1 Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London
WC2A 3PX, UK
2 Department of Dermatology, Kurume University School of Medicine, 67
Asahimachi, Kurume, Fukuoka 830, Japan
* Author for correspondence (e-mail: fiona.watt{at}cancer.org.uk)
Accepted 23 September 2002
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Summary |
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Key words: Envoplakin, Periplakin, Intermediate filaments, Cornified envelope, Keratinocytes
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Introduction |
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Like the other conventional plakins, envoplakin and periplakin have an
N-terminal globular domain, a central rod domain comprising heptad repeats,
and a C-terminal globular domain (Ruhrberg
et al., 1996; Ruhrberg et al.,
1997
). When transfected into cells full length periplakin
localises to desmosomes, the interdesmosomal plasma membrane and intermediate
filaments, the N-terminal domain mediating the interaction with the plasma
membrane and the C-terminus associating with intermediate filaments
(DiColandrea et al., 2000
).
Full length envoplakin mainly accumulates in aggregates associated with
intermediate filaments. The envoplakin rod domain is required for aggregation
and the periplakin rod domain is necessary and sufficient to redistribute
envoplakin to desmosomes (DiColandrea et
al., 2000
). The observations on envoplakin aggregates support the
conclusion that envoplakin and periplakin can heterodimerise via their rod
domains (Ruhrberg et al.,
1997
).
The C-termini of the plakins contain a variable number of tandem repeats
(plakin repeats) that are predicted to fold into discrete subdomains,
designated A, B and C, consisting of helices separated by ß turns
(Sawamura et al., 1991
;
Wiche et al., 1991
;
Green et al., 1992
;
Ruhrberg and Watt, 1997
;
Kowalczyk et al., 1999
).
Desmoplakin has one A, one B and one C-type plakin repeat; plectin has five B
repeats and one C; and different isoforms of BPAG1 have either no repeats, one
A repeat or one B and one C repeat (reviewed by
Leung et al., 2002
). The
C-termini of envoplakin and periplakin are considerably shorter than those of
other plakins, with envoplakin having one C repeat, and periplakin no repeats
(Ruhrberg et al., 1996
;
Ruhrberg et al., 1997
;
Leung et al., 2002
).
The C-terminus of periplakin comprises only the L-subdomain (for `linker'),
which in the other plakins links the C repeat with the preceding subdomain or
with the rod domain (Ruhrberg et al.,
1997; Leung et al.,
2001
; Leung et al.,
2002
). The L-subdomain is the region of highest sequence
conservation amongst different plakin family members and is also the most
highly conserved between mouse and human
(Mahoney et al., 1998
;
Määttä et al.,
2000
). It is lacking in the more distantly related proteins
epiplakin (Fujiwara et al.,
2001
) and MACF/ACF-7, the mammalian homologue of
Drosophila Kakapo (Gregory and
Brown, 1998
; Strumpf and Volk,
1998
; Leung et al.,
1999
; Karakesisoglou et al.,
2000
; Leung et al.,
2001
; Leung et al.,
2002
). In transient transfection experiments the C-terminus of
periplakin associates with intermediate filaments, whereas the envoplakin
C-terminal C box, lacking the linker motif, has a punctate distribution
throughout the cytoplasm (DiColandrea et
al., 2000
). This led us to propose that the linker sequence
mediates intermediate filament binding
(DiColandrea et al., 2000
).
The association of other plakins with intermediate filaments has already
been examined in some detail, using a variety of experimental approaches.
Yeast two-hybrid analysis of the C-terminus of desmoplakin is consistent with
a role for the linker domain in the interaction of desmoplakin with vimentin;
however, a sequence within the C subdomain is implicated in the interaction of
desmoplakin with keratins, and peptide competition experiments suggest that
the equivalent region of envoplakin and plectin has the same function
(Meng et al., 1997). Recently
the A, B and C subdomains of desmoplakin have been purified and tested for
vimentin binding by co-sedimentation (Choi
et al., 2002
); these studies show that the linker region does not
contribute significantly to binding affinity, but may provide the flexibility
that allows the combination of B and C domains to bind more strongly than
either domain individually. The intermediate filament binding region of
plectin has been mapped to 50 amino acids linking the C-terminal repeat
domains 5 and 6 [box B and C in the nomenclature of Green et al.
(Green et al., 1992
)]; the
first 7 of these amino acids are within the B box, while the rest are within
the linker domain (Nikolic et al.,
1996
; Steinböck et al.,
2000
).
The aim of our experiments was to carry out a more detailed study of the interaction of envoplakin and periplakin with intermediate filaments. We have mapped regions of the linker motif that are required for periplakin to associate with intermediate filaments; we show that periplakin stabilises the interaction of envoplakin with intermediate filaments; and we demonstrate that some envoplakin and periplakin interactions are not dependent on an intact rod domain.
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Materials and Methods |
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All envoplakin cDNA constructs were tagged at the C-terminus with the FLAG
epitope and expressed in pCI-neo, as described previously
(DiColandrea et al., 2000).
