1 Laboratory of Physiological Chemistry and Centre for Biomedical Genetics,
University Medical Center Utrecht, Stratenum, Universiteitsweg 100, 3584 CG
Utrecht, The Netherlands
* Present address: Department of Biochemistry and Biophysics, UCSF, San
Francisco, CA 94143-0448, USA
Author for correspondence (e-mail:
b.m.t.burgering{at}med.uu.nl)
Accepted 25 July 2002
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Summary |
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Key words: PKB/Akt, Periplakin, Vimentin, Mitochondria, Insulin
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Introduction |
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Once activated, PKB can phosphorylate a range of proteins on either serine
or threonine residues contained within an RxRxxS/T motif. One of the first
identified substrates for PKB is glycogen synthase kinase-3 (GSK-3)
(Cross et al., 1995). PKB
phosphorylates Ser9 of GSK3ß and Ser21 of GSK3
both in vitro
(Cross et al., 1995
) and in
vivo (van Weeren et al.,
1998
), and this results in inactivation of GSK3 thereby regulating
glucose utilization and activation of glycogen synthesis
(Rylatt et al., 1980
).
PKB can also regulate transcription factor activity. Upon activation PKB
has been shown to translocate to the nucleus
(Andjelkovic et al., 1997;
Meier et al., 1997
), where it
can phosphorylate transcription factors such as the FOXO members of the
Forkhead family (reviewed by Kops and
Burgering, 1999
). This leads to nuclear export and inactivation of
these FOXO transcription factors. Regulation of FOXO transcriptional activity
has been implicated in cell cycle and cell death control by PKB. However,
besides FOXO regulation many other mechanisms have been described for
PKB-mediated protection from apoptosis. For example, direct phosphorylation of
proteins such as the pro-apoptotic Bcl-2 family member BAD
(Datta et al., 1997
), or
caspase-9 (Cardone et al.,
1998
). Irrespective of the multitude of potential protein targets
for PKB it is clear that PKB can protect from mitochondrial-dependent
apoptosis (Kennedy et al.,
1999
). Thus PKB activity can maintain mitochondrial membrane
stability and prevent cytochrome-C leakage under conditions of stress
(Gottlob et al., 2001
;
Plas et al., 2001
).
Periplakin belongs to the plakin family of cytolinker proteins that also
includes desmoplakin, envoplakin, plectin and bullous pemphigoid antigen 1
(BPAG1) (Ruhrberg et al.,
1997). It has an approximate molecular mass of 195 kDa, contains a
central rod dimerisation domain and its C-terminal region is involved in
intermediate filament binding, whereas its N-terminus has been shown to
interact with cortical actin (DiColandrea
et al., 2000
). Periplakin is expressed in epithelial cells
(Aho et al., 1998
), where it is
found to be, together with envoplakin, the precursor of the epidermal
cornified envelope (DiColandrea et al.,
2000
). Furthermore, periplakin has been shown to be a target
antigen in paraneoplastic pemphigus (de
Bruin et al., 1999
), and in keratinocytes it localizes at
desmosomes and the interdesmosomal plasma membrane
(Simon and Green, 1984
).
Although these observations suggest a specified role of plakin family members
in epidermal cornification there is substantial evidence that within other
cell types plakins may be involved in a variety of cellular processes. For
example, plectin, the best-studied member of the plakin family, has been
suggested to play a role in reorganization of microfilaments in apoptosis when
it is cleaved by caspase 8 (Stegh et al.,
2000
). A further role in this is suggested by the observation that
in muscle cells it is found associated with mitochondria and intermediate
filaments (Reipert et al.,
1999
). Also in endothelial cells plectin localizes to focal
contacts and here a role in adhesion is suggested
(Gonzales et al., 2001
).
In order to investigate PKB regulation through binding to other proteins in more detail we performed a yeast two-hybrid screen with a part of the PH domain of PKB. We found that periplakin is an interaction partner of PKB and determined the binding side within PKB. Furthermore we also show binding of periplakin to the intermediate filament protein vimentin. In addition to intermediate filament structures, periplakin localizes to the cell membrane, nucleus and mitochondria. Localization to these cellular compartments is in part influenced by the presence or absence of vimentin. These and other observations suggest that periplakin may act as a localization signal for PKB signalling.
