1 Unité 9003 du CNRS, Ecole Supérieure de Biotechnologie de
Strasbourg, Boulevard Sébastien Brant, 67400 Illkirch, France
2 Institut Curie, Section Recherche UMR 144 du CNRS, 26 Rue d'Ulm, F-75248
Paris, France
3 Institut de Biologie Moléculaire des Plantes, CNRS, 12 rue du General
Zimmer, 67084, Strasbourg, France
4 Institut Curie, Section Recherche UMR 147 du CNRS, 26 Rue d'Ulm, F-75248
Paris, France
5 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 163, 67400 Illkirch,
France
6 Pharmaceuticals Research, BASF AG, D-67056 Ludwigshafen, Germany
Author for correspondence (e-mail:
demurcia{at}esbs.u-strasbg.fr)
Accepted 2 January 2003
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Summary |
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Key words: Centrosome, NAD+ metabolism, DNA damage, G1/S cell cycle control, Midbody
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Introduction |
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A superfamily of PARP-domain-containing proteins has recently emerged
(Amé et al., 1999;
Jacobson and Jacobson, 1999
;
Smith, 2001
). From this
family, PARP-1 and PARP-2 were until now the only characterized enzymes whose
activity has been shown to be stimulated by DNA strand breaks
(Amé et al., 1999
;
Schreiber et al., 2002
).
Acting as survival factors in mammalian cells under genotoxic stress, they are
localized in the nucleus and possibly function as homo and/or heterodimers.
Murine fibroblasts carrying a targeted disruption of either the
mPARP-1 or mPARP-2 gene are defective in base excision
repair (BER), indicating that PARP-1 and PARP-2 can reciprocally, but
partially, compensate for the absence of each other
(Schreiber et al., 2002
).
VPARP (vault-PARP, PARP-4) was characterized through its interaction with
the MVP (major vault protein) in yeast in a two-hybrid screen. Vault particles
are large ribonucleoprotein complexes found in the cytoplasm of mammalian
cells (Kickhoefer et al.,
1999), which may have a transport function. VPARP
poly(ADP-ribosyl)ates MVP in purified vaults, but the consequences of this
modification on vaults properties remain elusive. Tankyrase-1 (PARP-5a) was
initially identified through its interaction with the telomeric protein TRF1,
a negative regulator of telomere length
(Smith et al., 1998b
). In
vitro poly(ADP-ribosyl)ation by tankyrase-1 inhibits TRF1 binding to telomeric
DNA, suggesting a role for tankyrase-1 in telomere function
(Smith and de Lange, 2000
).
Overexpression of tankyrase-1 has recently been found to release TRF1 from
telomeres, thus inducing their elongation
(Smith and de Lange, 2000
).
Tankyrase-2 (PARP-5b) appears to interact with many partners at discrete
subcellular locations, including the Golgi complex
(Chi and Lodish, 2000
) and
endosomes (Lyons et al.,
2001
). It also displays telomeric functions while interacting with
tankyrase-1 (Cook et al.,
2002
). During mitosis VPARP and tankyrase-1 are located at the
mitotic spindle (Kickhoefer et al.,
1999
) and in the pericentriolar region, respectively
(Smith and de Lange,
1999
).
A sequence encoding a 60 kDa protein with homology to PARP-1 and 2, called
PARP-3, was previously discovered in an EST library screening using the
catalytic domain sequence of hPARP-1
(Johansson, 1999). Using the
same approach, we cloned a different human cDNA encoding a version of PARP-3
that was seven amino acids longer. Here we report that recombinant hPARP-3 is
endowed with PARP activity in vitro. During the entire cell cycle, hPARP-3 is
localized to the centrosome, the microtubule organising centre of animal
cells, and resides preferentially in the daughter centriole. Given the
potential role of centrosomes in cell cycle regulation, we tested whether
hPARP-3 participates in the regulation of cell cycle checkpoints. Our results
argue that hPARP-3 negatively influences the G1/S cell cycle progression
without interfering with centrosome duplication. Moreover, we found that
hPARP-3 interacts with hPARP-1, which was previously shown to be present at
the centrosome as well (Kanai et al.,
2000
).