Full-length envoplakin (DiColandrea et al.,
2000
) was used as a template to generate E-1/4R+L+C in pCI-neo by
PCR. The primers used were 5'-ATGCTCAACAGGGAGCGCACGGCCCGGCAGGC-3'
and 5'-GGTCGTAAAGGCAAGTGCTGGCTCAGGCTGGGCTTGGC-3'. The C box was
deleted from E-1/4R+L+C (deletion mutant
AA 1836-2011;
E-1/4R+L
C) by PCR deletion mutagenesis using these primers:
5'-CCAGCGGCACTGGAGGGGTACCGCTGCTA-3' and
5'-CAGGAGCAGCAGGCCGCTCAGGGGGTCTTT-3'. The rod domain deleted
envoplakin construct (E
Rod) was made by PCR deletion mutagenesis using
full-length envoplakin cDNA as a template and these primers:
5'-CTGGCTGAGCTCCTCCCGGCTCACCTTGGCGTG-3' and
5'-GACTACAAGGACGACGATGACAAGTGA-3'. Full-length envoplakin was
digested with SacII, removing two fragments that were self-ligated to
yield E-
1/2N1/2R. All envoplakin and periplakin plasmid constructs were
verified by dye-terminator cycle sequencing (Applied Biosystems).
Transient transfection of cells
COS7 cells (African green monkey kidney derived) and HeLa cells were
maintained in DMEM supplemented with 10% FCS. The day before transfection,
cells were seeded on glass coverslips in 24-well plates at the density of
5x104 cells/well. Cells were treated with transfection
reaction mixture for 2-3 hours, washed with PBS, and transferred to DMEM
containing 10% FCS. The transfection mixture consisted of Superfect reagent
(Qiagen) combined with serum-free DMEM and plasmid DNA, according the
manufacturer's protocol. Primary human keratinocytes were cultured and
transfected as described previously
(DiColandrea et al., 2000).
Immunofluorescence analysis
The following antibodies were used: LP34 [mouse monoclonal antibody to
keratins 5, 6 and 18 (Lane et al.,
1985)]; LL001 [mouse monoclonal antibody to keratin 14
(Lane, 1982
)]; V9 (mouse
monoclonal antibody to vimentin; Novocastra); TUB 1A2 (mouse monoclonal
antibody to ß-tubulin; Sigma-Aldrich); rabbit anti-HA (Y-11; Santa Cruz),
rabbit anti-FLAG (Santa Cruz Biotechnology) and M2 (mouse monoclonal antibody
to FLAG; Sigma). Alexa-488- or Alexa-594-conjugated goat anti-rabbit or -mouse
IgG (Molecular Probes) was used as secondary antibody.
Cells on coverslips that were to be stained without prior saponin extraction were fixed in cold acetone/methanol 1:1 for 5 minutes on ice or in 4% paraformalehyde (Sigma)/PBS for 20 minutes at room temperature. Paraformaldehyde-fixed cells were subsequently permeabilized with 0.2% Triton X-100 (Sigma) for 5 minutes at room temperature. After fixation cells were washed in PBS and blocked in a 1:500 dilution of normal goat serum (Sigma) in PBS for 10 minutes at room temperature. Cells were incubated with primary antibodies for 45 minutes at room temperature, washed in PBS, and then incubated for a further 45 minutes at room temperature with the appropriate Alexa-conjugated secondary antibodies. Polymerised actin was detected with Alexa-594-conjugated phalloidin (Molecular Probes). Nuclear counter staining was performed with TOTO-3 (Molecular Probes). After further washing in PBS and distilled water, coverslips were mounted in Gelvatol (Monsanto) and examined using a laser scanning confocal microscope (LSM 510; Carl Zeiss).
To remove detergent-labile cytoskeletal components, cells on coverslips
were extracted with saponin prior to fixation. The methods of Svitkina et al.
(Svitkina et al., 1995;
Svitkina et al., 1996
) and
Herrmann and Wiche (Herrmann and Wiche,
1983
) were used with some modifications. Briefly, cells were
treated with 1% Triton X-100 or 0.6% saponin in imidazole buffer (50 mM
imidazole, pH 6.8, 50 mM KCl, 0.5 mM MgCl2, 0.1 mM EDTA, 1 mM EGTA)
containing 4% polyethelene glycol (Mr 8000: PEG8000)
(Sigma) for 30 minutes at room temperature. Extracted cells were washed gently
in PBS, fixed in 4% paraformaldehyde/PBS at 4°C for 30 minutes or
overnight and then processed as described above for immunofluorescence
staining or as described below for scanning electron microscopy.
Scanning electron microscopy
Non-extracted cells and cells that had been extracted in saponin were
gently washed in PBS and fixed in 4% paraformaldehyde in PBS overnight at
4°C. Fixed cells were dehydrated in graded ethanols up to 100%, then in
two changes of acetone. Critical point drying was performed using a Polaron
critical point dryer (Polaron, UK). Dried samples were rotary shadowed with
platinum in a Polaron sputter coater and examined in a JEOL 5600 scanning
electron microscope.