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Materials and Methods |
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Antibodies
Anti-periplakin C-terminal peptide antibody was raised by immunizing two
rabbits with a peptide encoding the last 12 amino acids of periplakin
(IQELAVLVSGQK) coupled to KLH (7445 and 7446). This peptide is fully conserved
between human and mouse periplakin. The anti-GST-c-ppl sera were raised by
immunizing two rabbits with purified GST-c-ppl (5117 and 5118). Anti-PKB (5179
and 5178) (Burgering and Coffer,
1995); anti-MAPK [124 (de
Vries-Smits et al., 1992
)]; anti-HA [12CA5
(Burgering and Coffer, 1995
)]
and anti-myc [9E10 (van Weeren et al.,
1998
)] were described previously. Anti-cytochrome c was kindly
provided by F. J. T. Zwartkruis, (UMCU, Utrecht, Netherlands). Anti-vimentin
was purchased from Oncogene Science and anti-cox4 was from Molecular Probes.
MAB1273, which recognizes a 65 kDa mitochondrial protein, was obtained from
Chemicon International, anti-pSer193 FOXO4 was from Cell Signaling, and
anti-actin was from Santa Cruz.
Cell culture
Rat-1, A14 and COS-7 cells were grown in Dulbecco's modified Eagle's
medium. 293T (human embryonic kidney 293 cells immortalised with SV40 large T
antigen), MCF-7 and MCF-7/FR (MCF-7 cells stably expressing the Fas receptor)
cells were grown in RPMI. Both types of media were supplemented with 10% fetal
calf serum (Bio-Whittaker, Belgium), 1% penicillin/streptomycin
(Bio-Whittaker) and 2 mM L-Gln (Bio-Whittaker). Cells were treated for 10
minutes with 1 µM insulin after overnight starvation in medium without
serum, unless otherwise indicated. Cells were transfected using the
Ca(PO4)2 procedure, except MCF-7 cells, which were
transfected with Fugene6 (Roche).
Yeast two-hybrid screen
Yeast two-hybrid screens were performed as described previously
(Wolthuis et al., 1996). An
oligo-dT primed 13.5 day mouse embryo cDNA library cloned into the pPC86 yeast
two-hybrid vector was used in all cases. To diminish background, 50 mM
3-amino-triazol was included in the screen and clones were picked after 3 and
4 days.
GST pull-down assay
For purification of GST-c-ppl, protein expression was induced in DH5
using 100 nM isopropyl-1-thio-ß-D-galactopyranoside for 20 hours at room
temperature. Bacteria were collected and lysed in ice-cold phosphate-buffered
saline containing 1% Triton X-100 and protease inhibitors. The lysates were
sonicated three times for 20 seconds at 60 Hz (UP200S GmbH) and centrifuged at
10,000 g for 20 minutes to remove insoluble material. GST-c-ppl was
purified from the cleared lysate by batchwise incubation with
glutathione-agarose beads (Sigma), and after washing the protein was eluted
from the beads in buffer containing 50 mM Tris pH 7.5, 100 mM NaCl, 10%
glycerol and 10 mM glutathione. The eluted protein was dialysed for 20 hours
in the same buffer without glutathione.
GST or GST-c-ppl attached to glutathione sepharose were incubated for 1 hour with extracts of A14 cells transiently transfected with either wild-type or kinase-dead HA-tagged PKB and washed four times in solubilisation buffer. Subsequently, bound protein was removed by elution with 40 mM reduced glutathione. Samples were analysed for the presence of PKB by western blotting using the anti-HA antibody.