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Materials and Methods |
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Fluorescence in situ hybridization (FISH) analysis
Human chromosomes were prepared from human peripheral blood lymphocyte
cultures after BrdU incorporation during the last 7 hours before harvesting.
Mouse chromosomes were prepared from normal mouse fibroblast cultures without
BrdU incorporation. The full-length cDNA sequence encoding the
hPARP-3 gene and a 2.8 kb mouse PARP-3 genomic clone (to be
described elsewhere) were labelled by nick-translation with biotin-11-dUTP. 20
ng/µl hPARP-3 probe was hybridized to human chromosomes in hybridization
buffer; the mouse PARP-3 probe was hybridized to mouse chromosomes at a
concentration of 15 ng/µl, as previously described
(Apiou et al., 1996). Detection
of hybridization was performed using a goat anti-biotin antibody (Vector
Laboratories, Peterborough, UK) and a rabbit anti-goat fluorescein
isothiocyanate-conjugated antibody (P.A.R.I.S., Compiègne, France).
Direct banding of 5-BrdU-substituted chromosomes stained with propidium iodide
was obtained by incubation in an alkaline solution of p-phenylenediamine
(PPD11) (Lemieux et al.,
1992
). Mouse chromosomes were stained with DAPI and identified by
computer-generated reverse-DAPI banding. Metaphases were observed under a
fluorescent microscope (DMRB, Leica, Germany). Images were captured using a
cooled Photometrics CCD camera and Quips-smart capture software (Vysis).
Overproduction and purification of h PARP-3
hPARP-3 cDNA was cloned into the baculovirus transfer vector pFASTBAC1
(Life Technologies, Invitrogen, Cergy Pontoise, France). Sf9 cell propagation
and protein production was performed according to Miranda et al.
(Miranda et al., 1997).
Purification of hPARP-3 was performed by affinity chromatography on
Affigel-3-Aminobenzamide as previously described
(Amé et al., 1999
;
Giner et al., 1992
).
Plasmids
cDNA encoding hPARP-3 or its N-terminal domain (54 amino-acids) were cloned
either into the eukaryotic expression vectors pBC
(Chatton et al., 1995) in frame
with GST, giving raise to pBC hPARP-3 and pBC N-ter hPARP-3, or into the pEGFP
vector (Clontech), giving raise to pEGFP-hPARP-3 and pEGFP N-ter hPARP-3.
Poly(ADP-ribosyl)ation assay, western blot, south-western blot and
GST pulldown
900 ng of purified hPARP-3 were incubated in 20 µl of buffer containing
100 mM Tris-HCl, pH 8.0, 10 µM NAD+ and 10 µCi
[32P] NAD+ (3 Ci/mmoles). After 15 minutes of incubation
at 25°C, the reaction was stopped by dilution in the Laemmli buffer.
Samples were analyzed on a 10% SDS-PAGE and blotted as described previously
(Mazen et al., 1989) and
autoradiographed on Kodak BiMax MS film. South-western blotting using
[32P]-labelled nick-translated activated DNA was performed as
described previously (Mazen et al.,
1989
).
Two peptides matching the hPARP-3 N-terminal region (amino-acid 25-37 and
8-22) were used to elicit hPARP-3 polyclonal antibodies in rabbits (Ab 1650)
and in mice (TJ56), respectively. Potential partners of hPARP-3 were isolated
using the GST-pulldown technique described previously
(Masson et al., 1998).
Cell culture and cell cycle analysis
Mammalian cell cultures were maintained at 37°C in 5% CO2.
The human lymphoblastic KE 37 cell line
(Moudjou and Bornens, 1994)
was grown in suspension in RPMI 1640 medium supplemented with 7% foetal calf
serum (FCS) (GIBCO) and gentamicin. HeLa, CHO, 3T3 and HeLa HC1 cells stably
expressing the fusion GFP-centrin (Piel et
al., 2000
) were cultured in DMEM containing 10% FCS and
gentamicin. For cell cycle analysis, HeLa cells were transiently transfected
by plasmids expressing GST alone or in fusion with hPARP-3 or N-ter hPARP-3.