Overlay binding assay
Labelled periplakin protein probes used in the overlay assays were
generated by in vitro transcription/translation using the TNT Quick Coupled
Transcription/Translation System or TNT Coupled Wheat Germ Extract System
(Promega, Madison, WI), in the presence of [35S]-methionine
(Amersham Pharmacia Biotech, Little Chalfont, UK). A partial desmoplakin I
cDNA with an intact C-terminus [DPNwt; a kind gift of Kathy Green,
Northwestern University Medical School
(Stappenbeck and Green, 1992
)]
was subcloned into Bluescript KS+ (Stratagene) prior to in vitro translation.
Human keratins 5 and 14 in pET vectors kindly provided by Elaine Fuchs
(University of Chicago) (Coulombe and
Fuchs, 1990
) were also used for in vitro translation. Intermediate
filament proteins were purified from cultured primary human keratinocytes and
COS7 cells essentially as described
(Steinert et al., 1982
).
The overlay assays were performed as described
(Merdes et al., 1991).
Isolated intermediate filaments were resolved on 1-mm-thick SDS-polyacrylamide
gels made using ultra-pure electrophoresis reagents (Bio-Rad Laboratories,
Hercules, CA). Part of each gel was stained with Coomassie Brilliant Blue to
visualise the proteins, and the remainder was transferred to nitocellulose
membrane. [35S]-methionine-labeled probes were diluted in gelatin
buffer (0.9% NaCl, 20 mM Tris-HCl, pH 7.3, 1 mM MgCl2, 1 mM DTT,
0.1% Tween 20, 0.2% boiled gelatin, and 0.2 mM PMSF) such that all probes had
the same cpm/unit volume. Blots were incubated with probes for 1 hour at room
temperature in gelatin buffer then washed five times with gelatin buffer at
room temperature. After the final wash, blots were incubated with Amplify
Fluorographic Reagent (Amersham Pharmacia Biotech) and exposed to X-ray film
(Bio Max MS, Kodak, NY).
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Results |
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As one method to evaluate the strength of association of the periplakin
constructs with intermediate filaments we compared the localisation of the
constructs in cells that had been fixed without prior extraction and cells
that had been extracted in saponin prior to fixation. The method we used was
based on those described previously that preserve the interaction between
plectin and intermediate filaments
(Herrmann and Wiche, 1983;
Svitkina et al., 1995
;
Svitkina et al., 1996
).
Scanning electron microscopy revealed that after extraction with 0.6% saponin
for 15 minutes there was significant retention of the plasma membrane
(Fig. 2A), but when the
extraction time was increased to 30 minutes
(Fig. 2B,C) most of the plasma
membrane was lost. This was confirmed by loss of the integral membrane protein
HB-EGF after a 30 minute saponin treatment (as judged by immunofluorescence
staining; data not shown). Cells extracted for 30 minutes no longer had an
intact microtubule network (green in Fig.
2D,E), but polymerised actin (red in
Fig. 2D-F) and intermediate
filaments (green in Fig. 2F)
were unaffected. The distribution of desmoplakin and plakoglobin was also
unaffected by extraction (data not shown). When 1% Triton X-100 was
substituted for saponin the effects on the cytoskeleton were similar (data not
shown).
|
The results of transient transfection experiments in COS7 cells are summarised in Fig. 1C and illustrated in Fig. 3. In Fig. 1C, `+' refers to colocalisation that was sufficiently complete to make the distribution of periplakin almost indistinguishable from intermediate filaments (see, for example, Fig. 3A-C). `+/-' refers to partial colocalisation in which the filamentous distribution of the periplakin construct was evident without the need to overlay the intermediate filament staining pattern (Fig. 3J-L). `-' refers to constructs that had no obvious filamentous distribution even if some of the periplakin staining did overlap with intermediate filaments (see, for example, Fig. 3F).
|
The intact C-terminus of periplakin on its own (P-L) or connected to part
of the rod domain (amino acids 1588-1645; P-1/4R+L) (green fluorescence)
showed extensive colocalisation with vimentin intermediate filaments (red
fluorescence) in both non-extracted (Fig.
3A,E) and saponin extracted
(Fig. 3B-D and data not shown)
cells. Deletion of the C-terminal 10 or 20 amino acids of periplakin had no
effect on this distribution (P-L-10AA, P-L-20AA;
Fig. 1C and data not shown).
When all of homologous box 1 was deleted (P-LBox1) there was no
association of the periplakin C-terminus with vimentin filaments and the
construct was completely extractable with saponin
(Fig. 1C and data not shown).
Deletion of only 5 amino acids within box 1 (P-L
DWEEI) had the same
effect (Fig. 1C; green
fluorescence in Fig. 3F-H).
Although homologous box 1 was required for the intermediate filament
association of the periplakin C-terminus, it was not sufficient. Deletion of
the first 8 amino acids of box 2 (P-L1/2Box2) had the same effect as
deletion of box 1 (Fig. 1C and
data not shown). When the whole of box 2 was deleted (P-L
Box2) most of
the intermediate filament association was lost, but a small amount of
vimentin-associated protein was retained after detergent extraction
(Fig. 1C; green fluorescence in
Fig. 3I,J,L). The distribution
of several of the constructs (P-1/4+L, P-L, P-L
DWEEI and
P-L
Box1) was examined in transiently transfected, non-extracted primary
human keratinocytes (which have keratin filaments but no vimentin) and found
to be the same as in COS7 cells (Fig.
