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Nuclear particles containing RNA polymerase III complexes associated with the junctional plaque protein plakophilin 2

Abstract and Figures

Plakophilin 2, a member of the arm-repeat protein family, is a dual location protein that occurs both in the cytoplasmic plaques of desmosomes as an architectural component and in an extractable form in the nucleoplasm. Here we report the existence of two nuclear particles containing plakophilin 2 and the largest subunit of RNA polymerase (pol) III (RPC155), both of which colocalize and are coimmunoselected with other pol III subunits and with the transcription factor TFIIIB. We also show that plakophilin 2 is present in the pol III holoenzyme, but not the core complex, and that it binds specifically to RPC155 in vitro. We propose the existence of diverse nuclear particles in which proteins known as plaque proteins of intercellular junctions are complexed with specific nuclear proteins.
Plakophilin 2 interacts with RPC155. ( a ) Coimmunoselection of RPC155 with plakophilin 2. Extracts from cultured A-431 cells were subjected to immunoselection with plakophilin 2 antiserum (lane 1; lane 2, control showing the proteins of the same antiserum bound to protein A-Sepharose), and the proteins selected were analyzed by SDS ͞ PAGE and stained with Coomassie brilliant blue (the sizes of reference proteins are indicated). The 155-kDa polypeptide (upper arrow) coselected with plakophilin 2 (lower arrow) was excised from the gel and identified by matrix-assisted laser desorption ionization analysis as the largest subunit of RNA polymerase III (RPC155). ( b ) Peptide sequences determined in the 155-kDa binding partner of plakophilin 2 by matrix-assisted laser desorption ionization analysis. In addition, myosin heavy chain and major vault protein (42) were identified as apparently nonspecifically bound coadsorbed proteins ( cf. ref. 7). ( c ) Blot-overlay assay of polypeptides of a chromatographic fraction enriched in pol III, separated by SDS ͞ PAGE, and visualized with Coomassie brilliant blue (lane 1) or transferred to nitrocellulose membranes (lanes 2 and 3). After incubation with in vitro translated 35 S-labeled plakophilin 2, the binding partners of plakophilin 2 within the pol III fraction were detected by autoradiography (lane 2). A 155-kDa protein, which was identified subsequently as the largest subunit of pol III by immunoblot analysis using RPC155-specific antibodies (lane 3), binds plakophilin 2 selectively. The position of reference proteins is indicated on the left.
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Nuclear particles containing RNA polymerase III
complexes associated with the junctional
plaque protein plakophilin 2
Claudia Mertens*
, Ilse Hofmann*, Zhengxin Wang
, Martin Teichmann
, Setareh Sepehri Chong
‡§
, Martina Schno
¨
lzer
,
and Werner W. Franke*
*Division of Cell Biology and
Peptide Analysis Facility, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany;
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10021; and
Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY 11724
Communicated by Howard Green, Harvard Medical School, Boston, MA, May 3, 2001 (received for review October 28, 2000)
Plakophilin 2, a member of the arm-repeat protein family, is a dual
location protein that occurs both in the cytoplasmic plaques of
desmosomes as an architectural component and in an extractable
form in the nucleoplasm. Here we report the existence of two nuclear
particles containing plakophilin 2 and the largest subunit of RNA
polymerase (pol) III (RPC155), both of which colocalize and are coim-
munoselected with other pol III subunits and with the transcription
factor TFIIIB. We also show that plakophilin 2 is present in the pol III
holoenzyme, but not the core complex, and that it binds specifically
to RPC155 in vitro. We propose the existence of diverse nuclear
particles in which proteins known as plaque proteins of intercellular
junctions are complexed with specific nuclear proteins.
I
n recent years, several components of the plaques of intercel-
lular junctions surprisingly have also been identified in cell
nuclei, suggesting their involvement in nuclear functions and
regulatory interactions between the cell periphery and the
nucleus. Such proteins include members of the arm-repeat
family, which are characterized by variable numbers of a 42-
amino acid motif (1). Among these, the plakophilins (PKPs) are
characteristic of desmosomes and also occur constitutively in the
nucleoplasm of a wide range of cells (2–11). These positively
charged proteins (isoelectric points ranging from 9.3 to 10.1)
represent the products of three genes, PKP1, PKP2, and PKP3,
and at least PKP1 and PKP2 occur in two splice variants a and
b (3–12). Both variants can also be detected in the nuclei of
various cells devoid of desmosomes and after cDNA transfection
have also been shown to accumulate in the nuclei (9).
The observation of plakophilins in the nucleus, however, has
been possible only with the use of appropriate immunocyto-
chemical protocols that minimize the extraction of soluble
nucleoplasmic proteins and particles (e.g., see refs. 6, 9, and
13–15). Moreover, in a wide range of different kinds of cells
lacking desmosomes including fibroblasts, lymphocytes, and
chicken erythrocytes, plakophilins have been detected exclu-
sively in the nucleus (6, 7, 10, 11). This finding suggests that
plakophilins are constitutive nuclear proteins that, in certain
pathways of cell differentiation, are recruited to a second specific
location in desmosomal plaques (6–9).
Plakophilins 2a (92.75 kDa) and 2b (97.41 kDa) are of special
importance, because they display the most widespread occurrence
in desmosomes of all simple and complex epithelia, in lower layers
of many stratified epithelia, in all carcinomas, in heart muscle, in
reticulum cells of lymph node follicles, and in early embryos (68).
