybrid system. The GFP constructs of all
domains were analyzed in parallel with nontagged or T7-
tagged constructs to verify that the GFP tag did not inter-
fere with intracellular sorting.
Rabbit polyclonal antibodies against the plakophilin 1
-terminal domain and the arm repeat domain were
generated and tested for their specificity by Western blot-
ting on total cellular extracts. Fig. 1 b shows that both anti-
bodies reacted with a single band of 80 kD, demonstrating
that they did not cross-react with related proteins, such as
plakophilin 2 (96 kD) and 3 (86 kD), p120
forms of 96–115 kD), or p0071 (130 kD). The majority of
the protein was detected in the insoluble protein fraction.
Wild-Type Plakophilin 1 and Its Head Domain
Associate with Desmosomes and Enhance Recruitment
of Desmosomal Proteins to the Plasma Membrane in
Since plakophilin 1 has been described as a protein with
dual localization in desmosomes and in the nucleus
(Schmidt et al., 1997), we analyzed intracellular targeting
of the protein after overexpression. A ttempts to obtain
clonal cell lines that strongly overexpress plakophilin 1, or
its head, or repeat domain, thus far, have been unsuccess-
ful. This may be due to the phenotype that is caused by
strong overexpression of plakophilin 1 or its fragments
(see below). Therefore, we have used transient transfec-
tion studies to analyze the function of plakophilin 1 and its
domains in a cellular context. Wild-type plakophilin 1,
which was overexpressed in HaCaT keratinocytes, local-
ized predominantly to the nucleus and to cell borders in
confluent monolayers (Fig. 2 a), which is in agreement
with the intracellular localization of the endogenous pro-
tein (Schmidt et al., 1997). The balance between nuclear
localization and plasma membrane association appeared
similar in transfected and nontransfected cells. Double la-
Figure 1. (a) Schematic representation of plakophilin 1 (PKP-1)
and its deletion mutant constructs used in this study. The PKP-1
head comprises amino acids 1–286; PKP-1 ?C1, amino acids 1–213;
PKP-1 ?C2, amino acids 1–168; PKP-1 ?N1, amino acids 70–286;
and PKP-1 ?N2, amino acids 147–286. The PKP-1 arm repeat do-
main contains amino acids 287–726, and the PKP-1 headless frag-
ment contains amino acids 224–726. The ?ENCMC fragments
carry an additional internal deletion comprising amino acids 422–
426. (b) Specificity of plakophilin head and arm repeat domain
antibodies. Total protein extracts (lanes 1 and 5) as well as Tri-
ton-soluble (lanes 2 and 6), high salt soluble (lanes 3 and 7), and
insoluble fractions (lanes 4 and 8) of HaCaT keratinocytes were
separated on 8% SDS gels, transferred to nitrocellulose, and
probed with the plakophilin head (lanes 1?–4?) and repeat do-
main antibodies (lanes 5?–8?). Both antibodies reacted with a sin-
gle band of 80 kD in total cell extracts (lanes 1? and 5?). The ma-
jority of the protein was in the insoluble fraction (lanes 4? and 8?).
The Journal of Cell Biology, Volume 149, 2000
beling with desmoplakin antibodies revealed a strong in-
crease of endogenous desmoplakin at the plasma membrane
in the transfected cells compared with nontransfected cells
(Fig. 2 a
To identify the domains that target plakophilin 1 to
desmosomes and to the nucleus, the head and the arm re-
peat domains of plakophilin 1 were expressed separately.
Whereas the arm repeat domain colocalized with the actin
cytoskeleton (see below), the head domain, like the full-
length protein, was detected in the nucleus and along the
cell periphery (Fig. 3, a–e) and strongly enhanced recruit-
ment of desmoplakin to the plasma membrane (Fig. 3 a
Costaining for other desmosomal proteins revealed that
recruitment of Dsg (Fig. 3 b
lesser extent, plakoglobin (Fig. 3 d
The amount of recruited protein roughly correlated with
the size of the membrane pool of plakophilin 1. In cells
with a large membrane pool of plakophilin 1, recruited
proteins were detected continuously along the plasma
membrane (Fig. 3, a–d’). In other cells, the typical punc-
tate pattern of individual desmosomes was retained (Fig.
4). Costaining for keratins showed colocalization of a
small pool of these proteins to plasma membrane patches
), Dsc (Fig. 3 c
) was also enhanced.
) and, to a
enriched for plakophilin 1 (Fig. 3, e and e
demonstrate that plakophilin 1 is able to recruit various
desmosomal plaque proteins to the plasma membrane,
and that this effect is mediated by its head domain.
In addition to its plasma membrane association, the
head domain showed very strong nuclear localization. Sur-
prisingly, some desmoplakin, Dsg, and Dsc were also de-
tected in the nucleus, suggesting that plakophilin 1 coim-
ported a fraction of these proteins into the nucleus.