Immunoprecipitations
Non-confluent cells were lysed in 0.5 ml RIPA buffer (20 mM Tris pH 8.0, 1%
Triton X-100, 0.5% Na-DOC, 0.1% SDS, 10 mM EDTA, 150 mM NaCl, 1 mM NaF, 1 mM
sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin
and 2 µg/ml aprotinin) except for the endogenous Ppl-PKB and vimentin
co-immunoprecipitations (1% Triton X-100, 50 mM Tris pH 7.5, 150 mM NaCl, 10
mM EDTA, 10 mg/ml trypsin inhibitor, 1 µg/ml leupeptin and 2 µg/ml
aprotinin). Lysates were incubated for 2 hours at 4°C with 10 µl
antibody and 100 µl pre-washed protein-A beads. The immunoprecipitations
were washed four times with the used lysis buffer before being taken up in
Laemmli buffer.
Immunoblot analysis
Protein samples in Laemmli buffer were separated by SDS-PAGE on 6%
(periplakin) or 10% (others) gels and transferred to PVDF membrane (NEN).
Western blots were blocked overnight at 4°C in phosphate-buffered saline
(PBS) containing 2% nonfat dried milk (Protifar, Nutricia) and 0.5% bovine
serum albumin (Sigma). The western blots were then incubated for 2 hours with
the indicated primary antibodies in PBS containing 0.1% Tween 20 using the
dilutions recommended by the manufacturers or 1:8000 for the self-generated
antibodies. After washing four times for 5 minutes with PBS/0.1% Tween-20
blots were incubated with secondary antibodies anti-mouse HRP and anti-rabbit
HRP (1:10,000) for 1 hour at 4°C. Blots were washed again four times for 5
minutes with PBS/0.1% Tween-20 and analysed with chemiluminescence (ECL
(NEN)).
Immunofluorescence staining
Cells on coverslips were fixed in 4% paraformaldehyde for 30 minutes at
4°C and then permeabilised with 0.1% Triton X-100 in the presence of 0.5%
BSA for 30 minutes at 4°C. Before fixation, cells were incubated with 100
nM Mitotracker (Molecular Probes) for 30 minutes. Cells were incubated with
primary antibodies for 2 hours at 4°C, washed in PBS with 0.1% Triton
X-100 and 0.01% BSA, and then incubated further for 1 hour with the
appropriate conjugated secondary antibody. After further washing, coverslips
were mounted in Immu-mount (Shandon) and examined using a 63x planapo
objective on a Leitz DMIRB fluorescence microscope (Leica, Voorburg, the
Netherlands) interfaced with a Leica TCS4D confocal laser-scanning microscope
(Leica, Heidelberg, Germany). Digital images were recorded using Leica TCS NT
version 1.6.587.
As primary antibodies, the anti-vimentin (Oncogene), anti-periplakin anti-serum 5117 (3rd boost) or non-immune antiserum were used; FITC-conjugated anti-rabbit, anti-mouse or anti-goat antibodies were used as secondary antibodies.
Vimentin extraction
A14 cells were lysed with 0.5% Triton X-100 in CSK-buffer (10 mM Pipes pH
6.8, 250 mM sucrose, 3 mM MgCl2, 150 mM KCl, 1 mM EGTA and 1 mM
PMSF) for 10 minutes (fraction 1: total cells). Lysates were centrifuged at
14,000 g for 10 minutes (supernatant, fraction 2: membrane
proteins, cytosol, tubulin). The pellet was further extracted with 0.3 M KI in
CSK-buffer for 5 minutes and centrifuged at 14,000 g for 30
minutes (pellet, fraction 4: nuclei, intermediate filaments). Subsequently,
fraction 3 (actin) was isolated by dialyzing the supernatant against
CSK-buffer for 16 hours and centrifuging at 14,000 g for 30
minutes. The pellet was resuspended in 0.3 M KI in CSK-buffer and incubated
for 10 minutes after which the same dialysis was performed.
Percoll gradient-based cell fractionation
MCF-7, Rat-1 and COS-7 cells were fractionated using a percoll
gradient-based assay as described (Baumann
et al., 2000).
Affinity purification of mitochondria
Mitochondria from MCF-7 and Rat-1 cells were isolated as described
(Herrnstadt et al., 1999).