3M-P and data not shown).
Direct binding of the periplakin C-terminus to keratins and
vimentin
The immunofluorescence data suggested that the C-terminus of periplakin,
corresponding to the linker domain in other plakin family members, was
sufficient to mediate the association of periplakin with intermediate
filaments and that amino acids within the two regions that show highest
homology with the other plakins were necessary for this association. To
investigate whether the intermediate filament association was direct or
indirect we carried out overlay assays with several of the constructs
(Fig. 4; summarised in
Fig. 1C).
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|
Intermediate filaments were isolated from cultured human keratinocytes and COS7 cells. Coomassie Brilliant Blue (CBB in Fig. 4B) staining of keratinocyte intermediate filament preparations resolved by SDS-PAGE revealed two doublets, corresponding to the major keratins expressed by keratinocytes in culture (K5, K14, K6 and K16; data not shown). The major intermediate filament proteins expressed by COS7 cells are vimentin, keratins 8 and 18 (data not shown); these bands migrated as a broad doublet, with vimentin and keratin 8 in the upper band and keratin 18 in the lower band (Fig. 4B).
The intermediate filament preparations were transferred to nitrocellulose
membranes and incubated with equal amounts of radioactively labelled in vitro
translated proteins, washed extensively and subjected to fluorography
(Fig. 4B). Full length
periplakin (P-full), K5 and K14 and the desmoplakin construct (DP-C) all bound
to the keratinocyte and COS7 cell intermediate filament preparations, whereas
P-R and pCI-neo did not. In keratinocyte and COS7 extracts the binding of
P-1/4R+L was equal to P-full and stronger than that of the C-terminal deletion
constructs, P-LDWEEI, P-L
1/2Box2, P-L
Box2. The three
deletion constructs were indistinguishable in the overlay assays, whereas in
transfection experiments only P-L
Box2 showed any colocalisation with
intermediate filaments (see Fig.
1C, Fig. 3). All of
the constructs tested bound preferentially to the upper doublet in the
keratinocyte preparations, but bound equally to both bands in the COS7 cell
preparations, possibly indicating different affinities for different
intermediate filament proteins.
We conclude that the association of the periplakin C-terminus with intermediate filaments observed by immunofluorescence (Fig. 3) involves direct binding to intermediate filament proteins and that amino acids within the two homology boxes are required for maximal binding.
Interactions of periplakin and envoplakin constructs containing rod
domain deletions
Envoplakin and periplakin are known to associate via their rod domains
(Ruhrberg et al., 1997;
DiColandrea et al., 2000
) and
we wanted to investigate whether the two proteins could still interact when
part or all of each rod domain was deleted. When full length envoplakin is
transfected into cells it accumulates in aggregates with associated
intermediate filaments, whether simple or complex keratins, vimentin or
nuclear lamins (DiColandrea et al.,
2000
). Co-transfection with full length periplakin or its isolated
rod domain prevents envoplakin aggregates from forming and targets both
proteins to their correct subcellular locations, including desmosomes and
intermediate filaments (DiColandrea et al.,
2000
). Removal of the rod domain of envoplakin or periplakin
(E
Rod and P
Rod in Fig.
5) resulted in each protein having a diffuse, partially
filamentous, distribution throughout the cytoplasm and nucleus (green in
Fig. 6A,B). Each protein was
completely lost from the cytoplasm of cells treated with saponin prior to
fixation, although some protein remained in the nucleus (data not shown).
Co-transfection of the two rod domain deleted constructs (E
Rod,
P
Rod) resulted in the same distribution as when either construct was
transfected individually (Fig.
6C). The constructs in the cytoplasm of doubly transfected cells
were also completely extractable with saponin, although transfected cells
could be detected by residual nuclear staining
(Fig. 6D).
|
As expected, co-transfection of rod deleted periplakin (PRod) with
full length envoplakin (E-full) failed to prevent envoplakin from forming
detergent insoluble aggregates (Fig.
6E-G; E-full is shown in green in each panel and P
Rod in
red). In most cells the rod deleted periplakin showed extensive colocalisation
with envoplakin in aggregates (Fig.
6E,F). Since the aggregates have previously been shown to contain
intermediate filament proteins (DiColandrea
et al., 2000
) periplakin could be binding via its C-terminus to
intermediate filaments in the aggregates. However, in some doubly transfected
cells the envoplakin aggregates were smaller (e.g.
Fig. 6H; E-full is shown in
green and P
Rod in red) than in singly transfected cells and these small
aggregates were detergent sensitive (see
Fig. 6I in which only large
envoplakin aggregates, shown in green, remain in a detergent extracted cell;
red indicates vimentin filaments). Thus P
Rod could partially alter the
distribution of full length envoplakin by reducing the size and increasing the
detergent solubility of envoplakin aggregates.
The effect of full length periplakin on the distribution of ERod was
more pronounced. In the presence of P-full, E
Rod showed increased
colocalisation with intermediate filaments (compare
Fig. 6A with 6J-L; envoplakin
signal is green; P-full is red in 6K,L). The intermediate filament association
of E
Rod under those circumstances was, however, detergent sensitive
(data not shown). These experiments demonstrate that full length periplakin
could recruit E
Rod to intermediate filaments, suggesting that
interactions between the two proteins can occur outwith the envoplakin rod
domain.