After 24 hours, cells were mock-treated or treated with 1 mM N-methyl
N-nitrosourea (MNU) for 20 hours and trypsinized. Cell suspensions were washed
twice in PBS containing 1% glucose, 1 mM EDTA and fixed with cold 70% ethanol
in PBS for 2 hours. The fixed cells were then washed once with PBS, 1%
glucose, 1 mM EDTA, once with PBS, 1% glucose, 1 mM EDTA, 0.1% Triton X100 and
incubated for 45 minutes at room temperature with a monoclonal anti-GST
antibody diluted 1:400. Cells were washed twice with PBS, 1% glucose, 1 mM
EDTA, 0.1% Triton X100 and incubated with FITC-conjugated anti-mouse antiserum
(Cappel) for 45 minutes at room temperature. After a last wash with PBS, 1%
glucose, 1 mM EDTA, cells were incubated with 100 µg/ml RNaseA for 30
minutes at room temperature, stained with propidium iodide and analyzed with a
FACScan flow cytometer using a gate on FITC-positive cells.
Isolation of centrosomes
Purified centrosomes were isolated from KE 37 cells according to the method
described by Moudjou and Bornens (Moudjou
and Bornens, 1994). The centrosome fractions were analyzed by
immunofluorescence as described previously
(Bornens and Moudjou,
1999
).
Indirect immunofluorescence
Cells (5x104) grown on coverslips were transfected or not
with the plasmids expressing GST- or GFP-fusion proteins and subsequently
untreated or treated with various DNA-damaging agents, using either
-irradiation (10 Grays, 1.0 Gy/minute delivered by a 60Co
source) or a treatment with 1 mM N-methyl-N-nitrosourea (MNU) for 30 minutes
or 1 mM H2O2 for 10 minutes. Amplification of
centrosomes was evaluated in CHO cells treated with 4 mM Hydroxyurea (HU) for
48 hours prior to processing for immunochemistry
(Meraldi et al., 2002
;
Meraldi et al., 1999
).
Following fixation with 100% methanol for 5 minutes at 20°C, cells
were washed three times with PBS supplemented with 0.1% Tween (v/v). Cells
were incubated overnight at 4°C or for 2 hours at room temperature, with a
primary antibody the polyclonal antibody anti-hPARP-3 (1650) (1:100),
a monoclonal IgG2a antibody anti-p34cdc2 (1:200, Sigma), a
monoclonal IgG2b antibody anti-acetylated
-tubulin (1:1000, Sigma) or a
monoclonal antibody anti-glutamylated tubulin (Gt 335) (1:2000) or a
monoclonal antibody anti-hPARP-1 (F1-23, 1:100). After washing, cells were
incubated for 2 hours at room temperature with the appropriate conjugated
secondary antibody: a Texas-Red-conjugated anti-rabbit antiserum (1:400,
Sigma), a sheep FITC-conjugated anti-mouse anti-serum (1:400, Sigma) or an
Alexa Fluor (568 or 488) goat-anti-mouse IgG (1:1000, Molecular Probes). DNA
was counterstained with DAPI. Immunofluorescence microscopy was performed
using a Zeiss Axioplan equipped with a DP50 chilled CCD camera (Olympus) and
the capture software ViewFinder Lite (Olympus). Alternatively, observations
were made with a confocal microscope equipped with an argon/krypton laser and
suitable barrier filters (Leica TCS4D, Heidelberg, Germany).
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Results |
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Analysis of the 5' end of the human PARP-3 gene (accession
number: GenBank AY126341) revealed the presence of two potential splicing
acceptor (AS) sites, AS1 and AS2 (Fig.
1C), which give rise to two proteins differing by seven amino
acids at the N-terminus. A sequence of hPARP-3 has been previously published
by Johansson (Johansson, 1999)
that lacks the first seven N-terminal amino acids. In order to assess the real
occurrence of the longest version, we used a PCR strategy to detect the
5' end of hPARP-3 reverse-transcribed mRNA from normal human lung tissue
(Fig. 1D). Primers were
designed to match the nucleotide sequence encoding the longest form of hPARP-3
(PCR1). Under high stringency PCR conditions, the sense primer can hybridize
only when AS2 is selected. As a PCR control, we used a second sense primer
(PCR2) that matches hPARP-3 mRNA downstream of the splicing site whatever the
type of mRNA in the cell. As shown in Fig.
