1 Institute of Physiological Chemistry, Medical Faculty of the University of
Halle, 06097 Halle/Saale, Germany
2 Departments of Pathology and Dermatology and the Robert H. Lurie Cancer
Center, Northwestern University Medical School, Chicago, IL 60611, USA
* Author for correspondence (e-mail: mechthild.hatzfeld{at}medizin.uni-halle.de)
Accepted 15 November 2002
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Summary |
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Key words: p0071, Plakophilin, Plakoglobin, Cadherin, Desmosome
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Introduction |
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In adherens junctions, ß-catenin or -catenin (plakoglobin), the
vertebrate homologues of armadillo, link
-catenin to the intracellular
domain of cadherins (Aberle et al.,
1996
; Gumbiner and McCrea,
1993
; Kemler,
1993
; Obama and Ozawa,
1997
). P120ctn, another armadillo repeat protein
located in cell contacts, binds to E-cadherin simultaneously with
ß-catenin or
-catenin (Aghib
and McCrea, 1995
; Daniel and
Reynolds, 1995
; Ohkubo and
Ozawa, 1999
; Reynolds et al.,
1996
; Shibamoto et al.,
1995
; Staddon et al.,
1995
). The ß-catenin-binding site maps to the C-terminal
region of cadherins, whereas p120ctn binds the juxtamembrane
region, which plays an important role in cadherin clustering and regulating
adhesion. ARVCF and NPRAP/
-catenin, two p120ctn-related
armadillo repeat proteins, can bind to the same cadherin domain
(Kaufmann et al., 2000
;
Lu et al., 1999
;
Mariner et al., 2000
;
Paulson et al., 2000
;
Waibler et al., 2001
). These
proteins are much less abundant than p120ctn and their role in
regulating adherens junction function is not well understood.
Desmosomes contain two types of cadherins, the desmogleins (dsgs) and
desmocollins (dscs). The three different desmoglein and desmocollin genes
(dsg 1-3 and dsc 1-3) are differentially expressed
(Koch and Franke, 1994;
Schmidt et al., 1994
). The
intracellular domains of the desmosomal cadherins associate with plakoglobin,
which also binds desmoplakin. Direct interactions between desmosomal cadherins
and desmoplakin have been reported in vitro
(Smith and Fuchs, 1998
) but it
appears that plakoglobin is necessary to link these proteins in vivo
(Kowalczyk et al., 1996
;
Kowalczyk et al., 1997
).
Desmoplakin binds to plakoglobin through its N-terminal domain and to
intermediate filaments through its C-terminal domain and connects desmosomes
to the cytoskeleton (Stappenbeck et al.,
1993
; Stappenbeck et al.,
1994
). The importance of plakoglobin and desmoplakin in desmosome
function became evident in vivo. In mice, lack of plakoglobin results in an
embryonic lethal phenotype due to reduced intercellular adhesion between
cardiomyocytes (Bierkamp et al.,
1996
; Ruiz et al.,
1996
). A C-terminal truncation of plakoglobin has been described
in arrhythmogenic right ventricular cardiomyopathy with palmoplantar
keratoderma and woolly hair (Naxos disease), which suggests that plakoglobin
and perhaps other proteins involved in cell-cell adhesion play an important
part in maintaining myocyte integrity
(McKoy et al., 2000
). Mice
lacking desmoplakin expression in their skin revealed reduced intercellular
adhesion upon mechanical stress and showed defects in epithelial sheet
formation (Vasioukhin et al.,
2001
). A mutation in the desmoplakin gene, resulting in a null
allele and haploinsufficiency, was observed in patients with striate
palmoplantar keratoderma. Affected skin demonstrated loosening of
intercellular connections, disruption of desmosome-keratin interactions and
rudimentary desmosomal structures
(Armstrong et al., 1999
). A
generalized striate keratoderma particularly affecting the palmoplantar
epidermis, woolly hair, and a dilated left ventricular cardiomyopathy was
described in patients with a recessive mutation in the desmoplakin gene.
Histology of the skin revealed large intercellular spaces with a perinuclear
localization of keratin filaments in suprabasal keratinocytes, suggesting a
collapsed intermediate filament network
(Norgett et al., 2000
).
Additional components of the desmosomal plaque are plakophilins 1-3
(Bonne et al., 1999;
Hatzfeld, 1997
;
Hatzfeld et al., 1994
;
Heid et al., 1994
;
Mertens et al., 1996
;
Schmidt et al., 1999
;
Schmidt et al., 1997
).