To further examine the role of the rod domain in periplakin/envoplakin
interactions we transfected COS7 cells with E-1/2N1/2R. This construct
consists of the first half of the envoplakin-N terminal domain, the C-terminal
half of the rod domain and the complete C-terminus
(Fig. 5). E-
1/2N1/2R
formed cytoplasmic aggregates (Fig.
7A-D; envoplakin shown in green, vimentin in red) that were
largely removed by saponin extraction (Fig.
7E-H; envoplakin in green, vimentin in red), in contrast to the
aggregates formed by full length envoplakin
[(DiColandrea et al., 2000
) and
data not shown]. Co-transfection with full length periplakin resulted in the
envoplakin construct colocalising with intermediate filaments and becoming non
extractable in saponin (Fig.
7K,L; envoplakin is green in each panel). Some bundling of
intermediate filaments was observed in doubly transfected cells
(Fig. 7K,L) that was not seen
in cells transfected with full length periplakin alone.
Fig. 7I,J shows full length
periplakin (green) colocalising with vimentin (red, panel I) or keratins (red,
panel J).
|
We conclude that removal of part of the N-terminus and rod domain does not prevent envoplakin from aggregating, nor from being rescued by full length periplakin. All of the observations made in non-extracted COS7 cells using the constructs shown in Fig. 5 were confirmed in HeLa cells and primary human keratinocytes (data not shown).
Periplakin stabilises the association of envoplakin with intermediate
filaments
The effects of P-full on ERod
(Fig. 6) and E-
1/2N1/2R
(Fig. 7) suggested that
periplakin could affect the association of envoplakin with intermediate
filaments. In addition to containing the linker motif, the C-terminus of
envoplakin contains one plakin repeat, designated a C subdomain
(Ruhrberg et al., 1996
;
Ruhrberg and Watt, 1997
). We
have previously reported that when transiently transfected into keratinocytes
the C subdomain of envoplakin is distributed in a punctate pattern throughout
the cytoplasm and does not show any colocalisation with the cytoskeleton
(DiColandrea et al., 2000
).
When the entire envoplakin C-terminus, comprising the linker and C box,
coupled to part of the rod domain, was transfected into COS7 cells (E-1/4R+L+C
in Fig. 5; see
Fig. 8A-C) its distribution was
primarily punctate in the cytoplasm but showed partial colocalisation with
intermediate filaments (envoplakin is green in panels A,B; vimentin is red in
A,C). This protein was completely extracted by saponin treatment
(Fig. 8D; note that there is no
residual green fluorescence attributable to the envoplakin construct).
Deletion of the C box had no effect on the distribution
(Fig. 8E-G) or detergent
solubility (Fig. 8H) of the
envoplakin C-terminus (green in panels E,F,H; vimentin is red). However, when
E-1/4R+L+C was co-transfected with P-1/4R+L the envoplakin protein showed
extensive colocalisation with intermediate filaments
(Fig. 8I; green fluorescence
denotes envoplakin construct; red fluorescence denotes periplakin construct)
and could not be extracted with saponin
(Fig. 8J-L; green: envoplakin;
red: periplakin).
|
These experiments confirm that envoplakin and periplakin can still interact when most of their rod domains have been deleted and suggest that periplakin enhances and stabilises the interaction of envoplakin with intermediate filaments.
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Discussion |
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We have previously shown that the C subdomain of envoplakin does not
colocalise with intermediate filaments in transiently transfected cells
(DiColandrea et al., 2000); the
same is true of the isolated C box of desmoplakin
(Stappenbeck et al., 1993
).
Different regions of the desmoplakin C-terminus interact with keratins and
vimentin (Stappenbeck et al.,
1993
; Meng et al.,
1997
) and in assays with purified recombinant proteins, the
desmoplakin C-terminus is reported to interact directly with keratin 5, but
not with simple type II keratins, vimentin or type I keratins
(Kouklis et al., 1994
). Our
overlay data support a preferred interaction of desmoplakin with keratin 5
rather than 14; however, we also saw some binding to COS7 cell intermediate
filaments (predominantly vimentin and keratins 8 and 18).
Our understanding of the interaction of desmoplakin with intermediate
filaments has been greatly increased by the recent studies of Choi et al.
(Choi et al., 2002). They
compared the binding of the C-terminal A, B and C subdomains of desmoplakin to
vimentin in a co-sedimentation assay and also obtained the crystal structures
of the B and C subdomains. All three subdomains bound vimentin when added in
molar excess. A construct spanning domains A and B did not bind more strongly
than either subdomain alone, whereas the combination of B and C did show
enhanced binding. The B and C subdomains are connected by the linker sequence;
since the combination of B plus linker did not increase vimentin binding the
linker may contribute indirectly by providing the flexibility to allow more
than one subdomain to bind simultaneously
(Choi et al., 2002
). The
crystal structures of subdomains B and C suggest that a basic groove within
the desmoplakin C-terminal domain may form the intermediate filament binding
site (Choi et al., 2002
).