Therefore, we have decided to characterize the plakophilin 2-con-
taining structures of nuclei. Here we show plakophilin 2 in com-
plexes with the largest subunit of RNA polymerase (pol) III and
other pol III polypeptides, and we describe nucleoplasmic particles
combining plakophilin 2 with pol III proteins.
Materials and Methods
Cell Cultures. The following human cell culture lines were used:
HaCaT keratinocytes (16), vulvar squamous carcinoma-derived
A-431, hepatocellular carcinoma PLC, mammary gland carci-
noma MCF-7, cervical adenocarcinoma HeLa, HEK (embryonic
kidney), acute myeloid leukemia KG1a, and promyelocytic
leukemia HL60 (all from the American Type Culture Collec-
tion). In addition, several cells and tissues of human and other
vertebrate origins were examined including chicken erythrocytes
and Xenopus laevis ovary (7). Several of these cell lines, notably
HaCaT cells, were also used for the isolation of nuclei according
to the method of Lee and Green (17)
Antibodies. Immunoselections were performed with guinea pig
antibodies specific for plakophilin 2 (6) and rabbit antisera
against one of the following components of human pol III:
hRPC155 (CSH499), hRPC39, hRPC82, hTFIIIB90, and
hTFIIIC63 (18–21). Guinea pig antibodies specific for RPC155
were generated by immunization with the synthetic carboxyl-
terminal peptide PKRPLIFDTNEFHIPLVT (residues 1374
1391) coupled to keyhole limpet hemocyanin and affinity-
purified. Immunoblotting for plakophilin 2 was performed with
the monoclonal antibodies PP2–150, PP2–86 (68), or plako-
philin 2a (Becton Dickinson and Transduction Laboratories,
Lexington, KY).
Sucrose Gradient Centrifugation and Gel Filtration Chromatography.
For sucrose gradient centrifugation, cells grown on 10-cm plates
were lysed with either 0.6 ml of ice-cold 5:1 buffer [83 mM KCl, 17
mM NaCl, 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 3 mM DTT, 1
mM PMSF, and 1Complete protease inhibitors (Roche Molec-
ular Biochemicals)] or physiological salt buffer (140 mM NaCl, 1.5
mM MgCl
2
, 15 mM Hepes, pH 7.4, and 1 Complete), both
containing 0.1% Triton X-100. Cells were scraped off with a rubber
policeman, resuspended by pipetting, and transferred to a 1.5-ml
tube. To analyze the influence of nucleases, extracts were incubated
with either 16
gml RNase A (Roche) or 416
gml DNase for
15 min on ice. Digestion with DNase was performed with cell
extracts in 2 mM MgCl
2
instead of 5 mM EDTA. After centrifu-
gation at 15,000 g and 4°C for 10 min, the supernatant was
centrifuged for1hat100,000 g. Then, 0.5-ml aliquots were
layered on linear 1040% (in 10 mM Tris-HCl, pH 7.5) or 1060%
(in 5:1 buffer without detergent) sucrose gradients and subjected to
centrifugation for 16 h at 23,000 rpm (1040%) or for 18 h at 35,000
rpm (1060%) in an SW40 rotor (Beckman Instruments, Munich).
Fractions of 0.4 ml (from 1040% gradients) or 0.8 ml (from
1060% gradients) were collected from top to bottom, and proteins
Abbreviations: PKP, plakophilin; pol, RNA polymerase.
§
Present address: Bone and Mineral Centre, University College London, London, United
Kingdom.
To whom reprint requests should be addressed. E-mail: w.franke@dkfz.de.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
www.pnas.orgcgidoi10.1073 PNAS
July 3, 2001
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CELL BIOLOGY
were analyzed by SDSPAGE directly or after immunoselection. In
parallel gradients, BSA (Sigma), catalase, thyroglobulin (Amer-
sham Pharmacia), or ribosomal subunits from X. laevis ovaries were
used as reference proteins or particles.
For gel filtration, cell extracts prepared by lysing cells from
three confluent 10-cm plates in a final volume of 0.6 ml of 5:1
buffer containing 0.1% Triton X-100 and 5 mM
-mercapto-
ethanol instead of DTT were centrifuged at 15,000 g for 10
min, followed by 100,000 g for 1 h. Then, 0.2 ml of the
supernatant was loaded on a Superose 12-h 1030 column
(Amersham Pharmacia), and proteins were eluted with 5:1
buffer at a flow rate of 0.2 mlmin. Proteins from 0.4-ml
fractions were precipitated with 3 volumes of absolute methanol
and analyzed by SDSPAGE.
Protein Fractions Containing Pol III. HeLa cells stably transfected
with flag epitope-tagged RPC53 (line BN51) were used. Pol III
was affinity-purified from S100 extracts by using BC buffer (20
mM Hepes, pH 7.9, 20% glycerol, 0.5 mM EDTA, 1 mM DTT,
and 0.5 mM PMSF) containing 300 mM KCl and 0.1% Nonidet
Fig. 1. Sucrose gradient sedimentation of plakophilin 2-containing particles extracted with 5:1 buffer (a) or physiological salt buffer (b) from human
keratinocyte cultures (HaCaT) or from HaCaT nuclei (c) in a linear 10 40% sucrose density gradient. Fractions were collected from top to bottom (1–30) and
analyzed by SDSPAGE and immunoblotting for plakophilin 2, which is recovered in fractions corresponding to S values of 11, 30–35, and 50 –55 S (a), 40 and
55S(b), and 55– 60 S (c). L, loading sample. Size references: catalase, 11 S; X. laevis ribosomal subunits, 40 and 60 S.