Plakophilin 3 is widely expressed in all desmosomes, whereas plakophilin 1 and
2 show more restricted expression patterns and are found mainly in stratified
or simple epithelia, respectively. Plakophilin 1 interacts with the suprabasal
desmoglein isoform dsg1 and the desmoplakin N-terminal domain
(Hatzfeld et al., 2000
), and
plakophilin 2 binds to dsg1 and 2, as well as plakoglobin and desmoplakin
(Chen et al., 2002
). An
essential function of plakophilin 1 in stabilizing intercellular adhesion has
been suggested by the phenotype caused by lack of plakophilin 1
(McGrath, 1999
;
McGrath et al., 1997
).
Null-mutations in the plakophilin 1 gene cause a genetic skin disease in
humans called skin fragility syndrome. Desmosomes in the skin from patients
are small and poorly formed with widening of keratinocyte intercellular spaces
and perturbed desmosomekeratin filament interactions. Moreover, desmoplakin
was found predominantly cytoplasmic suggesting a role for plakophilin 1 in
organizing desmosomal plaque proteins. Ectopic expression of plakophilin 1
induced recruitment of desmosomal proteins to the plasma membrane through its
interaction with several desmosomal proteins and suggests a function of
plakophilin 1 in determining desmosome size and organization
(Hatzfeld et al., 2000
;
Kowalczyk et al., 1999
).
The ability of cells to organize desmosomal proteins into a functional
structure critically depends on the presence of adherens junctions.
Plakoglobin, so far the only protein common to both types of cell contacts,
has been implicated in crosstalk between adherens junctions and desmosomes
(Lewis et al., 1997).
p0071 is another armadillo repeat protein located in intercellular
junctions (Hatzfeld and Nachtsheim,
1996). It is most closely related to NPRAP/
-catenin and
p120ctn, both of which associate with classical cadherins
(Hatzfeld, 1999
). In contrast,
a dual localization pattern in adherens junctions and desmosomes depending on
the cell type has been reported for p0071
(Hatzfeld and Nachtsheim,
1996
). In order to characterize the function of this protein in
intercellular adhesion we have characterized its intracellular localization
and show that the N-terminal head domain associates with desmosomes, whereas
the armadillo repeat domain colocalizes with classical cadherins and has the
capacity to recruit cadherins to the plasma membrane. A direct interaction
between the head domain and desmocollin 3a, desmoplakin and plakoglobin was
consistent with targeting of this domain to desmosomes. In contrast, the
armadillo repeats differentially interact with several non-desmosomal
cadherins. These findings indicate that p0071, like plakoglobin, can associate
with both types of intercellular junctions and may have a function in
crosstalk between these adhesive structures.
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Materials and Methods |
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Plasmids and cDNA constructs
RNA was prepared according to the LiCl/urea extraction method
(Auffray and Rougeon, 1980) and
cDNA synthesized by rtPCR using expand reverse transcriptase and high fidelity
or long template polymerase (Roche Diagnostics, Mannheim, FRG). Suitable
restriction sites for cloning were included in the primer sequences. PCR
products were ligated into the PCR2.1 vector using TOPO TA cloning
(Invitrogen, Karlsruhe, FRG). All PCR products were sequenced completely.
The following p0071 constructs were generated with a 5' MunI or EcoRI restriction site and a 3' SalI or XhoI restriction site and cloned into the pEGFP c2 vector (BD Clontech, Heidelberg, Germany): p0071 wt (aa 1-1193, short splice variant of the tail domain lacking aa 1043-1085); p0071 N-terminal head (aa 1-509); head aa 149-509; p0071 headless (aa 510-1193, short splice variant); p0071 tail (aa 989-1193, short splice variant); p0071 arm repeats (aa 510-988); arm repeats 1-5 (aa 510-745); arm repeats 2-10 (aa 552-988); and arm repeats 4-10 (aa 639-988). The same constructs including an additional Kozak sequence between the 5'-MunI restriction site and the start codon and lacking a stop codon were cloned into the EcoRI and XhoI sites of pcDNA4/TO/myc-His (Invitrogen) and into the EcoRI and SalI sites of pDsRed1-N1 (BD Clontech), which had been mutagenized to obtain the correct reading frame (Quick change mutagenesis kit, Stratagene, Amsterdam, The Netherlands).
The p0071 head and arm repeat domain constructs were also cloned into the yeast two-hybrid vectors pGBKT7 and pAS-2-1 (BD Clontech). Head domain clones showed autoactivation of the His and LacZ reporter genes except for one clone in pAS2-1. Sequence analysis revealed that this clone contained an internal deletion leaving an N-terminal head domain fragment of aa 1-198. The largest N-terminally deleted head domain construct without autoactivation (aa 209-509) and the p0071 arm repeats (repeats 1-10, 1-5, 2-10 and 4-10) were cloned into pGBKT7.