To analyze the recruitment of desmosomal plaque pro-
teins to the plasma membrane in more detail, we used la-
ser scanning microscopy on HaCaT cells expressing the
head domain of plakophilin 1. Whereas plakophilin 1 and
E-cadherin staining overlapped only very little (Fig. 4 a), a
high degree of overlap was found between plakophilin 1
and desmoplakin staining (Fig. 4 b), demonstrating that
the major portion of overexpressed plakophilin 1 head do-
main does not localize to adherens junctions. These data
indicate that plakophilin 1–mediated recruitment of pro-
teins occurs primarily in desmosomes. To investigate the
effect of the recruitment on desmosome size and number,
we quantitated desmoplakin staining at cell borders by
scanning along plasma membrane stretches (Fig. 4, b
, arrows). The data are displayed as fluorescence inten-
sity profiles below the corresponding image. The number
and size of the peaks within these profiles were signifi-
cantly higher when recorded along cell borders of two
transfected cells (Fig. 4 b
), compared with the cell border
between transfected and nontransfected cells (Fig. 4 b
A ssuming that each peak represents a desmosome or a
group of desmosomes, these data indicate that plakophilin
1–mediated recruitment of plaque proteins might result in
the generation and enlargement of desmosomes.
To determine the region within the plakophilin 1 head
domain responsible for desmosome association, several
fragments were constructed (Fig. 1 a). Whereas all of them
were still able to associate with desmosomes in HaCaT
cells (Fig. 5), only the
capable of significantly enhancing the recruitment of en-
dogenous desmoplakin (Fig. 5) and other desmosomal
plaque proteins (data not shown) to the cell membrane.
C2 fragment did not recruit endogenous desmo-
somal proteins, although it associated with desmosomes.
A major portion of the
cytoplasmic (Fig. 5). A ll fragments were still able to enter
the nucleus, but nuclear targeting was more efficient with
C2 constructs. These experiments show that
at least one region mediating plasma membrane targeting
of plakophilin 1 as well as a signal directing nuclear local-
ization is retained in all head deletion constructs.
). These data
C1 fragments were
C2 fragments remained
Wild-Type Plakophilin 1 and Its Head Domain
Accumulate in the Nucleus of HeLa Cells
When overexpressed in simple epithelial HeLa cells, pla-
kophilin 1 accumulated in the nucleus, but was not re-
cruited to the plasma membrane (Fig. 6 a), suggesting that
the nuclear function of plakophilin 1 is conserved among
all cells, whereas its function in stabilizing intercellular
junctions is restricted to certain cell types. The lack of des-
mosome association of plakophilin 1 in HeLa cells may be
due either to the lack of an appropriate binding partner
Figure 2. Expression of full-length plakophilin 1 in HaCaT cells.
Cells were fixed in methanol 30 h after transfection, and double
labeled with the plakophilin 1 head domain antibody (a) and the
desmoplakin 2.15 antibody (a?). In confluent monolayers, plako-
philin 1 accumulated in the nucleus and at the plasma membrane,
and desmoplakin was recruited to the plasma membrane of the
transfected cells. Labeling of endogenous desmoplakin in non-
transfected adjacent cells was comparatively weak. Bar, 20 ?m.
Hatzfeld et al.
Function of Plakophilin 1
such as cell type–specific Dsg and/or Dsc isoforms, or to
different regulatory mechanisms that control modification
and/or assembly of desmosomal proteins in HeLa cells. In
addition to its nuclear localization, plakophilin 1 was
found along actin filaments, as demonstrated by double la-
beling with phalloidin (Fig. 6, a and a
Transfection studies with the plakophilin 1 head domain
in HeLa cells showed almost exclusive nuclear localization
of the fragment (Fig. 6 b). Decoration of actin filaments
was not observed, suggesting that the binding site for di-
rect or indirect actin filament association is located in the
arm repeat domain (see below). Desmoplakin staining was
strong in the transfected cells, but it appeared in a punc-
tate pattern in the cytoplasm rather than in membranes
(Fig. 6 b
in mitotic cells (Fig. 6 b
proteins have been internalized in vesicles. Nontrans-
fected, nonmitotic cells revealed the punctate staining pat-
tern along the plasma membrane, which is typical of des-
mosomes (Fig. 6 b
, arrows). The extent of cytoplasmic
staining of desmoplakin seemed to correlate with plako-
philin 1 expression levels. The cytoplasmic staining could
be due to internalization of desmosomes and/or enhanced
synthesis and assembly of desmosomal proteins in the cy-
toplasm (Demlehner et al., 1995). The
N2 (not shown) constructs showed almost ex-
clusive nuclear localization with the same effect on des-
moplakin distribution as described above.
). A similar distribution of desmoplakin was seen
arrowheads), where desmosomal
C1 (Fig. 6 c),
Figure 3. Expression of the plakophilin 1 head
domain in HaCaT cells. Plasmid DNA s encod-
ing the plakophilin 1 head domain in pCMV 5
were transfected into HaCaT cells. Cells were
fixed in methanol and extracted in Triton X -100
and double labeled with the plakophilin 1 head
domain antibody (a–e) and antibodies against
desmoplakin (a?), desmoglein (b?), desmocollin
(c?), plakoglobin (d?), and keratin (e?). In a and
b, single transfected cells are in the center; ar-
rows in c–e denote the plasma membranes be-
tween two transfected cells. The plakophilin 1
head domain was found in the nucleus and at
cell borders; it enhanced the recruitment of des-
moplakin (a?), desmoglein (b?), desmocollin (ar-
rows, c?) and, to a lesser extent, of plakoglobin
(arrows, d?) to the plasma membrane. Keratins
colocalized with plakophilin 1 at the borders of
transfected cells (arrows, e?). Bar, 20 ?m.