1D, the PCR2 control product was 141 bp, as expected, and a unique
band of 162 bp was detected in PCR1, which is in good agreement with the
presence of the longest PARP-3 encoding mRNA. Therefore, we concluded
that a human PARP-3, which is seven residues longer than the amino-acid
sequence published by Johansson
(Johansson, 1999
), is
expressed in human cells. Nevertheless we cannot exclude the possibility that
both sequences are present in cells, depending upon physiological
conditions.
|
The chromosomal localization of the hPARP-3 gene was identified by using FISH on human chromosomes using a human cDNA PARP-3 probe. Consistent signals on chromosome 3 band 3p21.1 to 3p21.31 were identified (Fig. 1E) and on chromosome 9, band F1-F2 in mouse (Fig. 1F-G), which confirms that hPARP-3 is a novel member of the PARP family encoded by a specific gene. As it is already the case for the PARP-1 and PARP-2 genes, a synteny was noticed between the human and mouse chromosomal regions coding for PARP-3 genes.
Full-length hPARP-3 was overexpressed in the baculovirus/Sf9 system and
purified by affinity chromatography on Affigel-3AB
(Giner et al., 1992), yielding
a polypeptide with an apparent molecular mass of 67 kDa
(Fig. 2A, lane b). We took
advantage of the unique N-terminal domain of hPARP-3, which has no counterpart
in PARP-1 and PARP-2 to generate two polyclonal antibodies 1650 and TJ56. As
shown in Fig. 2B (lanes f to
j), anti-PARP-3 antibody 1650 recognizes the recombinant hPARP-3 as well as
hPARP-3 in HeLa cell extracts. As expected, these antibodies did not
crossreact with either hPARP-1 or hPARP-2 (data not shown).
|
The ability of hPARP-3 to synthesize ADP-ribose polymers in an autopoly(ADP-ribosyl)ation reaction was examined in an in vitro PARP activity assay using [32P] NAD+ as a substrate; the optimal concentration of 10 µM NAD+ was determined. As shown in Fig. 2A (lane d), PARP-3 automodification occurs and is specifically inhibited by the competitive inhibitor 3-Aminobenzamide (3-AB) (lane e). Interestingly, a slight increase in enzymatic activity (two-fold) was repeatedly observed in the presence of nicked DNA, in accord with the capacity of the N-terminal domain to bind DNA in a south-western assay (Fig. 2A, lane c). Altogether, these results clearly indicate that PARP-3 is a bona fide poly(ADP-ribose) polymerase.
hPARP-3 localizes preferentially to the daughter centriole throughout
the cell cycle
To establish the subcellular localization of hPARP-3, exponentially growing
HeLa cells were stained with the purified hPARP-3 antibody 1650. hPARP-3
localized to two closely spaced dots resembling centrosomes, usually located
close to the nuclear envelope. As a control, we performed a double
immunofluorescence experiment using the anti-hPARP-3 antibody 1650 and an
antibody raised against p34cdc2, as
p34cdc2 has been previously shown to localize to
centrosomes (Bailly et al.,
1989; Pockwinse et al.,
1997
). As shown in Fig.
3A, the immunostaining of hPARP-3 was clearly associated with the
centrosomal staining of p34cdc2. To verify this
observation, HeLa HC1 cells constitutively expressing the centrosomal protein
centrin in fusion with GFP (Piel et al.,
2000
; White et al.,
2000
) were immunostained with the anti-hPARP-3 antibody 1650. The
colocalization of hPARP-3 with GFP-centrin is clearly visible in
Fig. 3B and confirms the
association of PARP-3 with the centrosome. A similar result was obtained with
the mouse polyclonal TJ56 antibody (data not shown). Staining of centrosomes
was independent of the fixation method, as it was observed following
procedures based on either aldehyde or organic solvent fixation. Finally, cell
treatment with a microtubule-depolymerizing drug such as nocodazole or with a
microtubule-stabilizing drug such as taxol did not displace the centrosomal
signal emerging from the anti-PARP-3 antibodies (data not shown),
demonstrating that the localization of hPARP-3 to the centrosome is
independent of microtubule dynamics.
|
Strikingly, a preferential colocalization of hPARP-3 with one of the two
centrioles was repeatedly noticed (Fig.