Vectors encoding cytoplasmic domains of human desmogleins (dsg) 1-3 and
desmocollins (dsc) 1a-3b have been described
(Hatzfeld et al., 2000). To
exclude false-negative results due to low expression levels in the pGAD424
vector used earlier, all fragments were subcloned into the pGADT7 vector,
which allows high protein expression (BD Clontech). The human E- (aa 734-884),
N- (aa 746-906) and OB-cadherin/cadherin 11 (aa 638-797) cytoplasmic tails
were cloned into pGADT7. All dsgs, dscs and cadherin cytoplasmic domains were
also inserted in the pcDNAmom-flag vector as described
(Kaufmann et al., 2000
). The
E-cadherin cytoplasmic domain was also fused to the 4A6-birch profilin
antibody epitope (4A6-tag) (Kaufmann et
al., 2000
).
An N-terminal deleted plakoglobin clone in pACT lacking the head portion
and repeats 1-2 (pg-3-13+C, aa 230-745, GenBank accession number Z68228) was
isolated in a two-hybrid screen for p0071-repeat-domain-interacting proteins.
Candidate clones were isolated from a Hela cell cDNA library (BD Clontech).
Plakoglobin constructs in pACT containing the N-terminus through arm repeat 6
(residues 1-375) with a C-terminal myc tag (pg-N+1-6) and arm repeats 4-9
(residues 249-490, pg-4-9) were generated by PCR and cloned into pACTII for
expression in yeast. Full length myc-tagged plakoglobin (pg-myc) and the N-
and C-terminally deleted plakoglobin (pg-N
C) in a eukaryotic
expression vector under the control of a ß-actin promotor were generated
as previously described (Kowalczyk et al.,
1994
; Palka and Green,
1997
). A plakoglobin construct lacking the majority of the central
arm repeat region (pg-
Sac) was constructed by partial digestion of
pg-wt with SacI and ligation in frame to yield a plasmid containing
residues 1-146 fused to residues 668-745.
The p120ctn (isoform A) and NPRAP/-catenin tail domains
were amplified by PCR from Hela cDNA or a human brain cDNA library
(Stratagene), respectively. The fragments were cloned into the pRSET vector
containing an N-terminal His-tag sequence (Invitrogen) and expressed in
BL21DE3 bacteria. The PKP1 repeat construct (including the short tail) has
been described before (Hatzfeld et al.,
2000
).
Antibodies and immunofluorescence
Rabbit polyclonal anti-p0071 arm repeat (serum 678) and rabbit polyclonal
anti-plakophilin 1 antibodies have been described
(Hatzfeld et al., 2000;
Hatzfeld and Nachtsheim,
1996
). The monoclonal antibody 6D1-10 was produced by immunizing
mice with the recombinant p0071 tail domain (aa 989-1193, short splice
variant, Davids Biotechnologie, Regensburg, Germany) expressed from the pRSET
vector. Protein expression was performed in BL21DE3 bacteria using standard
methods and His-tagged recombinant protein purified using Ni-NTA resin
(Qiagen, Hilden, FRG). Rabbit antiserum 616 against desmoplakin was prepared
by immunizing a rabbit with gel-purified bovine desmoplakin. Desmosomes were
prepared from bovine snout epithelia. Desmosomal proteins were separated by
SDS-PAGE, protein bands cut out of the gel and the protein eluted by diffusion
in 0.01% SDS (Hatzfeld et al.,
1994
). Antibody 4A6 against the birch profilin tag was kindly
provided by B. M. Jockusch.
The following commercial antibodies were used: mouse monoclonal anti-desmoplakin 1+2; anti-desmoglein 1+2; anti-plakophilin 2; anti-E-cadherin (Progen, Heidelberg, Germany); rabbit polyclonal anti-Pan-cadherin; mouse monoclonal anti-plakoglobin; anti-c-myc clone 9E10; anti-flag M2 (Sigma, Taufkirchen, FRG); monoclonal anti-p120 (Transduction, BD Pharmingen, Heidelberg, FRG); and mouse monoclonal anti-His-tag (Qiagen). The monoclonal antibody against plakophilin 3 (clone 23E3) was kindly provided by S. Bonné and F. van Roy (Ghent, Belgium). Secondary antibodies used in this study were: Cy3 donkey anti-rabbit and anti-mouse IgG; Cy3 goat anti-mouse IgG+IgM (Dianova, Hamburg, FRG); Alexa 488 goat anti-mouse and goat anti-rabbit IgG; Alexa 350 goat anti-rabbit IgG (Molecular probes, MoBiTec Göttingen, FRG); alkaline-phosphatase (AP)-coupled goat anti-mouse and monoclonal mouse anti-rabbit IgG (Sigma); and AP anti-mouse IgM (Jackson/Dianova, Hamburg).