The Journal of Cell Biology, Volume 149, 2000
Since wild-type plakophilin 1 decorated actin filaments
in transfected HeLa cells, we analyzed plakophilin 1 local-
ization more carefully in nontransfected cells to distin-
guish whether this was an artifact due to heavy overexpres-
sion that disturbed the intracellular sorting mechanisms, or
whether it was connected to a novel function of plakophi-
lin 1. In a wound healing experiment with HaCaT cells,
colocalization of actin filaments and plakophilin 1 was ob-
served at the tips of cellular protrusions (Fig. 6, d and d
suggesting a role for plakophilin 1 in regulating actin fila-
ment organization. A ssociation with stress fibers was not
The Plakophilin 1 Head Domain Binds to
Desmoglein1, Desmoplakin, and Keratins in the
Yeast Two-hybrid Assay
Plakophilin 1 has been shown to bind to Dsg1, Dsc1a, and
desmoplakin in in vitro overlay assays (Smith and Fuchs,
1998), and to Dsg1 and desmoplakin in the two-hybrid sys-
tem (Kowalczyk et al., 1999). To localize the binding sites
of these proteins in plakophilin 1, the cytoplasmic domains
of Dsgs1-3 and Dscs1a,b-3a,b and the NH
desmoplakin were tested in the yeast two-hybrid system.
From all the desmosomal cadherins, only Dsg1 interacted
with the plakophilin 1 head domain (Fig. 7 a) and with all
head domain deletion constructs (Fig. 7, b and c), although
C2 constructs appeared somewhat less effi-
cient in reporter gene activation, suggesting that the Dsg1
binding site was not completely retained in these con-
structs. Desmoplakin binding was retained in the
C2 fragments (Fig. 7 b), but not in the
fragments (Fig. 7 c), demonstrating that desmoplakin
binds close to the NH
terminus of plakophilin 1. These re-
sults suggest that desmoplakin and Dsg1 do not compete
for the same binding site in the plakophilin 1 head.
Since plakophilin 1 and plakoglobin (Troyanovsky et al.,
1993; Chitaev and Troyanovsky, 1997) both bind to des-
moplakin and Dsg1, we wanted to analyze if these two
proteins provide alternative links between the cadherins
and the cytoskeleton, or if plakophilin 1 stabilizes the Dsg-
plakoglobin-desmoplakin interaction through additional
protein interactions. Therefore, we determined the plako-
philin 1 binding site in the Dsg1 cytoplasmic domain. The
plakophilin 1 head domain interacted with the intact Dsg1
cytoplasmic domain, the Dsg
N1 and N2
CS domain and, although
pressing the plakophilin 1 head domain. (a) Cells were stained
with the plakophilin 1 head domain antibody (red fluorescence)
and the anti–Pan-cadherin antibody (green fluorescence). Over-
lay of both fluorescence signals showed only little overlap along
the plasma membrane, demonstrating that the major portion of
plakophilin 1 does not localize to adherens junctions. (b) Cells
were stained with the plakophilin 1 head domain antibody (green
Laser scanning microscopy analysis of HaCaT cells ex-
fluorescence) and an antidesmoplakin antibody (red fluores-
cence). A high degree of colocalization is visible along cell bor-
ders of transfected cells (arrows), demonstrating that plakophilin
1 is recruited primarily to desmosomes. To test whether the size
or the number of desmosomes is affected by this recruitment, we
recorded fluorescence intensities in the desmoplakin channel by
scanning along two defined plasma membrane stretches (arrows,
). The scan results are displayed as intensity profiles be-
low the corresponding image. In the profile recorded along a cell
border between two transfected cells (b
ber are increased when compared with the profile recorded along
a cell border between a transfected and a nontransfected cell
). Note that photobleaching during the scan accounts for the
slight differences in the two fluorescent pictures. Bars, 10
), the peak size and num-
Hatzfeld et al.
Function of Plakophilin 1
Figure 5. Expression of plakophilin 1 head domain fragments in
HaCaT cells. Plasmid DNA s encoding the GFP-tagged plakophi-
lin 1 head domain fragments were transfected into HaCaT cells,
and their ability to recruit desmoplakin to the cell membrane was
analyzed by immunofluorescence. Whereas desmosome localiza-
tion was found with all fragments, nuclear staining was strong
only with ?N2 and ?C2. ?N1 and ?C1 showed reduced nuclear
staining. Desmoplakin recruitment to the plasma membrane, as
revealed by continuous labeling along the cell periphery, was en-
hanced with ?N1, ?N2, and ?C1. In ?C2-overexpressing cells,
desmoplakin staining showed no considerable increase. Bar, 20 ?m.
Figure 6. Plakophilin 1 associates with the actin cytoskeleton in
HeLa (a–c) and HaCaT (d) cells. Plasmids encoding wild-type
plakophilin 1 (a), the head domain (b), and the ?C1-GFP fusion
construct (c) were transfected into HeLa cells, and the cells were
fixed in formaldehyde (a) or methanol (b and c) and processed
for immunofluorescence. (a) Wild-type plakophilin 1 was pre-
dominantly in the nucleus and decorated actin filaments as re-
vealed by double labeling with FITC-phalloidin (a?). (b) The
head domain was almost exclusively in the nucleus. In transfected
cells, desmoplakin revealed a punctate staining pattern in the cy-
toplasm. A similar distribution of desmoplakin was seen in mi-
totic cells (arrowheads). In contrast, desmoplakin showed the
punctate staining pattern along the plasma membrane that is typ-
ical of desmosomes in other nontransfected cells (arrows, b?). (c)
The ?C1 construct showed a similar distribution as the head do-
main. It was almost exclusively nuclear, whereas desmoplakin la-
beling was cytoplasmic (c?). (d) A wound was inserted into a con-
fluent monolayer of HaCaT cells, and cells were fixed in
formaldehyde and processed for immunofluorescence after 24 h.