3C). The nature of the two centrioles can be distinguished by
using immunocytochemistry in mouse 3T3 cells
(Chang and Stearns, 2000;
Lange and Gull, 1995
). In G1
phase, the mother centriole of these cells grows a primary cilium, which is
partially made of an acetylated form of
-tubulin. As shown in
Fig. 3D, the signal
corresponding to hPARP-3 antibodies cannot be superimposed on the signal from
anti-acetylated
-tubulin antibodies, therefore suggesting that hPARP-3
colocalizes preferentially with the daughter centriole. A similar conclusion
could be reached from colocalization experiments using HeLa HC1 cells, where
the reproducible association of hPARP-3 with the `smaller' (daughter)
centriole was clearly visible (Fig.
3C).
We next examined the stage of the cell cycle that hPARP-3 associates with the centrosome. Following immunostaining of exponentially growing HeLa HC1 cells, hPARP-3 could unequivocally be identified at the centrosome in >90% of cells, independently of the antibody used. Moreover, confocal microscopy revealed a colocalization of hPARP-3, mostly with one of the two centrioles, most probably the daughter one, in G2 and throughout mitosis from early prophase to telophase (Fig. 3E). Taken together, these data demonstrate that hPARP-3 is a core component of the centrosome and is preferentially associated with the daughter centriole at all stages of the cell cycle.
Purified centrosomes are enriched in hPARP-3
To further substantiate the cellular distribution of hPARP-3, centrosomes
were isolated from KE 37 cells (Moudjou
and Bornens, 1994) and examined by indirect immunofluorescence
(Fig. 4A). Centrosomes were
spun down on coverslips and co-stained with the
anti-p34cdc2 antibody, to label centriole doublets, and
with the purified polyclonal anti-PARP-3 antibody 1650. Again, a specific
staining of one of the two centrioles was observed, in agreement with the
above results obtained with whole cells.
|
The presence of hPARP-3 in the centrosome was also confirmed biochemically
(Fig. 4B). Low-speed Triton
X-100 soluble and insoluble fractions of unsynchronized KE 37 cell lysates
were prepared as described previously
(Tassin and Bornens, 1999) and
submitted to western blot analysis together with centrosome sucrose gradient
preparations (Moudjou and Bornens,
1994
) and recombinant hPARP-3. Proteins were probed with the
affinity-purified anti-hPARP-3 antibody 1650
(Fig. 4B). A band at 67 kDa is
observed in enriched centrosomes, as well as in the Triton-insoluble fraction
and to a lesser extent in the Triton-soluble fraction of KE 37 cells.
DNA damage does not affect hPARP-3 localization
Sato et al. have previously reported that -irradiation of U2-OS
osteosarcoma cells or HeLa cells results in centrosome overduplication
(Sato et al., 2000
). Moreover,
we and others have demonstrated that radiation-induced DNA strand-breaks
activate PARP-1 and PARP-2 in the nucleus. We thus examined the hPARP-3
localization in HeLa HC1 cells exposed to various DNA-damaging agents
including
-radiation, N-methyl-N-nitrosourea (MNU) or
H2O2 treatment. Asynchronous HeLa HC1 cells were
irradiated at a single dose of 10 Gy or treated with 1 mM MNU or 1 mM
H2O2; the centrosome number and hPARP-3 subcellular
localization were subsequently determined by immunofluorescence at various
time points following DNA damage (Fig.
5 and data not shown). Although the abnormal cells with more than
two centrioles were less than 5% of the population in untreated cells, this
population increased up to 70% by 120 hours post-irradiation or 72 hours
post-MNU treatment as previously (Sato et
al., 2000
). Whatever the type of DNA injury, multipolar spindles
(Fig. 5A-C) and coalescence of
centrosomes (Fig. 5D-F) were
frequently observed, as already described
(Brinkley, 2001
). However,
co-staining of treated cells with the affinity-purified anti-hPARP-3 antibody
and the anti-p34cdc2 antibody revealed that hPARP-3 was always present in the
centrosome even under DNA damage conditions. Therefore, the localization of
hPARP-3 is not affected by centrosome dynamics in response to DNA-damaging
agents.
|
Overexpression of hPARP-3 or its N-terminal domain interferes with
the cell cycle progression at the G1/S transition
Given the tight link between centrosome homeostasis and cell cycle
regulation, we tested whether hPARP-3 participates in cell cycle regulation in
mock or DNA-damage-exposed cells. We transiently expressed in HeLa cells the
full-length hPARP-3 or its N-terminal domain as a glutathione S-transferase
(GST)-fusion protein, and the cell cycle distribution of the GST-expressing
cells was analyzed. As displayed in Fig.