For immunofluorescence analysis cells were either fixed in 3.7% paraformaldehyde for 15 minutes at room temperature or in methanol at -20°C for 10 minutes, followed by permeabilization in 0.5% Triton in PBS for 20 minutes. For immunolabeling of endogenous p0071, cells were washed in PBS and extracted in 0.5% Triton in PBS for 20 minutes at 4°C. Cells were fixed in 3.7% paraformaldehyde for 20 minutes at room temperature and washed in PBS again. After fixation and permeabilization, cells were incubated in PBS containing 1% nonfat milk for 30 minutes, incubated with the primary antibody either for 2 hours at room temperature or overnight at 4°C, washed three times with PBS and treated with secondary antibodies for 1 hour at room temperature. Coverslips were mounted in Mowiol and analyzed with a Nikon Eclipse E600 microscope equipped with a Nikon FDX-35 camera.
Gel electrophoresis and western blotting
Total extracts from E. coli BL21DE3 cells were prepared by boiling
cell pellets in SDS-sample buffer. Rat kidney was homogenized in Tris buffer
(10 mM Tris pH 7.5, 5 mM EDTA, 2 mM EGTA, 150 mM NaCl). SDS and
ß-mercaptoethanol were added to a final concentration of 5% and 10%,
respectively and the sample was centrifuged at 12,000 g for 15
minutes. Samples were separated on 15% or 7% SDS-gels. Electrophoresis and
blotting was performed following standard protocols.
Yeast two-hybrid assay
The yeast strain YRG2 (Stratagene) was transformed by electroporation.
Double transformants were grown on plates lacking leucine and tryptophane.
Expression of the His-reporter gene was analyzed on plates lacking histidine
in addition to leucine and tryptophane. LacZ reporter gene expression was
analyzed with the colony-lift filter assay and quantitated using the ONPG
(onitrophenyl-ß-D-galactopyranoside) substrate as described in the yeast
protocols handbook (BD Clontech).
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Results |
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Transfected wt p0071 associates with cell contacts and recruits
non-desmosomal cadherins to the plasma membrane of MCF-7 cells
Overexpressed wild-type (wt) p0071 with a DsRed tag at its C-terminus
exhibited a prominent membrane association in MCF-7 cells. In contrast to the
endogenous protein, exogenous p0071 distributed in a linear pattern along the
plasma membrane and overlapped only partially with desmoplakin
(Fig. 2). Moreover, desmoplakin
staining was excluded from membrane regions highly enriched in p0071
(Fig. 2). This could be due to
either epitope masking resulting from the high density of cell contact
proteins recruited to the membrane or a displacement of desmosomal proteins
from regions highly enriched in p0071. Since desmoplakin labeling was not
recovered after extraction with detergent buffers prior to fixation, and
immunodetection of dsg and dsc was also lost, we conclude that the effect is
probably not caused by epitope masking. Double labeling of p0071 wt
DsRed-expressing cells with cadherin antibodies revealed colocalization of
these proteins along the plasma membrane. In addition, regions rich in p0071
wt protein were also enriched in E-cadherin and plakoglobin (not shown)
implying that p0071 either recruits cadherins to the plasma membrane or
stabilizes membrane-associated cadherins by clustering
(Fig. 2).
|
Since localization of exogenous p0071 differs from that of endogenous p0071, we used constructs with different tags in order to exclude the possibility that the tag influenced localization of p0071. P0071 wt containing either a C-terminal myc tag (not shown) or an N-terminal EGFP tag (Fig. 2) showed the same intracellular distribution as DsRed-tagged p0071 wt. As before, p0071 was preferentially found along the plasma membrane and colocalized with non-desmosomal cadherins, which suggests that p0071 intracellular distribution is not determined by the tag sequence and that high expression levels favor association with adherens junctions.
The p0071 head and tail domains are responsible for desmosomal
targeting
In order to gain insight into the regulation of intracellular localization
and membrane association of p0071, we generated constructs comprising the
N-terminal head domain and several fragments thereof, a headless mutant, the
armadillo repeat domain and the C-terminal tail domain.
Fig. 3A gives an overview of
the p0071 constructs used in this study.
|
Ectopic expression of the p0071 head domain resulted in a punctate staining
pattern along the plasma membrane and some punctate cytoplasmic staining
(Fig. 4A) resembling the
localization pattern of the endogenous protein (see
Fig. 1C). Double labeling with
desmoplakin antibodies showed that these proteins colocalized, suggesting that
protein interactions of the p0071 head domain are capable of targeting the
protein to desmosomes. Colocalization with desmosomes was also observed with
the p0071 head domain mutant comprising aa 149-509. This construct lacks the
N-terminal conserved coiled-coil motif characteristic of
p120ctn-related proteins
(Anastasiadis and Reynolds,
2000), indicating that this motif is not essential for plasma
membrane targeting (Fig. 4B).