The intermediate filament binding domain of plectin binds to and crosslinks
all types of intermediate filament, but shows a higher affinity for vimentin
than keratins 5 and 14 (Steinböck et
al., 2000). One role of the C-terminal plakin repeats may be to
modulate the affinity of the linker for particular classes of intermediate
filament (Meng et al., 1997
;
Steinböck et al., 2000
).
It will be particularly interesting to know whether epiplakin, which consists
of 13 B subdomains, but lacks the conserved linker motif, has any intermediate
filament association (Fujiwara et al.,
2001
). Phosphorylation of plectin and desmoplakin regulates their
interaction with intermediate filaments
(Foisner et al., 1991
;
Stappenbeck et al., 1994
;
Wiche, 1998
;
Kowalczyk et al., 1999
), but
it remains to be determined whether this is also the case for envoplakin and
periplakin.
The ability of the envoplakin C-terminus to colocalise with intermediate
filaments was enhanced in the presence of the C-terminus of periplakin
(Fig. 8), suggesting that
periplakin not only mediates the association of envoplakin with the
interdesmosomal plasma membrane
(DiColandrea et al., 2000) but
also regulates its association with intermediate filaments. As overlay assays
were not performed with envoplakin constructs it is possible that rather than
periplakin enhancing direct binding of envoplakin to intermediate filaments
envoplakin binds to periplakin and only periplakin binds intermediate
filaments. This seems, however, unlikely, given that envoplakin aggregates
associate with intermediate filaments in the absence of periplakin
(DiColandrea et al., 2000
) and
that all of the other conventional plakins bind intermediate filament proteins
directly (reviewed by Leung et al., 2000). The envoplakin linker construct
(E-1/4R+L
C in Fig. 8)
did show some colocalisation with vimentin filaments; however, it is
surprising that it was not as strong as that of the periplakin linker
(Fig. 3), given that the
sequence conservation of the periplakin linker is highest with envoplakin
(Ruhrberg et al., 1997
;
Mahoney et al., 1998
;
Määttä et al.,
2000
). One way to investigate this further would be by generating
point mutations in the periplakin linker to systematically convert the
sequence to that of envoplakin and test the effects on intermediate filament
binding.
The envoplakin and periplakin C-terminal constructs tested for interaction
(E-1/4R+L+C and P-1/4R+L) each contained a partial rod domain and this may
have been sufficient for heterodimerisation. Alternatively the two C-termini
may interact directly, by analogy with the ability of the plectin intermediate
filament binding domain to self-associate
(Wiche et al., 1993;
Steinböck et al., 2000
).
We did not observe any collapse of the intermediate filament network in the
presence of the periplakin C-terminus, in contrast to the effects of the
plectin intermediate filament binding domain
(Steinböck et al., 2000
).
However, the bundling of filaments in cells transfected with
E-
1/2N
1/2R and full length periplakin
(Fig. 7K,L) would suggest that
envoplakin and periplakin can, like plectin, influence filament dynamics and
crosslinking (Steinböck et al.,
2000
).
While the rod domains are undoubtedly important for the interaction of
periplakin and envoplakin, most likely mediating heterodimer formation
(Ruhrberg et al., 1997;
DiColandrea et al., 2000
), our
data suggest that the proteins may be able to interact even when there are
deletions within each rod domain. Thus full length periplakin could prevent an
envoplakin construct lacking the N-terminal half of the rod domain from
forming aggregates and facilitated its association with intermediate filaments
(Fig. 7), and P-1/4R+L
influenced the distribution and extractability of E-1/4R+L+C
(Fig. 8). Perhaps most
surprising was the finding that the two proteins appeared able to interact
even when one was completely lacking the rod domain; this was most striking in
cells co-transfected with E
Rod and P-full, where the presence of P-full
allowed recruitment of E
Rod to intermediate filaments
(Fig. 6). There is good
evidence that plakins can form higher order complexes than dimers, although
the nature of the interactions involved is currently unclear (reviewed by
Ruhrberg and Watt, 1997
;
Wiche, 1998
; Kowalzcyk et al.,
1999). For example, plectin can self-associate in vitro and forms filamentous
side arms that emerge from vimentin filaments
(Foisner et al., 1988
;
Svitkina et al., 1996
;
Wiche, 1998
). Acidic and basic
residues are distributed periodically along the rod domains of plakins and
ionic interactions could play a role in formation of higher order filamentous
structures (Green et al.,
1992
). In the case of envoplakin and periplakin constructs lacking
the rod domains other types of association must be involved and it is
attractive to suggest a role for the linker domains, by analogy with the
ability of the plectin intermediate filament binding domain to self-associate
(Wiche et al., 1993
;
Steinböck et al.,
2000
).
In conclusion, we have shown that conserved sequences within the periplakin
C-terminus mediate the association of periplakin with intermediate filaments,
demonstrated that periplakin regulates the association of envoplakin with
intermediate filaments, and obtained evidence to suggest that the interactions
between envoplakin and periplakin extend to regions outwith their rod domains.
The challenge now is to uncover the role of these interactions in the context
of intact tissues, in particular the epidermis, where envoplakin and
periplakin are early precursors in the assembly of the cornified envelope
(Kalinin et al., 2001).