Endogenous plakophilin 1 (d) colocalized with actin (d?) at the
tips of cellular protrusions of cells next to the wound. Bars, 10 ?m.
The Journal of Cell Biology, Volume 149, 2000
to a somewhat lesser extent, with the Dsg domain alone
(Fig. 7 d), indicating that the plakophilin 1 binding site dif-
fers from the plakoglobin binding site in the CS domain.
The requirement of the CS domain for strong binding sug-
gests that the plakophilin 1 binding site is close to the pla-
koglobin binding site, and that simultaneous binding might
be prevented because of steric hindrance.
Since plakophilin 1 has been shown to bind keratins in
vitro (Kapprell et al., 1988; Hatzfeld et al., 1994; Smith and
Fuchs, 1998), we also examined several keratin constructs
for their interaction with the plakophilin 1 head domain. A s
shown in Fig. 7 e, the type I keratins K17 and K18 strongly
interacted with the plakophilin 1 head, whereas of the type
II keratins tested, only K8 showed a weak interaction. Inter-
action studies with the head fragments revealed binding of
K17 and K18 to the ?C1 and ?C2 constructs, but not to
?N1 and ?N2. The K8 binding site appeared to differ since
binding was retained in the ?N1, ?C1, and ?C2 constructs,
but was lost in the ?N2 fragment (not shown).
Interactions between the plakophilin 1 head fragments
and desmoplakin and Dsg1 were quantitated by measuring
LacZ reporter gene activation with the ONPG substrate.
A s shown in Fig. 7 g, the desmoplakin–plakophilin 1 and
the Dsg1–plakophilin 1 interactions were much stronger
with the plakophilin 1 head in the pBD vector compared
with the pA S2-1 vector, although this vector allows high
protein expression levels as verified by Western blotting
with Gal4 and plakophilin 1–specific antibodies (Fig. 8 a).
The high protein expression could either interfere with
correct folding of plakophilin 1, or the desmoplakin and
Dsg1 binding sites are masked by inter- or intramolecular
interactions after expression in the pA S vector. Dsg1 in-
teracted most strongly with the ?N1 construct, which lacks
the desmoplakin binding site. Interaction with the ?C1
construct was somewhat weaker. In contrast, the ?C2 and
?N2 constructs showed considerably reduced reporter
gene activation, suggesting that these constructs did not
contain the entire Dsg1 binding site. Desmoplakin inter-
acted most strongly with the ?C2 construct, which lacks
part of the Dsg1 binding site. The interaction was lost with
the ?N1 and ?N2 constructs, indicating that the binding
site is in the NH2-terminal region.
Since all previous experiments had shown interactions
between cadherins and the arm repeat domains, but not the
end domains of arm proteins, we also analyzed whether the
headless plakophilin 1 or the repeat domain interacted with
any of the desmosomal cadherins. A s shown in Fig. 7 f, none
of the desmosomal cadherins interacted with headless pla-
kophilin 1. The same result was obtained with the arm do-
main construct. Expression of the headless fragment in
yeast cells was verified by Western blotting with anti-Gal4
antibodies (Fig. 8 a, lane 7?). Here, the headless fragment
gave the strongest signal, indicating that a lack of protein
expression did not account for the lack of binding.
The armadillo Repeat Domain of Plakophilin 1
Associates with Actin and Induces the Formation of
Filopodia and Long Cellular Protrusions
The colocalization of wild-type plakophilin 1 with stress fi-
bers as well as actin-rich structures at the tips of filopodia
(Fig. 6) pointed to a possible role of plakophilin 1 in regu-
lating the actin cytoskeleton. Therefore, we expressed the
plakophilin 1 arm repeat and the headless domain in HeLa
and HaCaT cells and verified expression by Western blot-
ting (Fig. 8 b). Cells with high levels of these plakophilin 1
fragments displayed a highly unusual morphology with
formation of filopodia, lamellipodia, or long protrusions
(Fig. 9, a–d), which interfered with normal monolayer for-
mation. The transfected cells often sat on top of the other
cells. Cells with lower expression levels still displayed a
normal cell morphology (Fig. 10, a–e). However, double
labeling with desmoplakin antibodies showed that des-
moplakin had been internalized, and disintegration of
junctions had already begun (Fig. 10, e and e?). The plako-
philin 1 arm repeat domain colocalized with actin in lamel-
lipodia (Fig. 10, a, c, and d) and sometimes stress fibers
(Fig. 10, c and c?), suggesting a role in regulating actin po-
lymerization and filopodia formation. This phenotype was
observed in HeLa and HaCaT cells.
The Phenotype Produced by the Plakophilin 1 Arm
Domain and Its Capacity to Associate with Actin
Filaments Critically Depend on a Conserved Motif
A similar phenotype, the formation of long dendritelike
cellular protrusions, had been observed in transfection
Figure 7 (continues on facing page).
Hatzfeld et al. Function of Plakophilin 1
studies with full-length p120ctn (Reynolds et al., 1996) and
?-catenin (Lu et al., 1999). This suggested that the pheno-
type is conserved among p120ctn family members, and
might depend on the interaction with a common binding
partner that is involved in regulating actin filament organi-
zation. To characterize this binding site in plakophilin 1,
we constructed a deletion mutant that lacks a central pen-
tapeptide motif conserved among all p120ctn family mem-
bers. The motif (ENCM/V C) is specific for this family and
not detected in other arm related proteins. Transfection
studies with this mutant construct (plakophilin 1 arm
?ENCMC) showed that the mutant had lost its capacity to
induce changes in cell morphology and no longer associ-
ated with actin filaments in filopodia (Fig. 10, f and f?). In-
stead, it accumulated in the cytoplasm, sometimes in an
aggregated form (Fig. 10 f).