The p0071 tail domain localized in the nucleus of all transfected cells. In
addition, it was found to varying extents in a diffuse-cytoplasmic- and a
punctate-membrane-associated pattern. Double labeling with E-cadherin and
desmoplakin antibodies showed that the p0071 tail, like the head domain,
colocalized with desmosomes (Fig.
4C,D).
|
Headless p0071 and the armadillo repeat domain colocalize with
non-desmosomal cadherins
Ectopic expression of p0071 headless revealed a linear staining pattern
along the plasma membrane that colocalized exactly with E-cadherin
(Fig. 4E,F). Similarly to p0071
wt, the headless fragment recruited non-desmosomal cadherins to sites of cell
cell contact (Fig. 4F). Double
labeling with desmoplakin antibodies showed that p0071 headless was targeted
to structures different from desmosomes. Membrane regions highly enriched in
p0071 headless showed reduced staining for desmosomal markers
(Fig. 4G).
|
The armadillo repeat domain showed localization along the membrane similar to that of the headless construct, but in most cells a large portion of the protein was cytoplasmic, which suggests that the tail domain contributes to membrane targeting (Fig. 4H). Membrane-associated p0071 repeats colocalized with E-cadherin but not with desmoplakin (Fig. 4H,I,J). Again, cadherin was recruited to the plasma membrane of transfected cells with high expression levels (Fig. 4I). Recruitment of cadherin was observed with all constructs containing arm repeats, and strong recruitment was always accompanied by the exclusion of desmosomal markers from the respective regions.
The p0071 head domain binds directly to dsc3a, plakoglobin and the
DP-N-terminus whereas the repeat domain interacts with non-desmosomal
cadherins
Direct interactions between p0071 domains and putative binding partners in
cell cell contacts were examined by yeast two-hybrid analysis. Since the head
domain showed autoactivation in this system we used two fragments coding for
aa 1-198 and 209-509 of the head, respectively.
p0071 head 1-198 interacted with the dsc3a intracellular domain and with a plakoglobin fragment lacking the N-terminus including repeats 1-2 (pg-3-13+C). It did not interact with plakoglobin head + repeats 1-6 (pg-N+1-6) or repeats 4-9 (pg-4-9), suggesting that the p0071 N-terminal fragment binds to the plakoglobin tail (for an overview of pg-constructs see Fig. 3B). An interaction with desmoplakin was detected when analyzing activation of the His-reporter gene, but could not be confirmed by analyzing lacZ reporter gene activation (Fig. 5A).
|
p0071 head (aa 209-509) interacted with the desmoplakin N-terminal domain but again activation of the lacZ reporter gene was weak. In addition a strong interaction with pg-3-13+C but not with pg-N+1-6 or pg-4-9 was observed (Fig. 5A). No interaction was detected between p0071 head domain fragments and non-desmosomal cadherins, desmogleins or desmocollin isoforms other than dsc3a.
The repeat domain of p0071 interacted with all non-desmosomal cadherins analyzed (E-, N-, OB-cadherin/cadherin 11, Fig. 5A). Deletion of p0071 arm repeats 1 or 1-3 resulted in a loss of E-cadherin interaction, whereas repeats 1-5 were sufficient to mediate the p0071-E-cadherin interaction, which suggests that repeat 1 is important for cadherin binding. Moreover, p0071 repeats also interacted with plakophilin 2 (Fig. 5A), pg-3-13+C and with pg-N+1-6 but not with pg-4-9, suggesting that the binding site of the p0071 head and repeat domains in plakoglobin differ from each other and that p0071 repeat binding depends on arm repeat 3 of plakoglobin, a region that is involved in plakoglobin-desmoglein interactions. The results of p0071 two-hybrid interactions are summarized in Table 1.
|
E-Cadherin is the typical classical cadherin of all normal epithelial cells and tissues. However, several carcinoma cells express more than one cadherin and N-cadherin has been detected in several tumor cell lines. To investigate whether p0071 preferentially interacts with one of the cadherins we quantitated p0071-cadherin interactions. Whereas the p0071-E-cadherin and OB-cadherin/cadherin 11 interactions were very similar, p0071-N-cadherin was more than three times as efficient in activating the lacZ reporter gene (Fig. 5B). Whether this preference for N-cadherin observed in the two-hybrid system is of physiological significance remains to be determined in other experimental systems.
The finding that the p0071 arm repeat domain interacts with non-desmosomal cadherins is consistent with its colocalization with E-cadherin in transfected cells and with its capacity to recruit non-desmosomal cadherins to sites of cell cell contact.