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
A recent study demonstrated a specific interaction of the periplakin linker
domain with keratin 8 and vimentin
(Kazerounian et al.,
2002).
![]() |
References |
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Aho, S., McLean, W. H., Li, K. and Uitto, J. (1998). cDNA cloning, mRNA expression, and chromosomal mapping of human and mouse periplakin genes. Genomics 48,242 -247.[CrossRef][Medline]
Choi, H. J., Park-Snyder, S., Pascoe, L. T., Green, K. J. and Weis, W. I. (2002). Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure. Nat. Struct. Biol. 9, 612-620.[Medline]
Coulombe, P. A. and Fuchs, E. (1990) Elucidating the early stages of keratin filament assembly. J. Cell Biol. 111,153 -169.[Abstract]
DiColandrea, T., Karashima, T., Määttä, A. and
Watt, F. M. (2000). Subcellular distribution of envoplakin
and periplakin: insights into their role as precursors of the epidermal
cornified envelope. J. Cell Biol.
151,573
-585.
Foisner, R., Leichtfried, F. E., Herrmann, H., Small, J. V., Lawson, D. and Wiche, G. (1988) Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins. J. Cell Biol. 106,723 -733.[Abstract]
Foisner, R., Traub, P. and Wiche, G. (1991). Protein kinase A- and protein kinase C-regulated interaction of plectin with lamin B and vimentin. Proc. Natl. Acad. Sci. USA 88,3812 -3816.[Abstract]
Fuchs, E. and Yang, Y. (1999). Crossroads on cytoskeletal highways. Cell 98,547 -550.[Medline]
Fujiwara, S., Takeo, N., Otani, Y., Parry, D. A. D., Kunimatsu,
M., Lu, R., Sasaki, M., Matsuo, N., Khaleduzzaman, M. and Yoshioka, H.
(2001). Epiplakin, a novel member of the plakin family originally
identified as a 450-kDa human epidermal autoantigen. Structure and tissue
localization. J. Biol. Chem.
276,13340
-13347.
Green, K. J., Virata, M. L., Elgart, G. W., Stanley, J. R. and Parry, D. A. (1992). Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: members of a new gene family involved in organization of intermediate filaments. Int. J. Biol. Macromol. 14,145 -153.[Medline]
Gregory, S. L. and Brown, N. H. (1998).
kakapo, a gene required for adhesion between and within cell layers
in Drosophila, encodes a large cytoskeletal linker protein related to
plectin and dystrophin. J. Cell Biol.
143,1271
-1282.
Herrmann, H. and Aebi, U. (2000). Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics. Curr. Opin. Cell Biol. 12,79 -90.[CrossRef][Medline]
Herrmann, H. and Wiche, G. (1983). Specific
in situ phosphorylation of plectin in detergent-resistant
cytoskeletons from cultured Chinese hamster ovary cells. J. Biol.
Chem. 258,14610
-14618.
Imai, Y., Matsushima, Y., Sugimura, T. and Terada, M. (1991). A simple and rapid method for generating a deletion by PCR. Nucleic Acids Res. 19, 2785.[Medline]
Kalinin, A., Marekov, L. N. and Steinert, P. M.
(2001). Assembly of the epidermal cornified cell envelope.
J. Cell Sci. 114,3069
-3070.
Karakesisoglou, I., Yang, Y. and Fuchs, E.
(2000). An epidermal plakin that integrates actin and microtubule
networks at cellular junctions. J. Cell Biol.
149,195
-208.
Kazerounian, S., Uitto, J. and Aho, S. (2002). Unique role for the periplakin tail in intermediate filament association: specific binding to keratin 8 and vimentin. Exp. Dermatol. 11,428 -438.[CrossRef][Medline]
Kouklis, P. D., Hutton, E. and Fuchs, E. (1994). Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins. J. Cell Biol. 127,1049 -1060.[Abstract]
Kowalczyk, A. P., Bornslaeger, E. A., Norvell, S. M., Palka, H. L. and Green, K. J. (1999). Desmosomes: intercellular adhesive junctions specialized for attachment of intermediate filaments. Int. Rev. Cytol. 185,237 -302.[Medline]
Lane, E. B. (1982). Monoclonal antibodies provide specific molecular markers for the study of epithelial tonofilament organization. J. Cell Biol. 92,665 -673.[Abstract]
Lane, E. B., Bartek, J., Purkis, P. E. and Leigh, I. M. (1985). Keratin antigens in differentiating skin. Annu. New York Acad. Sci. 455,241 -258.[Medline]
Leung, C. L., Sun, D., Zheng, M., Knowles, D. R. and Liem, R.
K. (1999). Microtubule actin cross-linking factor (MACF): a
hybrid of dystonin and dystrophin that can interact with the actin and
microtubule cytoskeletons. J. Cell Biol.
147,1275
-1286.
Leung, C. L., Liem, R. K., Parry, D. A. and Green, K. J.
(2001). The plakin family. J. Cell Sci.
114,3409
-3410.