6A, in untreated cells the expression of both hPARP-3 or its
N-terminal domain caused an imbalance in normal cell cycle distribution
characterized by an increase in the fraction of cells in G1/S compared to
cells expressing GST alone.
|
DNA damage checkpoints arrest the cell cycle at the G2/M boundary to allow DNA repair, thus preventing progression of cells into mitosis. Following DNA base damage induced by a treatment with MNU, HeLa cells expressing only GST showed, as expected, a prominent accumulation at the G2/M boundary (39%). In contrast, the proportion of cells at the G2/M boundary was markedly decreased in cells expressing GST-N-ter hPARP-3 or GST-hPARP-3 (13% and 22% respectively); instead the GST fusion protein accumulated at the G1/S boundary. Together, these results imply that hPARP-3 acts at the G1/S cell cycle transition and that this function is carried out by its N-terminal domain but not by its catalytic domain.
To better correlate the biological function of hPARP-3 with its
localization at the centrosome, we transiently expressed in HeLa cells the
full-length hPARP-3 or its N-terminal domain as GFP fusions and analyzed their
subcellular distribution using anti--tubulin antibodies as centrosome
markers. As shown in Fig. 6B,
hPARP-3 (panel d-f), and more precisely its 54 amino-acid N-terminal domain
(panel a-c), contains a motif responsible for centrosomal retention. This
centrosomal localisation paralleled the G1/S cell cycle accumulation, as the
exon-1-deleted version of the hPARP-3 N-terminal domain in fusion with GST did
not target to the centrosome and did not induce a G1/S accumulation when
overexpressed in HeLa cells (data not shown).
hPARP-3 overexpression does not interfere with centrosome
overduplication induced by hydroxyurea treatment
We considered the possibility that the G1/S block observed in cells
expressing GST-N-ter hPARP-3 or GST-hPARP-3 is caused by an inhibition of
centrosome duplication. To test this hypothesis, the GFP-hPARP-3 or GFP-N-ter
hPARP-3 constructs were tested for their ability to interfere with centrosome
duplication in an assay developed by Balczon et al.
(Balczon et al., 1995). This
assay is based on the observation that hydroxyurea (HU) treatment of CHO cells
blocks DNA replication but allows multiple rounds of centrosome replication to
occur. Following 40 hours of treatment, more than 50% of cells contain more
than two centrosomes (Balczon et al.,
1995
; Matsumoto et al.,
1999
; Meraldi et al.,
1999
). As displayed in Fig.
7E-G, CHO cells expressing GFP-hPARP-3 were not affected by
centrosome overamplification (Fig.
7C,D). Similar results were obtained with CHO cells expressing the
GFP-N-ter hPARP-3.
|
To quantify the amplification of centrosomes, the number of spots obtained
after anti--tubulin staining were counted, and the mean
values±s.d. for at least 50 cells were calculated and plotted on a
histogram (Fig. 7H). For cells
expressing GFP in fusion with hPARP-3 or its N-terminal domain, we detected,
respectively, 4.3±1.6 (range 1 to 9) and 4.2±1.5 (range 2 to 7)
centrosomes. This number is slightly decreased compared with non-transfected
cells, which display 5.4±1.6 (range 2 to 10) centrosomes, but quite
similar to the number (4.1±1.3, range 2 to 7) of centrosomes detected
in cells expressing GFP alone. Overall, these results suggest that the G1/S
cell cycle block mediated by hPARP-3 overexpression is not due to direct
inhibition of centrosome duplication.