In vivo interactions of p0071 head and p0071 armadillo repeat
domains
In order to verify the two-hybrid interaction data we used an intracellular
targeting assay to investigate protein-protein interactions in mammalian
cells. The cytoplasmic domains of dsg 1-3, dsc 1a-3b and E-cadherin were fused
to the TOM70 mitochondrial membrane anchor, thus replacing the transmembrane
domains of the full length proteins
(Kaufmann et al., 2000). The
constructs also contained a flag tag to facilitate detection. Owing to the
TOM70 membrane anchor, these proteins are expressed on the cytoplasmic surface
of mitochondria. Double transfection experiments with the
mitochondria-anchored cadherin cytoplasmic domains and the p0071 head domain
showed no colocalization with E-cadherin,
(Fig. 6; Table 2), the desmogleins 1-3,
desmocollins 1b, 2b and 3b (not shown) and desmocollins 1a and 2a
(Fig. 6). In contrast, the
desmocollin 3a domain was able to recruit the p0071 head domain to
mitochondria and both proteins colocalized
(Fig. 6). This finding is in
agreement with the two-hybrid interaction data, where an interaction between
the p0071 head domain and desmocollin 3a but not with other desmocollin or
desmoglein isoforms or non-desmosomal cadherins was observed.
|
|
We also examined interactions between the p0071 repeat and tail domains with desmoglein and desmocollin isoforms and E-cadherin. In contrast to the head and tail domains p0071 repeats were recruited to E-cadherin coated mitochondria (Fig. 6), indicating that the E-cadherin interaction is exclusively mediated by the arm repeat domain in vivo. Again, this finding is consistent with the two-hybrid interaction data. No recruitment was detected for the desmoglein and desmocollin isoforms nor the arm repeat or the tail domain of p0071 (data not shown). Interactions between p0071 and plakoglobin or the DP-N-terminus could not be analyzed by this assay since these proteins and protein fragments contained intracellular targeting signals that were dominant over the TOM70 membrane anchor.
Interactions between p0071 and plakoglobin
All p0071 domains interacted with plakoglobin in the yeast two-hybrid assay
and use of plakoglobin fragments suggested an interaction of the p0071 head
with the plakoglobin tail. In contrast, the p0071 repeat interaction with
plakoglobin did not depend on the end domains. To verify the p0071-plakoglobin
interactions in mammalian cells we cotransfected p0071 domains with
plakoglobin fragments and analyzed the extent of colocalization. Transfection
of p0071wt together with plakoglobin (pg-myc) resulted in colocalization of
both proteins along the plasma membrane. The overlap observed was not
identical with desmosomal labeling (Fig.
7). P0071 headless colocalized with N- and C-terminally deleted
plakoglobin (pg-N
C), whereas a plakoglobin fragment lacking
almost the entire repeat region (pg-
Sac) distributed in a diffuse
pattern in the cytoplasm and the nucleus and was not recruited to the
membrane. This supports our conclusion that the p0071 repeat-plakoglobin
interaction relies on the presence of the plakoglobin armadillo domain. In
contrast, cotransfection of the p0071 head domain with pg-
N
C
revealed no, or very restricted, colocalization of these two polypeptides.
This plakoglobin fragment showed predominantly a punctate distribution in the
cytoplasm with poor membrane association in MCF-7 cells. The plakoglobin
fragment lacking most of its repeat region (pg-
Sac) showed partial
membrane association when cotransfected with the p0071 head domain, consistent
with the finding in the yeast two-hybrid assay.
|
![]() |
Discussion |
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P0071 is both a desmosomal protein and an
adherens-junction-associated protein
Although p0071 is by its sequence more closely related to
NPRAP/-catenin, p120ctn and ARVCF, its intracellular
localization resembles more that of plakophilins 1-3, since in most cells
analyzed p0071 colocalized with desmosomal markers
(Hatzfeld and Nachtsheim,
1996
). In order to exclude that this observation was based on
antibody crossreactivity with plakophilins we have generated a new monoclonal
antibody directed against the p0071 tail domain that shows no sequence
homology to plakophilins. The 6D1-10 monoclonal antibody showed no
crossreactivity with either the desmosomal plakophilins 1-3 or
p120ctn but it also reacted with the closest relative of p0071,
NPRAP/
-catenin. Since this protein is expressed only in neuronal cells
and was not detected at the RNA or protein level in MCF-7 cells (M.H.,
unpublished), we conclude that in epithelial cells the signal obtained is
specific for p0071. By using this new monoclonal antibody we confirmed our
earlier results and showed colocalization of endogenous p0071 with desmosomes
in MCF-7 epithelial cells.
In contrast to endogenous p0071, overexpressed p0071wt colocalized with
non-desmosomal cadherins and was able to recruit cadherins to the plasma
membrane. The same phenotype has been described for NPRAP/-catenin
(Lu et al., 1999
), the closest
relative of p0071. Thus, at high expression levels p0071 behaves like
NPRAP/
-catenin, ARVCF and p120ctn
(Anastasiadis and Reynolds,
2000
). Desmosomal markers were displaced from plasma membrane
sites highly enriched in p0071, which suggests that unregulated p0071
expression interferes with the normal balance between desmosomes and adherens
junctions. Therefore, it is conceivable that the level of p0071 expression not
only determines its intracellular localization but also influences number and
size of both adhesive structures.