Fig. 2. Plakophilin 2 interacts with RPC155. (a) Coimmunoselection of RPC155 with plakophilin 2. Extracts from cultured A-431 cells were subjected to immunos-
election with plakophilin 2 antiserum (lane 1; lane 2, control showing the proteins of the same antiserum bound to protein A-Sepharose), and the proteins selected
were analyzed by SDSPAGE and stained with Coomassie brilliant blue (the sizes of reference proteins are indicated). The 155-kDa polypeptide (upper arrow) coselected
with plakophilin 2 (lower arrow) was excised from the gel and identified by matrix-assisted laser desorption ionization analysis as the largest subunit of RNA polymerase
III (RPC155). (b) Peptide sequences determined in the 155-kDa binding partner of plakophilin 2 by matrix-assisted laser desorption ionization analysis. In addition, myosin
heavy chain and major vault protein (42) were identified as apparently nonspecifically bound coadsorbed proteins (cf. ref. 7). (c) Blot-overlay assay of polypeptides of
a chromatographic fraction enriched in pol III, separated by SDSPAGE, and visualized with Coomassie brilliant blue (lane 1) or transferred to nitrocellulose membranes
(lanes 2 and 3). After incubation with in vitro translated
35
S-labeled plakophilin 2, the binding partners of plakophilin 2 within the pol III fraction were detected by
autoradiography (lane 2). A 155-kDa protein, which was identified subsequently as the largest subunit of pol III by immunoblot analysis using RPC155-specific antibodies
(lane 3), binds plakophilin 2 selectively. The position of reference proteins is indicated on the left.
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P-40 (20). Pol III holoenzyme was enriched from nuclear extracts
and immunopurified in BC buffer containing 100 mM KCl and
0.05% Nonidet P-40 (19).
Immunoisolation and Matrix-Assisted Laser Desorption Ionization
Analysis.
Confluent A-431 cells of 200 Petri dishes (10-cm
diameter) were lysed (0.6 ml per dish) in ice-cold modified RIPA
buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1%
Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM
DTT, 1 mM PMSF, and 1 Complete protease inhibitors) and
homogenized in a Dounce-type glass homogenizer (all proce-
dures were performed at 4°C). After centrifugation for 1 h at
100,000 g, the supernatant was incubated for2hwith20mg
of protein A-Sepharose beads (Amersham Pharmacia) and
cleared by centrifugation. The lysate was incubated overnight
with the beads bound with 200
l of plakophilin 2 antibodies, and
the selected protein complexes were washed four times in RIPA
buffer and analyzed by SDSPAGE. Bands seen after Coomassie
blue staining were excised and digested in the gel for peptide
fingerprinting by matrix-assisted laser desorption ionization
mass spectrometry (22). For sequence comparisons, the pro-
grams Peptide Search (European Molecular Biology Labora-
tory), MS-Fit (University of California, San Francisco), and
ProFound (Rockefeller University, New York) were used.
Immunoselections. Immunoselected complexes from sucrose gradi-
ent fractions were obtained with material from five parallel gradi-
ents (1060%). The fractions were pooled, adjusted by adding the
double volume of 2 RIPA buffer, and obtained after preclearing
with protein A-Sepharose for 2 h. Samples were incubated over-
night with protein A-Sepharose beads coated with plakophilin 2
antibodies. Before analysis by SDSPAGE, the immunoselected
material was washed four times with RIPA buffer.
Extracts from lysed MCF-7 cells were prepared by immuno-
selection buffer (20 mM Hepes, pH 7.9, 10% glycerol, 2 mM MgCl
2
,
100 mM KCl, 0.1% Triton X-100, 1 mM DTT, 1 mM PMSF, and
1 Complete protease inhibitors). After centrifugation at 15,000
g for 20 min, the supernatants were preincubated with Sepharose
beads and subjected to immunoselection as described by using
rabbit antibodies against one of the pol III components. In all
washes, the buffer was the same as that used for lysis.
Blot Binding Assays. A pol III-enriched fraction was prepared
from HeLa S100 extracts (18–20), separated by SDSPAGE, and
transferred to nitrocellulose membrane sheets. [
35
S]methionine-
labeled plakophilin 2 was obtained by coupled in vitro transcrip-
tion-translation of plakophilin 2 cDNA or a subclone encoding
the amino-terminal domain only (residues 1–367; ref. 6) using
the coupled transcription and translation reticulocyte lysate
system (Promega). Binding assays were as described (7).
Fig. 3. Gel filtration of plakophilin 2 and RPC155. Triton X-100-soluble extracts from human PLC cell cultures were chromatographed on a Superose 12 column,
and individual fractions were analyzed by immunoblotting with antibodies against plakophilin 2 and RPC155. The elution peaks of the size markers (kDa) Dextran
blue 2000 (2,000), thyroglobulin (669), catalase (232), and BSA (66) are indicated by arrows. Plakophilin 2 and RPC155 co-elute in a position corresponding to
1,500 kDa (fractions 6 8). L, loading sample.