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Results |
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Binding of periplakin to PKB can influence PKB function in several ways. To
examine the consequence of periplakin binding on PKB activation, myc-c-ppl and
HA-PKB were co-expressed in A14 cells. A14 cells are NIH3T3 cells
overexpressing the human insulin receptor and treatment of these cells with
insulin leads to a rapid and strong increase in PKB activity
(Burgering and Coffer, 1995).
However, overexpression of myc-c-ppl did not affect activation of HA-PKB by
insulin (Fig. 2F). This
strongly suggests that binding of periplakin does not interfere in
growth-factor-induced activation of PKB.
Binding interface of PKB with periplakin
Having established that PKB and periplakin interact, but that this
interaction does not affect activation of PKB by growth factors, we next
wanted to investigate in more detail the binding of PKB to periplakin.
Therefore we analysed binding of a series of HA-PKB mutants to periplakin.
HA-PKB mutants were co-transfected with myc-c-ppl, immunoprecipitated and
binding was determined by immunoblotting. In keeping with the performed yeast
two-hybrid, in which part of the PH domain was used as a bait, the binding
site within full length PKB could be defined to a short stretch of nine amino
acids within the N-terminal part of the PH domain
(Fig. 3A,B), as deletion
mutants encompassing this region (30-130 and
30-214), and the
mutant
9 no longer bound to myc-c-ppl. The results obtained by
immunoblot analysis were confirmed by analyzing binding in the yeast
two-hybrid system. The structure of PH domains present within a variety of
different proteins has been determined and consists of seven ß-sheets
followed by one
-helix. The presence of the ß-sheets and
-helix in the PH domain of PKB is indicated in
Fig. 3C and shows that the
9 mutant lacks the last part of the 5th flexible loop and most of the
6th ß-sheet. Therefore, this deletion is expected to have an effect on
the 3D structure of the PH domain. To restore at least the spacing of the
amino acid residues within the pH domain, we added back to the
9 mutant
a stretch of nine glycine residues. However, this did not restore binding to
myc-c-ppl. To see whether any distortion of the PH domain structure would
result in loss of myc-c-ppl binding we also analysed binding of myc-c-ppl to
the W99A mutant of HA-PKB. In this mutant the residue (W99) conserved in all
known PH domains was mutated and this is likely to result in a structural
change of the PH domain, as W99A is catalytically inactive (data not shown).
Nevertheless, myc-c-ppl did bind to this mutant, suggesting that the loss of
myc-c-ppl binding to the
9 mutant is not necessarily due to a
conformational change of the PH domain, but that myc-c-ppl probably binds to
the 5th flexible loop.
|
Periplakin binds the intermediate filament vimentin
As periplakin expression does not interfere in growth-factor-induced PKB
activation, we started to address the possibility that binding between
periplakin and PKB may serve as a localization signal for PKB. Therefore, we
first studied the cellular localization of periplakin in detail. All members
of the plakin family have been shown to bind cytoskeletal proteins. Thus, to
investigate which cytoskeletal proteins periplakin binds to, we performed a
yeast two-hybrid analysis with the C-terminal part of periplakin on a 13.5 day
mouse embryo cDNA library. This way we identified the intermediate filament
protein vimentin as a potential binding partner of periplakin. To further
establish periplakin binding to vimentin we co-expressed myc-tagged c-ppl with
HA-vimentin in COS-7 cells and observed co-immunoprecipitation when
precipitating myc-c-ppl in a stringent buffer and analysing by immunoblotting
for the presence of vimentin (Fig.
4A). For reasons that are not entirely clear to us, we could only
very faintly observe co-precipitation when precipitating vimentin and
analysing for the presence of myc-c-ppl. Therefore, we also analysed
co-localization by immunofluorescence in Rat-1 cells. In these experiments a
clear co-localization is observed between myc-c-ppl and the endogenous
vimentin network (Fig. 4B). A
fractionation on A14 cells transiently transfected with myc-c-ppl was
performed in which the vimentin network was solubilised and purified.
Fractions were analysed for actin, vimentin and myc-c-ppl, which was found to
be present in the vimentin fraction (Fig.