P0071 interactions with desmosomal and adherens junction proteins are
mediated by different domains
Binding partners of plakophilins, p120ctn, ARVCF and
NPRAP/-catenin are either desmosomal proteins [for plakophilins 1-3
(Chen et al., 2002
;
Hatzfeld et al., 2000
) S.
Bonné, B. Gilbert, M.H. et al., unpublished] or the non-desmosomal
cadherins [for p120ctn,
-catenin and ARVCF
(Kaufmann et al., 2000
;
Lu et al., 1999
;
Mariner et al., 2000
;
Reynolds et al., 1996
;
Shibamoto et al., 1995
;
Staddon et al., 1995
;
Thoreson et al., 2000
;
Waibler et al., 2001
)]. In
contrast, p0071 colocalizes with both types of junctions, which suggests
interactions with different types of junctional proteins. To elucidate this
seemingly contradictory behaviour of endogenous and exogenous p0071 we have
analyzed targeting of individual domains of p0071 and identified direct
binding partners in the yeast two-hybrid system.
Both N- and C-terminal domains were able to associate with desmosomes and
the N-terminal domain interacted with the desmosomal proteins dsc3a,
desmoplakin and plakoglobin but did not interact with adherens junction
proteins. Fig. 8A shows the
direct binding partners of p0071 domains,
Fig. 8B summarizes p0071
interactions in the desmosome. As anticipated, the N-terminal coiled-coil
motif conserved between p120ctn, NPRAP/-catenin, ARVCF and
p0071 but not the plakophilins 1-3
(Anastasiadis and Reynolds,
2000
) was not essential for desmosome targeting, which seems to be
a unique feature of p0071. In addition, head and tail domains of p0071 were
detected in the nucleus. In desmosome-bearing cells, the head domain
associated preferentially with desmosomes, whereas in cells lacking desmosomes
the head was not able to associate with the membrane and localized
predominantly in the nucleus (M.H., unpublished). As described for ARVCF
(Mariner et al., 2000
) and
plakophilin 1 (Hatzfeld et al.,
2000
) N-terminal sequences seem to be responsible for nuclear
targeting despite the presence of a putative nuclear localization signal in
the arm repeat region of all p120ctn family members. Although a
signaling function of p120ctn family members is generally accepted,
the extent of nuclear localization varies considerably between the members of
the family and nuclear binding partners have not yet been identified with the
exception of Kaiso, a transcription factor interacting with p120ctn
(Daniel and Reynolds,
1999
).
|
The tail domain localized preferentially in the nucleus in all cell types
analyzed and only minor amounts were cytoplasmic and desmosome-associated.
Since there is no classical nuclear localization signal (NLS) in the tail
domain the protein may be imported into the nucleus only after associating
with a bona fide nuclear protein. In addition, an interaction of the p0071
tail with the PDZ-domain proteins papin and erbin has recently been reported
(Deguchi et al., 2000;
Izawa et al., 2002
;
Jaulin-Bastard et al., 2002
;
Laura et al., 2002
). Erbin
seems to be able to associate with different types of junctions, adherens
junctions, desmosomes and hemidesmosomes
(Favre et al., 2001
) and
several lines of evidence support a role in appropriate organization of
cytoskeletal elements and epithelial cell polarity in a rho-dependent manner.
Recent data suggest that membrane association of papin and erbin does not
depend on p0071 but that these proteins come to the regions of cell contact
independently and interact with each other on the lateral membrane
(Ohno et al., 2002
).
In contrast to the end domains, the central arm repeat domain of p0071
behaves similarly to p120ctn, NPRAP/-catenin and ARVCF and
associates directly with various non-desmosomal cadherins of adherens
junctions (summarized in Fig.
8C). Deletion of p0071 repeat 1 abolished this interaction, which
suggests that this region is important for p0071-E-cadherin binding.
Interestingly, a splice variant in repeat 1 of mouse p0071 has been described
before (Hatzfeld and Nachtsheim,
1996
), and on the basis of preliminary observations a putative
correlation between expression of the longer splice variant and cadherin
association of p0071 had been suggested. The finding that repeat 1 is
important for the interaction is in agreement with the hypothesis of a
differential interaction of both splice variants with non-desmosomal
cadherins. However, we did not detect the longer rare splice variant in human
tumor cell lines derived from tissues corresponding to the mouse tissues
expressing this splice variant. Further investigation of the two splice
variants and their association with cadherins will be necessary to clarify
whether these alternative splice variants differ in their affinity for
cadherins and if this mechanism may contribute to the regulation of the
intracellular localization of p0071.