Fig. 4. Analysis of plakophilin 2: RPC155 complexes by ribonuclease treatment and immunoselection. (a) Sedimentation of plakophilin 2 and RPC155 in sucrose
density gradients (10 60%) from A-431 cell extracts without and with RNase A treatment. After centrifugation, proteins from individual fractions were
precipitated with absolute methanol, subjected to SDSPAGE, and analyzed by immunoblotting with antibodies against plakophilin 2 and RPC155. Size
references: BSA, 4.5 S; catalase, 11 S; and thyroglobulin, 16.5 S. (b) For coimmunoselection of plakophilin 2 and RPC155 from sucrose density gradient fractions
(no RNase treatment), fractions 9 and 12 (a) were subjected to immunoselection by using either antibodies specific for plakophilin 2 or unrelated antibodies
(Contr.). Fractions were analyzed by immunoblotting with antibodies against plakophilin 2 and RPC155. RPC155 coimmunoselects with plakophilin from both
fractions tested, whereas no signal is found in the negative controls.
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CELL BIOLOGY
Immunofluorescence Microscopy. For immunofluorescence mi-
croscopy, cultured cells grown on coverslips were briefly rinsed
in phosphate-buffered saline prewarmed to 37°C. Cells were
fixed at 20°C in methanol (for 5 min) and acetone (for 30 sec)
and air-dried. For double-staining experiments, guinea pig an-
tibodies against plakophilin 2 and rabbit antisera specific for
RPC155 were applied for 30 min followed by three phosphate-
buffered saline washes for 3 min each. Secondary antibodies
coupled to Texas red or fluorescein isothiocyanate (Dianova,
Hamburg, Germany) were applied for 30 min. After washing
with phosphate-buffered saline, the specimens were rinsed in
water, briefly dipped in ethanol, air-dried, and mounted with
Fluoromount (Biozol, Eching, Germany). Confocal laser scan-
ning immunofluorescence images were acquired with a Zeiss
LSM 410 microscope (Zeiss; ref. 68).
Results
Plakophilin 2-Containing Complexes. To study the native state of
nuclear plakophilin 2, rapid particle extracts from cultured cells in
Triton X-100 and EDTA were subjected to sucrose density gradient
centrifugation, and the fractions obtained were analyzed by SDS
PAGE and immunoblotting (Fig. 1a). Plakophilin 2 was recovered
in three peak fractions. The majority of the protein sedimented in
large complexes of 30–35 S (fractions 14–17) and 50–55 S (fractions
20–23), whereas a minor proportion appeared at 11 S (fractions
6 and 7). Corresponding sedimentation profiles were obtained with
extracts from various human cell lines such as HaCaT, A-431, or
PLC, although the relative recovery in the different peak fractions
varied somewhat.
Because plakophilin 2 is rather susceptible to proteolytic
breakdown, most of the experiments were performed with
rapidly isolated complexes. Under more protective conditions
(physiological salt buffer) without EDTA, the two major peaks
appeared at about 40 and 55 S (Fig. 1b). By contrast, when
isolated nuclei were washed and subjected to particle extractions,
only a single 55–60 S peak was predominant (Fig. 1c).
Plakophilin 2 in Complexes with the Largest Subunit of Pol III. When
extracts from A-431 cells were subjected to immunoselection
with antibodies against plakophilin 2, a 155-kDa polypeptide was
specifically coselected (Fig. 2a, arrow in lane 1; see also ref. 7).
On analysis by mass spectrometry and peptide fingerprinting,
this polypeptide was identified as the largest subunit of pol III
(Fig. 2b, RPC155; cf. ref. 18–20).
In Vitro
Binding of Plakophilin 2. The association between plako-
philin 2 and RPC155 was demonstrable also in vitro. When
fractions of nuclear proteins enriched in pol III were subjected
to SDSPAGE (Fig. 2c, lane 1), blotted onto nitrocellulose
membranes, and reacted with radioactively labeled plakophilin
2, specific and intense binding to the band containing RPC155
was noted (Fig. 2c, lanes 2 and 3).
Biochemical Characterizations. To examine the distribution of
particles containing plakophilin 2 and RPC155, extracts from
various cell cultures were subjected to gel filtration on a Super-
ose 12 column, and the fractions obtained were analyzed by
immunoblotting (Fig. 3). The majority of plakophilin 2 eluted in
a rather broad peak (fractions 4–11) briefly after the void
volume. In agreement with the sedimentation analysis (Fig. 1),
smaller amounts coeluted with the reference protein catalase
(fraction 21). RPC155 copurified with plakophilin 2 in peak
fractions 6–9, corresponding to a mean molecular mass of
1.5 10
6
. In addition, some RPC155 eluted as a distinct
complex with a relative weight of about 700,000, which corre-
sponds to the weight of the native catalytically active 16-subunit
pol III complex (23).
To characterize particles containing plakophilin 2 and
RPC155 further, they were subjected to centrifugation on 10
60% sucrose gradients. Both RPC155 and plakophilin 2 were
detected in fractions 9 and 12, corresponding to 30–35- and
50–55-S complexes, respectively (Fig. 4a, Left). Moreover, we
noted that treatment with RNase A had an effect on the
sedimentation behavior of the plakophilin 2-containing parti-
cles, which shifted to positions corresponding to smaller sizes
(Fig. 4a, Right). By contrast, DNase treatments did not alter the
sedimentation profiles (data not shown; ref. 7). These results may
suggest that plakophilin 2 and RPC155 are components of particles
that also contain some RNA of an as yet unknown nature.