4C). Thus from these experiments we conclude that periplakin binds
both PKB and vimentin through its C-terminus and is colocalised with vimentin.
To determine whether PKB and vimentin bind to different or overlapping sites
within the C-terminal part of periplakin we co-expressed, both in cells and in
the yeast two-hybrid system, deletion mutants of the C-terminal part of
periplakin together with either vimentin or PKB. However, in both cases we
were unable to identify within periplakin a small peptide sequence responsible
for vimentin or PKB binding. Although some difference was observed between PKB
and vimentin binding (Fig. 4D)
we could not establish whether PKB and vimentin bind different parts of
periplakin, or whether binding of PKB and vimentin is mutually exclusive.
|
Periplakin localizes to different cellular compartments
In tumor cell development, loss of epithelial morphology and acquisition of
mesenchymal characteristics is often correlated with increased expression of
vimentin (Dandachi et al.,
2001). As PKB function is also implicated in tumor development, we
were interested to know where periplakin would localize in cells that lack
vimentin expression and if differential localization, due to presence or
absence of vimentin, may influence PKB function. In contrast to many other
breast cancer cell lines, the breast epithelial carcinoma cell line MCF-7
expresses little or no vimentin (Stover et
al., 1994
; van de Klundert et
al., 1992
) but high levels of periplakin
(Fig. 2), and therefore this
cell line was used for confocal microscopy using the periplakin antibodies
developed. Endogenous periplakin was shown to be co-localizing with the cell
membrane, nucleus and mitochondria. The latter was demonstrated by co-staining
with a mitochondrial marker (Mitotracker). Also transfected HA-tagged
periplakin was shown to be co-localizing with a mitochondrial marker
(cytochrome c) in MCF-7 cells (Fig.
5A). Mitochondrial localization of periplakin was confirmed by
biochemical fractionation methods. First, various cellular fractions including
a mitochondrial fraction were isolated from MCF-7, COS-7 and Rat-1 cells by
fractionation on a Percoll gradient (Fig.
5B). In a second approach we used sorting by magnetic beads (MACS)
with a ferro-conjugated antibody (MAB 1273, see Materials and Methods) that
recognizes a 65-kDa mitochondrial membrane protein
(Herrnstadt et al., 1999
).
This method allows rapid single step purification of organelles
(Fig. 5C). Fractionation was
monitored in both cases by using COX4 as a mitochondrial marker and MAPK as a
non-mitochondrial marker. Both approaches revealed the presence of periplakin
in the mitochondrial fraction, confirming the immunofluorescent data.
Interestingly, we also demonstrated mitochondrial localization of periplakin
in COS-7 cells, albeit that the fraction of periplakin localized to
mitochondria appeared less compared with MCF-7. This observation suggests that
in vimentin-containing cells, such as COS-7, periplakin is bound to vimentin
and may function to recruit mitochondria to intermediate filament structures
whereas, in non-vimentin-containing cells, periplakin appears to localize more
clearly to mitochondria owing to the absence of clear filamentous
staining.
|
Periplakin expression can affect PKB-signalling by sequestration
Since periplakin binds to PKB but does not interfere with activation of PKB
and localizes to specific cellular compartments, we wanted to test whether
periplakin expression may affect the ability of PKB to generate specific
signalling outputs. Recently, we and others have shown that phosphorylation
and inactivation of FOXO transcription factors by PKB occurs within the
nucleus (Brownawell et al.,
2001; Brunet et al.,
2002
). Consequently, inhibition of PKB nuclear transport should
result in loss of FOXO regulation by PKB. To test this possibility we made use
of the observation that in vimentin-expressing cells ectopically expressed
myc-c-ppl localizes to intermediate filament structures, whereas little or no
expression in the nucleus is observed (Fig.
4B). As expected and reported previously, co-expression of PKB
resulted in inhibition of FOXO4 (AFX)-dependent transcription
(Fig. 6A). Interestingly both
full length and myc-c-ppl expression enhanced transcription by FOXO4. That
this is likely to be due to sequestration of PKB within the cytosol is
indicated by a lack of effect of myc-c-ppl expression on insulin-induced PKB
activity (Fig. 2F) and a
decrease of phosphorylation of the PKB site S193 of FOXO4
(Fig. 6B). Thus these results
suggest that periplakin can indeed act as a scaffold, as it can modulate
PKB-dependent signalling outputs without interfering with PKB activation
itself.