To analyze if the interaction between plakophilin 1 and
actin is direct, we used the two-hybrid system. These ex-
periments revealed no direct interaction between ?-actin
and the plakophilin 1 repeat domain (data not shown),
suggesting that the interaction either depends on an intact
microfilament or is mediated through an actin-associated
protein in vivo.
Plakophilin 1 was shown to localize to desmosomes and to
the nucleus, raising the possibility for a dual function in
cell adhesion and signal transduction (Schmidt et al.,
1997). In the present study, we have determined the re-
gions in plakophilin 1 responsible for binding of desmo-
somal proteins and provide a functional analysis of the
plakophilin 1 domains.
The Head Domain of Plakophilin 1 Mediates Binding to
Desmoplakin, Dsg1, and Keratins
Plakophilin 1 is a desmosome-associated protein and has
been shown to bind Dsg1, Dsc1a, and desmoplakin in vitro
(Smith and Fuchs, 1998) and desmoplakin in vivo (Kowal-
czyk et al., 1999). Using the yeast two-hybrid assay, we
have mapped the binding sites of desmosomal proteins
and keratins within plakophilin 1. We show that it is the
head domain that mediates the interactions between pla-
kophilin 1 and Dsg1, desmoplakin, as well as keratins.
Whereas desmoplakin binds close to the NH2 terminus be-
tween amino acids 1–70 of the head domain, the Dsg1
binding site is located between amino acids 70 and 213
(Fig. 11). Our data suggest that these two sites do not act
independently. Dsg1 bound most strongly to the deletion
construct lacking the desmoplakin binding site and vice
versa. This could be due to reduced accessibility of the
binding sites because of intramolecular interactions, which
are similar to those described for vinculin and ERM family
members (Winkler et al., 1996; Tsukita et al., 1997), or in-
teractions with other proteins. A similar observation was
Figure 7. Two-hybrid analysis of pro-
tein–protein interactions. (a) Y RG2
yeast cells were double transformed with
the plakophilin 1 head in pA S2-1 and
desmoplakin-NH2 terminus (DP-NTP,
1), Dsg1 (2), Dsg2 (3), Dsg3 (4), Dsc1a
(5), Dsc1b (6), Dsc2a (7), Dsc2b (8),
Dsc3a (9), and Dsc3b (10) intracellular
domains. A ll cells grew on selection
plates lacking tryptophan and leucine
(?TL), indicating that they contain both
plasmids. Histidine reporter gene activa-
tion was analyzed on plates lacking histi-
dine (?TLH) and LacZ reporter gene
activation in a filter lift assay. DP-NTP
and Dsg1 activated both reporter genes.
(b) Double transformations of the ?C1
construct in pA S2-1 and Dsg1 (1), Dsc1a (2), Dsc1b (3), and DP-NTP (4), and of ?C2 with DP-NTP (5), Dsc1b (6), Dsc1a (7), and Dsg1
(8). DP-NTP and Dsg1 interacted with the ?C1 and ?C2 constructs, although LacZ reporter gene activation seemed weaker with Dsg1 ?
?C2. (c) Double transformations of the ?N1 construct in pA S2-1 with Dsg1 (1), Dsc1a (2), Dsc1b (3), and DP-NTP (4) and of ?N2 with
DP-NTP (5), Dsc1b, (6), Dsc1a (7), and Dsg1 (8). ??1 and ?N2 reacted with Dsg1, whereas DP-NTP did not interact with the ?N1 and
?N2 constructs. Dsc1a and b did not interact with any of the head domain fragments. (d) Double transformants of the head domain with
Dsg1 deletion constructs containing the complete cytoplasmic domain (1), the IA domain (2), the CS domain (3), the IA and the CS do-
main (4), the Dsg domain (5), and the Dsg and CS domains (6). The head domain interacted strongly with the complete Dsg cytoplasmic
domain and with the Dsg?CS domain. The interaction with the Dsg domain alone was weaker. (e) Double transformations of the head
domain with K8 (1), K18 (2), K6 (3) and K17 (4) and of the arm domain with K8 (5), K18 (6), K6 (7) and K17 (8). The plakophilin 1
head domain interacted weakly with K8 and more strongly with K17 and K18. The arm repeats did not interact with any of the keratins
tested. (f) Double transformations of headless plakophilin 1 with the following intracellular domains: DP-NTP (1), Dsg1 (2), Dsg2 (3),
Dsg3 (4), Dsc1a (5), Dsc1b (6), Dsc2a (7), Dsc2b (8), Dsc3a (9), and Dsc3b (10). A lthough the His reporter gene was weakly activated
by some constructs, the LacZ reporter gene was not activated. (g) Interactions between the cytoplasmic domain of Dsg1 and DP-NTP
and the plakophilin 1 head domain fragments were quantitated using a ?-galactosidase assay and the ONPG substrate. The bars repre-
sent three independent experiments each performed in triplet. None of the plakophilin 1 constructs activated the LacZ reporter gene on
its own. Dsg1 interacted with all constructs tested. However, the ?C2 and ?N2 constructs showed a strong decrease in reporter gene ac-
tivation suggesting that these constructs do not contain the entire Dsg1 binding site. DP-NTP interacted most strongly with the ?C2
construct and revealed no interaction with the ?N1 and ?N2 constructs.