Leung, C. L., Green, K. J. and Liem, R. K. (2002). Plakins: a family of versatile cytolinker proteins. Trends Cell. Biol. 12,37 -45.[CrossRef][Medline]
Ma, A. S. and Sun, T. T. (1986). Differentiation-dependent changes in the solubility of a 195-kD protein in human epidermal keratinocytes. J. Cell Biol. 103, 41-48.[Abstract]
Määttä, A., Ruhrberg, C. and Watt, F. M.
(2000). Structure and regulation of the envoplakin gene.
J. Biol. Chem. 275,19857
-19865.
Mahoney, M. G., Aho, S., Uitto, J. and Stanley, J. R. (1998). The members of the plakin family of proteins recognized by paraneoplastic pemphigus antibodies include periplakin. J. Invest. Dermatol. 111,308 -313.[Abstract]
Meng, J. J., Bornslaeger, E. A., Green, K. J., Steinert, P. M.
and Ip, W. (1997). Two-hybrid analysis reveals functional
differences in direct interactions between desmoplakin and cell type-specific
intermediate filaments. J. Biol. Chem.
272,21495
-21503.
Merdes, A., Brunkener, M., Horstmann, H. and Georgatos, S. (1991). Filensin: A new vimentin-binding, polymerization-competent, and membrane-associated protein of the lens fiber cell. J. Cell Biol. 115,397 -410.[Abstract]
Nikolic, B., Nulty, E. M., Mir, B. and Wiche, G. (1996). Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions. J. Cell Biol. 134,1455 -1467.[Abstract]
Ruhrberg, C. and Watt, F. M. (1997). The plakin family: versatile organizers of cytoskeletal architecture. Curr. Opin. Genet. Dev. 7,392 -397.[CrossRef][Medline]
Ruhrberg, C., Hajibagheri, M. A. N., Simon, M., Dooley, T. P. and Watt, F. M. (1996). Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin. J. Cell Biol. 134,715 -729.[Abstract]
Ruhrberg, C., Hajibagheri, M. A. N., Parry, D. A. D. and Watt,
F. M. (1997). Periplakin, a novel component of cornified
envelopes and desmosomes that belongs to the plakin family and forms complexes
with envoplakin. J. Cell Biol.
139,1835
-1849.
Sawamura, D., Li, K., Chu, M. L. and Uitto, J.
(1991). Human bullous pemphigoid antigen (BPAG1). Amino acid
sequences deduced from cloned cDNAs predict biologically important peptide
segments and protein domains. J. Biol. Chem.
266,17784
-17790.
Simon, M. and Green, H. (1984). Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte. Cell 36,827 -834.[Medline]
Smith, E. A. and Fuchs, E. (1998). Defining the
interactions between intermediate filaments and desmosomes. J. Cell
Biol. 141,1229
-1241.
Stappenbeck, T. S. and Green, K. J. (1992). The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks when expressed in cultured cells. J. Cell Biol. 116,1197 -1209.[Abstract]
Stappenbeck, T. S., Bornslaeger, E. A., Corcoran, C. M., Luu, H. H., Virata, M. L. and Green, K. J. (1993). Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks. J. Cell Biol. 123,691 -705.[Abstract]
Stappenbeck, T. S., Lamb, J. A., Corcoran, C. M. and Green, K.
J. (1994). Phosphorylation of the desmoplakin C-terminus
negatively regulates its interaction with keratin intermediate filament
network. J. Biol Chem.
269,29351
-29354.
Steinböck, F. A., Nikolic, B., Coulombe, P. A., Fuchs, E.,
Traub, P. and Wiche, G. (2000). Dose-dependent linkage,
assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments
through plectin's intermediate filament-binding domain. J. Cell
Sci. 113,483
-491.
Steinert, P. M. and Marekov, L. N. (1999).
Initiation of assembly of the cell envelope barrier structure of stratified
squamous epithelia. Mol. Biol. Cell
10,4247
-4261.
Steinert, P. M., Zockroff, R., Aynardi-Whitman, M. and Goldman, R. D. (1982). Isolation and characterization of intermediate filaments. Methods Cell Biol. 24,399 -419.[Medline]
Strumpf, D. and Volk, T. (1998). Kakapo, a
novel cytoskeletal-associated protein, is essential for the restricted
localization of the neuregulin-like factor, vein, at the muscle-tendon
junction site. J. Cell Biol.
143,1259
-1270.
Svitkina, T. M., Verkhovsky, A. B. and Borisy, G. G. (1995). Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells. J. Struct. Biol. 115,290 -303.[CrossRef][Medline]
Svitkina, T. M., Verkhovsky, A. B. and Borisy, G. G. (1996). Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton. J. Cell Biol. 135,991 -1007.[Abstract]
Wiche, G. (1998). Role of plectin in cytoskeleton organization and dynamics. J. Cell Biol. 111,2477 -2486.
Wiche, G., Becker, B., Luber, K., Weitzer, G., Castanon, M. J., Hauptmann, R., Stratowa, C. and Stewart, M. (1991). Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a central alpha-helical coiled coil. J. Cell Biol. 114,83 -99.[Abstract]
Wiche, G., Gromov, D., Donovan, A., Castañón, M. J. and Fuchs, E. (1993). Expression of plectin mutant cDNA in cultured cells indicates a role of C-terminal domain in intermediate filament association. J. Cell Biol. 121,607 -619.[Abstract]