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Discussion |
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PKB binding to periplakin could be demonstrated by yeast two-hybrid
analysis as well as co-immunoprecipitations of ectopically and endogenously
expressed proteins. The binding region within PKB that is responsible for the
interaction with periplakin was narrowed down to a small region in the
C-terminal part of the PH domain (Fig.
3B). Alignment of the PH domain of PKB with PH domains of which
the 3D structure has been resolved indicates that binding to periplakin is
most likely mediated by the 5th flexible loop of the PH domain.
(Fig. 3C). Although it remains
possible that binding to the 9 mutant is lost owing to general
distortion of the 3D structure of the PH domain, we consider this unlikely for
several reasons. First, restoring at least the spacing by replacing the
deleted amino acids by glycine residues (
9-gly9) does not restore
binding. Second, as one can still argue that also
9-gly9 is no longer
properly folded, the presence of the binding site within a flexible loop would
at least suggest that the primary sequence rather than its folding determines
binding. In keeping with the latter conclusion would be the observations that
the yeast two-hybrid interaction was isolated with a truncated PH domain
construct and that myc-c-ppl still binds to the W99A mutant. With respect to
this mutant it should then be noted that this tryptophane residue is likely to
be an essential structural determinant for PH domains, since between all known
PH domains this residue is the only one that is actually conserved
(Musacchio et al., 1993
).
Indeed the W99A mutation results in a catalytically inactive mutant (not
shown) and on SDS-PAGE this mutant shows aberrant migration behavior
suggesting that this mutation distorts PH domain structure but does not affect
binding to myc-c-ppl.
Finally, as the residues involved in binding PtdIns3P lipids are primarily located within the first half of the PH domain, binding of periplakin to the 5th flexible loop would be expected to have little effect on PtdIns3P binding. Although not measured directly, the fact that we do not observe inhibition of insulin-induced activation of HA-PKB when co-expressing myc-c-ppl (Fig. 2F) corroborates this suggestion.
Plakin family members have been shown to bind or colocalize with
intermediate filaments (Ruhrberg et al.,
1997; DiColandrea et al.,
2000
) and indeed we also found in our yeast two-hybrid screen that
c-ppl binds to the intermediate filament vimentin. This interaction was
confirmed by co-immunoprecipitation and co-localization studies. The binding
to and colocalisation with vimentin in fibroblasts is in agreement with the
observations of DiColandrea et al., who also observe periplakin colocalisation
with vimentin in COS-7 cells. In addition, they observe in keratinocytes that
full-length and the C-terminal part of periplakin partially colocalise with
keratin filaments (DiColandrea et al.,
2000
). Thus, it appears that, depending on the cell type,
periplakin may localize to different intermediate filament networks.
Intermediate filaments are considered to function as scaffolding that
structures the cytoplasm and resists extracellular stresses
(Fuchs and Cleveland, 1998
).
The function of the binding of periplakin to vimentin is unclear and needs to
be further investigated, but we hypothesize that periplakin, through binding
to vimentin, correctly localizes PKB and other proteins within the cell.
Recently it was reported that PKB could interact with another intermediate
filament, keratin K-10, and that K-10 binding results in PKB inhibition and
keratin-induced cell cycle arrest (Paramio
et al., 2001
). As this study did not identify the nature of the
interaction between K-10 and PKB, our results indicate the possibility that
periplakin functions to bridge this interaction. Also, our observation that
periplakin expression enhances FOXO4 transcriptional activity is consistent
with the role of intermediate filaments in inducing cell cycle arrest as
proposed by Paramio et al. (Paramio et
al., 2001
). Previously we have shown that FOXO transcription
factors can cause cell cycle arrest in a p27kip-dependent manner
(Medema et al., 2000
). Since
activated PKB translocates to the nucleus, where it inactivates FOXO
transcription factors, sequestering PKB within the cytosolic compartment
through binding to periplakin/intermediate filaments would enhance FOXO
transcriptional activity and consequently stimulate cell cycle arrest.