The Journal of Cell Biology, Volume 149, 2000
made for plakoglobin, certain internal fragments of which
bound better to E-cadherin than the entire molecule (Chi-
taev et al., 1996). A lternatively, high expression of plako-
philin 1 could interfere with correct folding and thereby
prevent the interaction in an unspecified manner.
The localization of the plakophilin 1 binding site within
the Dsg1 cytoplasmic tail showed that it is distinct from
the reported plakoglobin binding site (Mathur et al., 1994;
Chitaev et al., 1996). However, the close proximity of the
two binding sites could prevent simultaneous binding. In
contrast to plakophilin 1, other arm family members in-
cluding ?-catenin, plakoglobin, and p120ctn associate with
classical or desmosomal cadherins through their arm re-
peat region (Hinck et al., 1994; Mathur et al., 1994; A ghib
and McCrea, 1995; Daniel and Reynolds, 1995; Sacco et al.,
1995; Shibamoto et al., 1995; A berle et al., 1996; Chitaev
et al., 1996; Reynolds et al., 1996; Troyanovsky et al., 1996;
Wahl et al., 1996; Witcher et al., 1996). Moreover, muta-
tional analysis has revealed that the arm repeat domains
of ?-catenin and plakoglobin were sufficient to direct nu-
clear localization (Funayama et al., 1995; Karnovsky and
Klymkowsky, 1995). It is interesting that both characteris-
tics, cell contact association as well as nuclear localization,
are conserved between ?-catenin, plakoglobin, and plako-
philin 1, but the domains responsible for these functions
are not in the conserved sequence region.
In our two-hybrid assay, we could not confirm the inter-
action between plakophilin 1 and Dsc1 reported by Smith
and Fuchs (1998) using an overlay assay. Since most of the
two-hybrid vectors allow only low protein expression we
were unable to detect expression of the Dsg, Dsc and kera-
tin fragments by Western blotting. Therefore, we cannot
unequivocally rule out the possibility that we might have
missed the plakophilin 1–Dsc1 interaction because of a lack
of protein expression or a lack of nuclear import of Dsc 1.
A lternatively, both proteins could associate in vitro after
their denaturation, but not under physiological conditions.
We also detected interactions between plakophilin 1
and keratins. We found a weak binding of K8 and strong
binding of K17 and K18, suggesting a preference for type I
keratins that had also been proposed on the basis of in
vitro overlay assays (Kapprell et al., 1988). In contrast,
Smith and Fuchs (1998) have reported that plakophilin 1
binds preferentially to type II keratins. The controversial
data may be either due to the analysis of different keratins
(K5 and K14 versus K8, K18, K6, and K17) or the use of
different assay systems (in vitro overlay versus in vivo as-
says). Since plakophilin 1 is expressed in suprabasal cells
Figure 9. Expression of the arm repeat domain in HaCaT (a and
b), L6 (c), and HeLa (d) cells. Cells were transfected with the
arm repeat domain in pCMV 5 (a, b, and d) or the headless con-
struct in pEGFP (c). Transfected cells were visualized with the
T7 antibody (a and b) or the plakophilin 1 repeat antibody (d) or
by GFP fluorescence (c). Transfected cells showed changes in
cellular morphology, with the development of filopodia and long
cellular protrusions. Bars: (a and b) 40 ?m; (c and d) 20 ?m.
Figure 8. Expression of plakophilin 1 constructs in yeast (a) and
HeLa cells (b). (a) Y east cell extracts were prepared as described
in Materials and Methods, and the cell extracts were stained with
Coomassie (lanes 1–7) or blotted with anti-GA L4 (lanes 1?–7?) or
plakophilin 1 head antibodies (lanes 2??–6??). (lane 1) Y RG2
yeast cells without plasmid; (lane 2) plakophilin 1 head domain;
(lane 3) ?C2; (lane 4) ?C1; (lane 5) ?N1; (lane 6) ?N2; and (lane
7) headless plakophilin 1. A rrows denote the plakophilin 1 head
fragments reacting with the GA L4 antibody. (b) Total extracts
from HeLa cells transfected with the plakophilin 1 head (lanes
1–1??) or the arm repeats (lanes 2–2??) were prepared in SDS
sample buffer and probed with the plakophilin 1 head and arm
repeat antibodies as indicated. Lanes 1 and 2 show the Ponceau
Hatzfeld et al. Function of Plakophilin 1
of stratified epithelia, its in vivo interaction partner is
probably one of the keratins specifically expressed in dif-
ferentiated keratinocytes such as K10.
Plakophilin 1 Enhances Recruitment of Desmosomal
Proteins to the Plasma Membrane
We have analyzed intracellular targeting of plakophilin 1
after overexpression. We chose two different cell lines for
our studies, HaCaT keratinocytes, which express endoge-
nous plakophilin 1 and consequently all its essential inter-
action partners, and simple epithelial HeLa cells. These
cells possess desmosomes and express the ubiquitous des-
mosomal proteins, but lack certain cell type–specific des-
mosomal proteins including Dsg1 and 3, Dsc1 and 3, and
plakophilin 1 (Schmidt et al., 1994). Moreover, desmo-
somes are less abundant and smaller in HeLa cells.