To characterize the particles containing plakophilin 2 and
RPC155 further, plakophilin 2 was immunoselected from the
peak sucrose gradient fractions (Fig. 4b presents fractions 9 and
12, for example). Immunoblot analysis demonstrated that
Fig. 5. Plakophilin 2 is a component of the pol III holoenzyme complex. (a) For
immunoselection, proteins extracted from human MCF-7 carcinoma cells were
reacted with antibodies against the pol III subunits RPC39, RPC82, and RPC155 as
well as with antibodies specific for the transcription factors TFIIIB (90-kDa sub-
unit) and TFIIIC (63-kDa subunit), respectively. For control, preimmune serum was
used. The immunoselected fractions were analyzed by immunoblotting using
antibodies against RPC155 and plakophilin 2. The result shows the presence of
plakophilin 2 in particles specifically selected with antibodies to different pol III
components (RPC39, RPC82, RPC155, and TFIIIB) but not to TFIIIC (note that here
the specific enrichment of RPC155 is too low to show plakophilin 2). (b) Immu-
noblot detection of plakophilin 2 in the purified pol III holoenzyme. Lane 1, S100
extract from human embryonic kidney (HEK) cells; lane 2, nuclear extract from
HEK cells; lane 3, 5
l of the FLAG-eluted pol III holoenzyme; lane 4, 5
lofthe
FLAG-eluted pol III core enzyme.
7798
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RPC155 was associated physically with plakophilin 2 (Fig. 4b),
indicating that both proteins are part of the same complex.
We also scored the sucrose gradient fractions for other known
desmosomal components. Desmogleins, desmoplakin, and pla-
koglobin appeared in fractions showing no cosedimentation with
plakophilin 2 (data not shown; ref. 7). Plakophilins 1 and 3 were
recovered in similarly sized particle fractions, which however did
not contain pol III.
The Pol III Holoenzyme Complex. The size of the complex contain-
ing plakophilin 2 and RPC155 corresponds to that of the pol III
holoenzyme, which consists of pol III and all basal factors
essential for transcription (23). To examine whether plakophilin
2 was also associated with other components of the pol III
holoenzyme, extracts prepared from MCF-7 cells were immu-
noselected with antibodies against other pol III components and
analyzed for the presence of RPC155 and plakophilin 2 by
immunoblotting (Fig. 5a). RPC155 and plakophilin 2 both
appeared together with the 39-kDa subunit (RPC39) in combi-
nation with the 82-kDa pol III subunit (RPC82) and were
associated also with transcription initiation factor TFIIIB. No
binding was found to the primary DNA-binding factor TFIIIC
(Fig. 5a).
Affinity-purification studies using nuclear extracts from HeLa
cells stably transfected with a FLAG-tagged form of the RPC53
subunit resulted in the recovery of a functional pol III-
containing complex, the holoenzyme, capable of transcription
initiation and consisting of a multitude of distinct factors (cf. refs.
18–21). Among them one was identified as a 100-kDa polypep-
tide corresponding in size to plakophilin 2 (68). Indeed,
immunoblot analysis (Fig. 5b) revealed a strong plakophilin 2
signal in fractions containing the affinity-purified pol III ho-
loenzyme (lane 3). By contrast, no plakophilin 2 reaction was
observed in fractions containing exclusively the 16-subunit pol
III, the so-called core enzyme (lane 4), indicating that plako-
philin 2 was associated with the pol III holoenzyme but not the
pol III core enzyme complex.
Immunolocalization Microscopy of the Nuclear Particles Containing
Plakophilin 2 and Pol III.
The nuclear particles containing plako-
philin 2 and RPC155 were visualized directly by double-label
immunofluorescence microscopy in several cell culture lines. For
example, the reaction on PLC cells is shown in Fig. 6. Plakophilin
2 and RPC155 displayed a granular nucleoplasmic immunostain-
ing and colocalized in distinct nuclear structures (Fig. 6c, yellow
dots). However, in addition to these particles containing both
proteins, each of them also occurred in separate granules as
indicated by the non-yellow dots. This finding suggests that only
a portion of nuclear plakophilin 2 is associated with complexes
containing RPC155 and that both proteins also exist in other
forms specific for either protein. Moreover, the plakophilin
2-positive dots were different from those immunostained for
plakophilins 1 and 3 (data not shown).
Discussion
We have begun to characterize the nucleoplasmic forms of
plakophilin 2, a protein also known as a major component of
desmosomes. In particle fractionation and immunoselection
experiments as well as in in vitro binding assays, plakophilin 2
has been detected in specific complexes with the largest
subunit of RNA polymerase III (RPC155), the pol III subunit
of 39 kDa and transcription factor TFIIIB and also in distinct
nucleoplasmic granules. Both plakophilin 2 and RPC155 are
also part of two large multisubunit complexes that appear,
depending on the specific extraction conditions, between
30–35 and 5060 S. Moreover, we have identified plakophilin
2 as one of the polypeptides enriched in affinity-purified
preparations of the pol III holoenzyme. This interaction
between plakophilin 2 and pol III components may provide a
clue to one of the hitherto unknown nuclear functions of a
junctional plaque protein.
Of the three nuclear RNA polymerases (I–III), pol III tran-
scribes genes encoding ribosomal 5S RNA, the tRNAs, signal
recognition particle RNA, U6 small nuclear RNA, and several
small viral RNAs (for review see ref. 23). The inclusion of
plakophilin 2 in nucleoplasmic assembly forms of the pol III
transcription machinery would be compatible with the wide-
spread and constitutive occurrence of plakophilin 2 in nuclei
(68), a situation that is fundamentally different from the
transient signal-induced nuclear translocation reported for other
proteins of the arm-repeat family such as
-catenin and plako-
globin (24–31) and suggests that it serves some general nuclear
function.