Although p120ctn and p120ctn-like proteins may share
redundant functions, it is clear that their roles in cell adhesion and
motility are not identical: whereas p120ctn and
NPRAP/-catenin overexpression produced a strong branching phenotype
(Lu et al., 1999
;
Reynolds et al., 1996
), this
effect is much weaker for p0071 and ARVCF
(Mariner et al., 2000
). The
strength of the phenotype also depended on the cell type analyzed and one
aspect possibly involved in mediating these differences is the composition of
junctional structures. In contrast to E-cadherin, which mediates strong
adhesion (Behrens et al., 1989
;
Behrens et al., 1993
;
Hermiston et al., 1996
;
Nabeshima et al., 1997
),
N-cadherin has been implicated in elevated cell motility and metastasis
(Cavallaro et al., 2002
;
Hazan et al., 2000
). It is not
known how adhesion is regulated by the interaction of p120ctn
family members with the juxtamembrane domain of cadherins. Conflicting results
have been described, since p120ctn either promoted or reduced
adhesion (Anastasiadis and Reynolds,
2000
; Aono et al.,
1999
; Chen et al.,
1997
; Lu et al.,
1999
; Mariner et al.,
2000
; Paulson et al.,
2000
; Thoreson et al.,
2000
; Yap et al.,
1998
; Horikawa and Takeichi,
2001
). Therefore, the mechanisms by which differential
interactions of p0071 with cadherin family members might modulate the balance
between adhesion and motility remain poorly understood.
P0071-plakoglobin interactions may be involved in regulating the
balance between adherens junctions and desmosomes
The head and arm repeat domains of p0071 interacted with plakoglobin, so
far the only known protein present in both adherens junctions and desmosomes
(Cowin et al., 1986). Using
plakoglobin deletion clones we present evidence that the p0071 head and arm
repeats may bind to different sites in plakoglobin. Since we could not confirm
these direct interactions in in vitro binding assays it is formally possible
that they are artefacts of the two-hybrid system. However, it is also possible
that interactions are stabilized in multiprotein complexes in vivo by
cooperative binding mechanisms that make it difficult to analyze the
individual interactions in vitro. Moreover, p0071 is not easily solubilized
from desmosome-containing cells complicating co-immunoprecipitation analyses
of such stable protein complexes. This explanation is supported by the results
of the transfection experiments with p0071 and plakoglobin fragments, which
are consistent with the two-hybrid data.
p0071 is possibly the first binding partner of the plakoglobin tail domain
that has been implicated in regulating its subcellular distribution and
desmosome size (Palka and Green,
1997). A role for the plakoglobin C-terminus in determining
desmosome size is based on the observation that expression of C-terminal
truncated plakoglobin in COS cells leads to striking alterations in desmosome
morphology with formation of extremely long junctions or groups of tandemly
linked desmosomes. Together with the data presented here, it seems plausible
that protein-protein interactions between the plakoglobin tail and p0071 may
contribute to this size-limiting function of the plakoglobin tail. Binding of
the p0071 head domain to plakoglobin leaves the plakoglobin-dsg binding site
accessible, which is consistent with the finding that the head domain targets
p0071 to desmosomes (see Fig.
8B). In contrast, the p0071 arm-repeat binding site in plakoglobin
overlaps with its dsg binding site so that binding of p0071 repeats could
prevent plakoglobin from interacting with desmosomal cadherins, which is
consistent with its role in targeting p0071 to adherens junctions (see
Fig. 8C). It is conceivable
that the interaction between plakoglobin and p0071 plays a role in targeting
these two proteins either to desmosmes or to adherens junctions. The
differential interaction of p0071 with plakoglobin described here might be one
mechanism required for the formation and regulation of the two types of
intercellular junctions.
A role for plakoglobin in regulating desmosome formation has been suggested
in an in vitro model using adhesion-defective cells
(Lewis et al., 1997). In this
system, plakoglobin had to be linked to E-cadherin before cells began to
assemble desmosomes. In vivo, the situation is more complex because
ß-catenin can partially compensate for the loss of plakoglobin
(Ruiz et al., 1996
;
Bierkamp et al., 1996
). In
general, formation of adherens junctions seems to preceed desmosome formation
and adherens junctions and desmosomes alternate in a regular pattern along the
plasma membrane of epithelial cells. A role of p0071 in regulating the balance
between desmosomal and cadherin-mediated adhesion is suggested by the
observation that plasma membrane sites highly enriched in p0071 not only
recruited non-desmosomal cadherins but also revealed displacement of
desmosomal markers. This suggests that unregulated p0071 expression interferes
with the normal balance between these structures. Elucidating the mechanisms
that direct p0071 to either desmosomes or adherens junctions will help to
further clarify its role in regulating cell cell adhesion.
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Acknowledgments |
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