In HaCaT cells, overexpressed plakophilin 1 was found
in the nucleus as well as plasma membrane associated, in
agreement with the intracellular localization of the endog-
enous protein (Schmidt et al., 1997). Using deletion clones
of plakophilin 1, we have determined which domains tar-
get plakophilin 1 to desmosomes (Table I). Whereas cell
contact association of other arm proteins including ?-cate-
nin, plakoglobin, and p120ctn is mediated by their arm re-
peat domain (Hinck et al., 1994; Mathur et al., 1994; A ghib
and McCrea, 1995; Daniel and Reynolds, 1995; Shibamoto
et al., 1995; A berle et al., 1996; Chitaev et al., 1996; Rey-
nolds et al., 1996; Troyanovsky et al., 1996; Wahl et al.,
1996; Witcher et al., 1996), we found that it is the head do-
main of plakophilin 1 that directs its localization to desmo-
somes as well as to the nucleus. This is consistent with the
localization of the binding sites for desmosomal proteins
determined in the two-hybrid system. The nuclear local-
ization was observed in all cell types examined, indicating
that the nuclear function is conserved among different cell
types. In contrast, desmosome association was restricted
to HaCaT cells, suggesting that binding to a cell type–spe-
cific desmosomal protein might be essential for targeting,
or that regulatory mechanisms prevent the cell contact as-
sociation of plakophilin 1 in simple epithelial HeLa cells.
A ll the head domain fragments were still able to associate
with desmosomes and to enter the nucleus. With the ?C1
and ?N1 fragments, desmosome association was preferred
over the nuclear localization. This may be due to better ac-
cessibility of desmosomal binding sites in these constructs
In HeLa cells, full-length plakophilin 1 also decorated
actin filaments. The head domain alone localized only to
the nucleus, indicating that it is the arm repeat domain
that mediates the association with the actin cytoskeleton.
In HaCaT cells, we also found colocalization of the plako-
philin 1 head domain with keratins along membrane
patches. This could be due either to the direct interaction
Figure 10. Expression of the
HaCaT and HeLa cells. (a)
HaCaT cells were trans-
fected with plakophilin 1 arm
repeats and processed for im-
munofluorescence after 20 h.
Cells were stained with the
plakophilin 1 repeat anti-
body (a) and FITC-phalloi-
din (a?). (b) HaCaT cells
transfected with the GFP-
tagged headless construct.
Cells were fixed 20 h after
transfection and labeled with
FITC phalloidin (b?). (c)
HeLa cells were transfected
with the GFP-tagged head-
less construct. Cells were la-
beled with TRITC-phalloidin
(c?). (d) HeLa cells trans-
fected with the arm repeats
were double stained with the
arm repeat antibody (d) and
HeLa cells transfected with
the arm repeats were double
stained with the arm repeat
antibody (e) and the des-
moplakin antibody (e?). (f)
HeLa cells transfected with
the rep?ENCMC construct
were double labeled with the
T7 tag antibody and FITC-
phalloidin (f?). Bar, 20 ?m.
The Journal of Cell Biology, Volume 149, 2000
between plakophilin 1 and keratins, as shown in the two-
hybrid system, or to recruitment via the keratin-binding
protein desmoplakin. Nevertheless, these data, together
with the results of the two-hybrid assay, suggest that pla-
kophilin 1 interacts with keratins in vivo. Recruitment of
desmoplakin and keratins to the plasma membrane has
also been described in cells overexpressing a plakoglobin-
synaptophysin chimera (Chitaev et al., 1996).
In a recent report, Kowalczyk et al. (1999) showed that
the desmoplakin NH2 terminus was recruited to the mem-
brane when overexpressed together with plakophilin 1 in
COS cells. We extended these experiments and analyzed
the recruitment of various endogenous desmosomal pro-
teins in HaCaT cells overexpressing plakophilin 1. A s
judged by immunofluorescence recruitment of desmoplakin,
Dsg and Dsc were strongly enhanced, and that of plako-
globin was slightly enhanced, suggesting a major role for
plakophilin 1 in desmosome assembly. In cells with high
plakophilin 1 expression, we observed nuclear localization
of other desmosomal proteins including desmoplakin,
probably due to coimport mediated by plakophilin 1. This
conclusion is supported by the fact that the ?N1 and ?N2
constructs, which lack the desmoplakin binding site, never
coimported desmoplakin into the nucleus (Fig. 5).
There are two ways in which additional desmosomal
proteins could be recruited to the plasma membrane. First,
plakophilin 1 could bind to endogenous desmosomal pro-
teins, target them to the plasma membrane, and thereby
dramatically increase their stability. This is consistent with
the finding that desmosomal proteins are usually synthe-
sized in excess, and their cytoplasmic pool is rapidly de-
graded (Pasdar and Nelson, 1988, 1989). In this model,
plakophilin 1 plays a structural role in desmosome assem-
bly. Second, plakophilin 1 might be directly involved in
regulating the synthesis of desmosomal proteins in the nu-
cleus. In this model, the induction of desmosomes would
depend on a putative signaling function of plakophilin 1.
Our experiments do not allow us to distinguish between
these two models, since all fragments that were able to in-
duce desmosome formation also revealed nuclear localiza-
tion and, therefore, might combine the signaling and struc-
tural functions. Further, both mechanisms might contribute
to the recruitment of endogenous desmosomal proteins.