Fig. 6. Colocalization of plakophilin 2 and RPC155 in nuclear particles. Cultured PLC cells were double-immunolabeled with antibodies against plakophilin2
(a, red) and RPC155 (b, green) and observed by confocal laser scanning microscopy. Both proteins exhibit a finely punctate nucleoplasmic immunostaining,
sparing the nucleoli. They colocalize in many nuclear particles as indicated (yellow) in the merged image (c, arrows). These structures are different from various
other nucleoplasmic granules examined in parallel (for antibodies used see refs. 43 and 44). In addition, one sees some dots mutually exclusive for pol III (green)
or plakophilin 2 (red). (Scale bar, 20
m.)
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The human pol III of 700 kDa representing the core complex
contains 16 subunits that remain tightly associated with each
other even under partially denaturing conditions (e.g., see refs.
18–21). Its largest subunit (RPC155), shown here in complexes
with plakophilin 2, has been highly conserved throughout evo-
lution (18, 23, 32), as have amino acid sequence motifs in other
pol III subunits (23, 33, 34). Accurate transcription by pol III
requires the initiation factors TFIIIB and TFIIIC (35) and an
ordered sequence of steps for their assembly into preinitiation
complexes. On the other hand, affinity purification of pol III
from nuclear extracts has revealed a preassembled nucleoplas-
mic particle (holoenzyme) containing the 16-subunit pol III and
all basal factors essential for transcription (19, 23, 35, 36). Our
results indicate that plakophilin 2 may be added to the list of
components that can be present in this pol III holoenzyme
complex.
The pol III complexes with plakophilin 2 seem to be located
free in interchromatin spaces of the nucleoplasm, although their
specific distribution in relation to transcriptionally engaged
chromatin sites (37) remains to be established. We think that the
particles described are not engaged in transcription but represent
preinitiation assembly or storage forms, from which complexes
active in transcription or RNA processing might be recruited (19,
23, 27, 38). A special kind of nucleoplasmic assembly granules
has been described in amphibian oocytes and shown to harbor
not only pol III but also other kinds of polymerases (transcrip-
tosomes) and to aggregate to very large sizes up to 20
min
diameter (Cajal bodies; refs. 3941). Clearly, future studies will
have to reveal the functional roles of both the Cajal bodies and
the much smaller plakophilin 2-containing granules of somatic
nuclei.
We thank Drs. H. Spring (German Cancer Research Center), N.
Hernandez (Cold Spring Harbor Laboratory), and R. G. Roeder (Rocke-
feller University) for valuable discussions and experimental help as well
as the German Research Foundation (Deutsche Forschungsgemein-
schaft) for financial support.
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www.pnas.orgcgidoi10.1073 Mertens et al.
... PKP1 localizes both in the cytoplasm and the nucleus and can bind to single-stranded DNA (Schmidt et al. 1997;Sobolik-Delmaire et al. 2010). Similarly, PKP2 was shown to translocate to the nucleus, where it associates with components of the pol III transcription complex (Mertens et al. 2001). Moreover, by using the developmental model animal, Xenopus laevis (Munoz et al. 2012), a positive functional interaction of PKP3 with the transcription factor ETV1 was reported (Munoz et al. 2014). ...
... This binding perturbs the influenza A virus polymerase complex and limits polymerase activity and thereby restricts viral infection. And this observation correlates with the association of PKP2 with the RNA polymerase III subunit TFIIIB suggesting important functions in RNA transcription (Mertens et al. 2001). ...
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Tissue homeostasis is maintained by several cellular mechanisms and an imbalance may lead to diseased states. Here, the plakophilins 1, 2 and 3 operate as structural components and stabilize desmosomal cell-cell contacts. In their non-junctional states, they serve as regulators of signaling programs and control varied cellular processes that range from transcription, mRNA abundance, protein synthesis, growth, proliferation, migration to invasion and tumor development. Accordingly, mutations in plakophilins 1 and 2 lead to skin or heart diseases. Corresponding to their strong impact on tissue homeostasis, the expression of plakophilins is specifically deregulated in various cancer types and can be correlated with patients’ survival. However, our understanding on how plakophilins contribute to tumor development, progression and metastasis in a given tumor is still in its infancy and further in-depth studies using patient-derived data together with in vitro data and animal models are required.
... PKP2 is a member of the armadillo family generally localized in the nucleus and cytoplasm of epithelial tissues and cardiomyocytes, which binds the desmosomal cadherins and desmoplakin [42,43] . Lately, PKP2 ...