In many cell types and under many conditions PKB signaling has been shown
crucial for providing cellular protection against apoptosis. Apoptosis is
often initiated by, or requires mitochondrial damage and leakage of,
cytochrome C (Adrain and Martin,
2001). Recent reports have shown that PKB signalling can maintain
mitochondrial membrane stability under conditions of stress
(Gottlob et al., 2001
;
Kennedy et al., 1999
;
Plas et al., 2001
). Therefore,
the clear colocalisation of periplakin with mitochondria combined with its
interaction with PKB suggests a possible role for periplakin in mediating
PKB-dependent protection. In keeping with such a model are reports that
vimentin is a substrate for caspase-9 and is cleaved at an early stage within
the apoptosis process (Byun et al.,
2001
; Nakanishi et al.,
2001
) and that caspase-resistant vimentin delays apoptosis
(Belichenko et al., 2001
).
Also, plectin is a substrate for caspase-8 and is cleaved early in apoptosis
(Stegh et al., 2000
). This
caspase cleavage site of plectin is conserved in periplakin. However
attractive, we have not been able to obtain clear evidence to support a role
for periplakin in PKB-mediated apoptosis protection. This could be due to, for
instance, redundancy in apoptosis protection signalling. Many cell types do
not, or only slightly, express periplakin and still depend on PKB-mediated
protection. Interestingly in this respect, plectin, a family member of
periplakin, has been shown to associate with mitochondria as well
(Reipert et al., 1999
). It is
possible that plectin and periplakin may act redundant in PKB-dependent
protection since plectin has been shown to have a ubiquitous expression
pattern (Wiche et al., 1983
).
Further, our observation that PKB localization to mitochondria, as determined
by biochemical fractionations, appears independently of periplakin and/or
vimentin expression indicates either redundant or alternative means of PKB
localization to this compartment. However, these biochemical fractionations do
not exclude the possibility that the presence of PKB is due to contamination
of this fraction. Unfortunately, with respect to PKB localization, we could
not complement the biochemical fractionation data with immunofluorescence
studies, as the quality of all different PKB antibodies we tested thus far
precluded this.
In addition to acting as a localization signal, periplakin may function as
a shuttle for delivery of PKB to the various cellular compartments. This
possibility derives primarily from the observation that periplakin localizes
to all sites where PKB is either observed or suspected to localize. Periplakin
localizes to the plasma membrane and PKB becomes activated at the plasma
membrane by PI 3-kinase-mediated production of phosphoinositol lipids
(Bos, 1995;
Burgering and Coffer, 1995
).
Following activation, PKB should be able to translocate to other cellular
compartments such as the nucleus and the vicinity of mitochondria, since
substrates for PKB present within these cellular domains have been identified.
For example, a fraction of PKB has been shown to translocate to the nucleus
(Andjelkovic et al., 1997
;
Brownawell et al., 2001
).
Within the nucleus PKB is thought to phosphorylate substrates such as the FOXO
family of transcription factors. We observe nuclear localization of periplakin
which, along with other members of the plakin family, contains a putative
bipartite nuclear localization (NLS). As other reports suggested that this NLS
sequence in other plakins [e.g. plectin
(Nikolic et al., 1996
)] is
also a part of the intermediate filament-binding region, it could be that
(induced) loss of intermediate filament binding unmasks the NLS and results in
nuclear translocation. These possibilities are currently under investigation.
Finally, although our experiments only start to delineate such a possibility,
it is interesting to note that such a function for periplakin and possibly
plakins in general is very similar to the function of other large cytolinkers
such as APC.
In conclusion, this study shows binding of periplakin to PKB and vimentin. The differential cellular localization of periplakin and its ability to affect PKB signalling when targeted to a specific localization suggest a role in determining cell-type-specific signalling by the PI3K/PKB pathway.
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Acknowledgments |
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