Plasma membrane association of desmoplakin and plako-
philin 1 after combined overexpression in COS cells (Kowal-
czyk et al., 1999) argues for a contribution of the recruit-
Using laser scanning microscopy, we demonstrate that
plakophilin 1 preferentially associates with and recruits des-
mosomal proteins, and that the recruitment of desmosomal
components might result in the generation and the enlarge-
ment of desmosomes. The possibility that expression of pla-
kophilin 1 enhances desmosome formation in keratinocytes
is consistent with the observation that desmosomes of su-
prabasal cells are larger than basal cell desmosomes, and
that desmosomes are more numerous in suprabasal cells.
Moreover, this finding explains why desmosomes were
small and rare in a patient lacking plakophilin 1 (McGrath
et al., 1997). Therefore, we propose that plakophilin 1 plays
an essential role in regulating desmosome organization and
size during keratinocyte differentiation.
The Arm Repeat Domain of Plakophilin 1
Associates with Actin Filaments and Induces
Formation of Filopodia
In HeLa cells, overexpressed plakophilin 1 associated with
actin filament, suggesting that it might be involved in regu-
lating the actin cytoskeleton. This association was also ob-
served in nontransfected cells, where plakophilin 1 colo-
calized with actin in normal cells at the tips of plasma
membrane protrusions. A similar localization has been de-
scribed for ?-catenin, which interacts with the actin fila-
ment bundling protein fascin through its arm repeat do-
main (Tao et al., 1996). A ctin filament association of
plakophilin 1 also appeared to be mediated by its arm re-
peats. When overexpressed at high levels in HeLa and
HaCaT cells, the arm repeat domain induced the forma-
tion of long cellular protrusions, supporting a possible role
in the regulation of cell motility. This phenotype inter-
fered with intercellular adhesion, and transfected cells
were separated from the monolayer. Since full-length pla-
Figure 11. Binding sites of plakophilin 1–associated proteins.
Table I. Intracellular Localization of Plakophilin 1 and Its Fragments
PKP1 head ?C1
PKP1 head ?C2
PKP1 head ?N1
PKP1 head ?N2
PKP1 arm repeat
PKP1 arm repeat ?ENCMC
*This localization is only seen in a few cells but not in the majority of transfected cells.
‡Cannot be determined due to the phenotype that is characterized by a loss of desmosomes.
Hatzfeld et al. Function of Plakophilin 1
kophilin 1 had no such effect, we conclude that desmo-
some association of the head domain is preferred. A simi-
lar phenotype has been described for p120ctn (Reynolds et
al., 1996) and ?-catenin (Lu et al., 1999) after expressing
the full-length protein. In the case of p120ctn, the arm do-
main was required for this effect (Reynolds et al., 1996),
suggesting that this function is conserved in the arm do-
main of p120ctn family members. We identified a 5–amino
acid motif (ENCMC) that is conserved among p120ctn fam-
ily members. Deletion of this motif in the plakophilin 1
arm repeat domain abolished the ability of the mutant to
associate with actin filaments and to induce the pheno-
type. This suggests that a protein–protein interaction me-
diated by this motif is responsible for this effect. Since we
were unable to detect a direct interaction between the pla-
kophilin 1 arm repeats and actin in the two-hybrid system,
the interaction either requires an intact microfilament, as
opposed to an actin monomer, or it is mediated by an ac-
The phenotype in patients lacking plakophilin 1 sug-
gested an important role for plakophilin 1 in stabilizing in-
tercellular adhesion, although the lack of hair follicles and
sweat glands suggests an additional role in certain differ-
entiation processes (McGrath et al., 1997). Our results
support the conclusion that plakophilin 1 has an important
structural function and explain the role of plakophilin 1 in
desmosome assembly at a molecular level. The localiza-
tion of desmosomal binding sites to the head domain cor-
relates with the finding that this domain recruits endoge-
nous desmosomal proteins to sites of cell contact, whereas
the arm repeat domain reduced cell contacts and induced
the formation of motility-associated structures. In conflu-
ent keratinocytes, localization of plakophilin 1 to desmo-
somes is preferred over association with adherens junc-
tions and actin filaments. This is consistent with strong
intercellular adhesion in these cells. However, in cells that
lack contact to adjacent cells, plakophilin 1 localizes to
filopodia. Here, it may have a function in inducing junc-
tion formation as soon as the tip of the cell contacts an op-
posing cell. This idea is consistent with the finding that for-
mation of actin-associated cell contacts precedes desmosome
formation and is a prerequisite for desmosome formation
(Lewis et al., 1994, 1997). Plakophilin 1 could play a role in
recruiting desmosomal proteins from the cytoplasm to the
plasma membrane at sites of newly formed cell contacts.
We are grateful to K. Green for providing the desmoplakin construct for
the two-hybrid studies and to F. Ramaekers for keratin antibodies. We
thank K. Green for many helpful suggestions and discussions and E.
Bornslaeger, K. Green (both from Northwestern University, Chicago, IL),
and M. Osborn (MPI-Biophysical Chemistry, Göttingen, Germany) for
critical reading of the manuscript. We would also like to thank C. Horn for
technical help, C. Nachtsheim for her contribution in the initial phase of
this study. We are also grateful to M. Iwig and the Zeiss company for help
with the confocal microscopy.
This work was supported by grants from the Deutsche Forschungsge-
meinschaft (Ha 1791/3-1 and 3-2, Ha 1791/5-1) and the BMBF.
Submitted: 18 A ugust 1999
Revised: 16 February 2000
A ccepted: 23 February 2000
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