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Background Desmosomes play a key role in intercellular adhesive, but also contribute to tumorigenesis. This study aimed to examine the differential expression of desmocollin2 (DSC2), desmoglein2 (DSG2), and plakophilin2 (PKP2) in the progression of reflux esophagitis to esophageal adenocarcinoma (EAC) in the rat model of reflux disease established by esophagogastroduodenal anastomosis (EGDA). Methods EGDA was performed on rats to induce gastroesophageal reflux leading to the development of EAC. All rats were randomly divided into four groups: group A rats received EGDA only (n=27); group B rats received EGDA and iron supplementation (n=28); group C received pseudo surgery only (n=20); group D received pseudo surgery and iron supplementation (n=20). Animals were randomly selected from each group and euthanized at 8 weeks and 32 weeks following EGDA. Esophageal tissues were harvested and divided into 4 types (normal esophagus, esophagitis, dysplasia, and EAC). On these tissue types, immunohistochemistry was performed to characterize the localization and distribution of DSC2, DSG2, and PKP2, while qRT-PCR and western blot were performed to detect the expression of DSC2, DSG2, and PKP2 at the gene and protein levels. Results At 8 weeks after surgery, 80% of rats in group A and 100% in group B had esophagitis. At 32 weeks, 29.41% and 17.65% of rats in group A developed dysplasia and EAC, respectively, while in group B, dysplasia and EAC accounted for 44.44% and 38.89%, respectively. The expression of DSC2, DSG2, and PKP2 at both the gene and protein levels increased progressively from esophagitis to dysplasia, and EAC. Of note, all of the three genes were significantly upregulated in EAC tissues compared with tissues of esophagitis. Conclusion DSC2, DSG2, and PKP2 may play an important role in the progression of esophagitis to EAC. Their expression levels may therefore be utilized as molecular biomarkers for early diagnosis and targeted therapy for EAC.
... [4][5][6][7] Pkp2 is important for desmosome assembly and is an essential morphogenic factor for, and architectural component of, the heart. [8][9][10] Pkp3 plays a role in both desmosome-dependent adhesion and signaling pathways. 11,12 Pkp4 is a component of desmosomal adhesion plaques that regulates junctional plaque organization and cadherin function. ...
... Consequently, more PKP-2 was found around the nucleus since this domain inhibits nuclear localization of PKP-2 (41). Moreover, since it's established that PKP-2 interacts with transcription factors (tfs) like TFIIIB and RNA polymerase III in the nucleus, we can say that such a localization is altering the gene expression of connexin43 (42). ...
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Cardiomyopathies (CMs) are a group of cardiac pathologies caused by an intrinsic defect within the myocardium. The relative contribution of genetic mutations in the pathogenesis of certain CMs, such as hypertrophic cardiomyopathy (HCM), arrythmogenic right/left ventricular cardiomyopathy (ARVC) and left ventricular non-compacted cardiomyopathy (LVNC) has been established in comparison to dilated cardiomyopathy (DCM) and restrictive cardiomyopathy (RCM). The aim of this article is to review mutations in the non-coding parts of the genome, namely, microRNA, promoter elements, enhancer/silencer elements, 3′/5′UTRs and introns, that are involved in the pathogenesis CMs. Additionally, we will explore the role of some long non-coding RNAs in the pathogenesis of CMs.
... The results in this report suggest that PKP3 physically forms a complex with p38 and inhibits the transport of p38 to the nucleus thereby affecting gene expression. Previous reports have demonstrated that in addition to being present at the cell border, PKP3 is also found in cytoplasmic granules [57][58][59]. We have not observed any co-localization between p38 and PKP3 either at the cell border or in the cytoplasm. ...
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An increase in tumour formation and metastasis are observed upon plakophilin3 (PKP3) loss. To identify pathways downstream of PKP3 loss that are required for increased tumour formation, a gene expression analysis was performed, which demonstrated that the expression of lipocalin2 (LCN2) was elevated upon PKP3 loss and this is consistent with expression data from human tumour samples suggesting that PKP3 loss correlates with an increase in LCN2 expression. PKP3 loss leads to an increase in invasion, tumour formation and metastasis and these phenotypes were dependent on the increase in LCN2 expression. The increased LCN2 expression was due to an increase in the activation of p38 MAPK in the HCT116 derived PKP3 knockdown clones as LCN2 expression decreased upon inhibition of p38 MAPK. The phosphorylated active form of p38 MAPK is translocated to the nucleus upon PKP3 loss and is dependent on complex formation between p38 MAPK and PKP3. WT PKP3 inhibits LCN2 reporter activity in PKP3 knockdown cells but a PKP3 mutant that fails to form a complex with p38 MAPK cannot supress LCN2 promoter activity. Further, LCN2 expression is decreased upon loss of p38β, but not p38α, in the PKP3 knockdown cells. These results suggest that PKP3 loss leads to an increase in the nuclear translocation of p38 MAPK and p38β MAPK is required for the increase in LCN2 expression.
... Nuclear PKP1 complexes with catenin and is found bound to single stranded DNA [47]. PKP2, which is more abundant in the lower density group, binds to catenin and complexes with the RNA polymerase III holoenzyme [48]. ...
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Background Vascular progenitor cells (VPCs) derived from embryonic stem cells (ESCs) are a valuable source for cell- and tissue-based therapeutic strategies. During the optimization of endothelial cell (EC) inductions from mouse ESCs using our staged and chemically-defined induction methods, we found that cell seeding density but not VEGF treatment between 10 ng/mL and 40 ng/mL was a significant variable directing ESCs into FLK1⁺ VPCs during stage 1 induction. Here, we examine potential contributions from cell-to-cell signaling or cellular metabolism in the production of VPCs from ESCs seeded at different cell densities. Methods Using 1D ¹H-NMR spectroscopy, transcriptomic arrays, and flow cytometry, we observed that the density-dependent differentiation of ESCs into FLK1⁺ VPCs positively correlated with a shift in metabolism and cellular growth. Results Specifically, cell differentiation correlated with an earlier plateauing of exhaustive glycolysis, decreased lactate production, lower metabolite consumption, decreased cellular proliferation and an increase in cell size. In contrast, cells seeded at a lower density of 1,000 cells/cm² exhibited increased rates of glycolysis, lactate secretion, metabolite utilization, and proliferation over the same induction period. Gene expression analysis indicated that high cell seeding density correlated with up-regulation of several genes including cell adhesion molecules of the notch family (NOTCH1 and NOTCH4) and cadherin family (CDH5) related to vascular development. Conclusions These results confirm that a distinct metabolic phenotype correlates with cell differentiation of VPCs.
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Vertebrate beta-catenin plays a key role as a transducer of canonical-Wnt signals. We earlier reported that, similar to beta-catenin, the cytoplasmic signaling pool of p120-catenin-isoform1 is stabilized in response to canonical-Wnt signals. To obtain a yet broader view of the Wnt-pathway's impact upon catenin proteins, we focused upon plakophilin3 (plakophilin-3; Pkp3) as a representative of the plakophilin-catenin subfamily. Promoting tissue integrity, the plakophilins assist in linking desmosomal cadherins to intermediate filaments at desmosome junctions, and in common with other catenins they perform additional functions including in the nucleus. In this report, we test whether canonical-Wnt pathway components modulate Pkp3 protein levels. We find that in common with beta-catenin and p120-catenin-isoform1, Pkp3 is stabilized in the presence of a Wnt-ligand or a dominant-active form of the LRP6 receptor. Pkp3's levels are conversely lowered upon expressing destruction-complex components such as GSK3β and Axin, and in further likeness to beta-catenin and p120-isoform1, Pkp3 associates with GSK3beta and Axin. Finally, we note that Pkp3-catenin trans-localizes into the nucleus in response to Wnt-ligand and its exogenous expression stimulates an accepted Wnt reporter. These findings fit an expanded model where context-dependent Wnt-signals or pathway components modulate Pkp3-catenin levels. Future studies will be needed to assess potential gene regulatory, cell adhesive, or cytoskeletal effects.
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Chapter
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RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.
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The cytokeratin-binding, basic 80.5 kDa polypeptide plakophilin 1 ("band 6 protein" of bovine muzzle desmosome fractions) has originally been described as a single molecular species, localized to desmosomal plaques of certain cell types, mostly stratified squamous epithelia and complex epithelia. We now report that this protein exists in at least two different isoforms: 726 amino acids (aa), plakophilin 1a; and 747 aa, plakophilin 1b. This reflects the splicing of the 21 aa-encoding exon 7 of the human plakophilin-1 gene and that each mRNA splice form can occur in two polyadenylation forms of 2.7 kb and 5.3 kb. Antibodies recognizing either isoform and/or others that are specific for the exon-encoded sequence of form 1b have allowed, in combination with immunolocalization protocols minimizing losses of diffusible proteins, the detection of both isoforms in the nucleoplasm of diverse kinds of cultured cells and tissues, including desmosome-forming cells as well as cells that never form desmosomes. The protein has also been identified in manually isolated nuclei (germinal vesicles) of Xenopus laevis oocytes. Plakophilin 1a accumulates in nuclei as shown by suitable immunolocalization protocols and upon overexpression following transfection with cDNAs, but is also located in desmosomes of stratified and complex epithelia. By contrast, isoform 1b has been found exclusively in nuclei, even in cells connected by desmosomes immunostained with plakophilin 1a-reactive antibodies. We conclude that plakophilins 1a and 1b are constitutive nuclear proteins encoded by the same gene, which is not expressed in relation to epithelial differentiation pathways, whereas the additional appearance of plakophilin 1a in desmosomal plaques of stratified and complex epithelia is regulated by an as yet unknown mechanism of differentiation-dependent topogenic recruitment. Possible functions of plakophilins are discussed in relation to recent reports of the involvement of other members of the armadillo/plakoglobin multigene family of proteins in cell surface-gene regulation signalling pathways.
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Transcription of small genes by RNA polymerase III or C (pol III) involves many of the strategies that are used for transcription complex formation and occasionally the same components as those used by RNA polymerase II or B (pol II). Transcription complex formation is a multistep process that leads to the binding of a single initiation factor, TFIIIB, which in turn directs the selection of pol III. The general transcription factor TFIID can be involved in both pol II and pol III transcription. These and other similarities point towards a unifying mechanism for eukaryotic transcription initiation.
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This chapter focuses on small-scale preparation of extracts from radiolabeled cells efficient in pre-mRNA splicing. By several criteria, nuclear extracts prepared by the small-scale procedure are comparable to extracts prepared by the conventional procedure. Miniextracts are fully active in pre-mRNA splicing and also efficiently transcribe class II promoters. Harvested cells are resuspended in a volume of buffer A equal to the packed cell volume, left to swell on ice for 15 rain, and then lysed with 30 strokes of a B-type Dounce homogenizer. The short dialysis period results in less precipitation of denatured protein and is sufficient to adjust the extract to the desired ionic conditions. Precipitate that forms during dialysis can be removed by centrifugation of the extract for 10 min at 2000 rpm in a Beckman J6B centrifuge using a JS 4.2 rotor. Monolayer cells at 80% confluence are harvested using a rubber policeman or by trypsinization. Cells are washed in 30 volumes of phosphate-buffered saline (PBS) and the packed cell volume determined by pelleting for 5 minutes at 1200 rpm in a J6B centrifuge using a JS 4.2 rotor. Packed cells are resuspended in one packed cell volume of buffer A and allowed to swell on ice for 15 min.