PARP-3 interacts with PARP-1 at the centrosome
Both the previously demonstrated localization of hPARP-1 to the centrosome
(Kanai et al., 2000) and the
ability of different PARPs to interact with each other
(Sbodio et al., 2002
;
Schreiber et al., 2002
)
prompted us to investigate the possibility of a contact between hPARP-3 and
hPARP-1. To this end, hPARP-3 or its N-terminal domain was transiently
overexpressed as a GST-fusion proteins in HeLa cells, and the interacting
proteins were analyzed by western blotting. As shown in
Fig. 8A, hPARP-1 was captured
by the GST-hPARP-3 fusion protein but not by GST alone or GST fused to the
N-terminal domain of hPARP-3. Moreover, the contact between hPARP-1 and
hPARP-3 could be substantially enhanced when poly(ADP-ribose) synthesis was
inhibited in the presence of the PARP inhibitor 3-aminobenzamide
(Fig. 8A, compare lanes b and
c), demonstrating an increased affinity of hPARP-1 and hPARP-3 for their
respective unmodified form.
|
Proof of this interaction was further strengthened by the detection of both
hPARP-1 and hPARP-3 in enriched fractions of purified centrosomes, which were
characterized by the presence of -tubulin, a major component of
centrosomes (Fig. 8B). Thus,
the probability that the two PARPs interact is thought to be highest in this
subcellular compartment. Indirect immunofluorescence experiments confirmed
that hPARP-1 also resides in the centrosome. Indeed, hPARP-1 colocalizes with
GFP-centrin in HeLa HC1 cells stably expressing this typically centrosomal
protein (Fig. 8C). Despite the
large nuclear staining of hPARP-1 and the close proximity of centrosomes to
the nucleus, which makes it harder to affirm the centrosomal localization of
hPARP-1, the colocalization of hPARP-1 and hPARP-3 could be performed using
their respective antibodies (Fig.
8D). Taken together, these results indicate that hPARP-1 is
present in both centrioles and interacts with hPARP-3, most probably at the
daughter centriole. The enzymatic activity of both enzymes at the centrosome
could be visualized in purified centrosome spreadings following incubation
with NAD+ and immunostaining with the anti-poly ADP-ribose antibody
(data not shown). Finally, using PARP-1 knockout cell lines, we could
demonstrate that the location of mPARP-3 at the centrosome was independent of
the presence of mPARP-1 (data not shown).
![]() |
Discussion |
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The gene encoding hPARP-3 has been localized to the short arm of chromosome
3. Interestingly, allele loss involving the 3p arm is one of the most frequent
and earliest known genetic events in lung cancer
(Wistuba et al., 2000).
Moreover, in the particular region 3p21.1 to 3p21.31 containing the
hPARP-3 gene a 600 kb region is most frequently undergoing allelic
loss in the bronchial epithelia of smokers. It is therefore important to test
whether hPARP-3 is present in lung epithelia, particularly in lung
cancers.
PARP-3 localizes to the daughter centriole throughout the cell
cycle
Indirect immunofluorescence experiments and subcellular fractionation
concur to demonstrate that hPARP-3 is a core component of the centrosome. This
particular localization, throughout the cell cycle, is independent of the
microtubule polymerization status. Our data show that hPARP-3 is
preferentially associated with the daughter centriole. Two independent lines
of evidence support this idea: (i) in HeLa cells expressing GFP-centrin,
PARP-3 immunostaining coincides with the smaller and less bright labelling
already identified as the daughter centriole
(White et al., 2000); and (ii)
in mouse 3T3 cells, PARP-3 is clearly not associated with the primary cilium
that identifies specifically the mature (mother) centriole during G1 phase
(Chang and Stearns, 2000
;
Lange and Gull, 1995
).
Therefore, hPARP-3 appears to be the first known marker of the daughter
centriole. What function does it exert here?
The mammalian centrosome is composed of two barrel-shaped centrioles, each
formed by nine triplets of short microtubules, surrounded by a fibrous
pericentriolar material. It is a vital organelle in animal cells as it directs
the nucleation and organization of microtubules
(Lange and Gull, 1996;
Tassin and Bornens, 1999
;
Urbani and Stearns, 1999
). As
a consequence, the centrosome is essential during interphase for intracellular
organelle transport, cell migration and the establishment of cell shape and
polarity. The interphase centrosome then duplicates only once per cell cycle,
thereby ensuring a strictly bipolar mitotic spindle axis
(Mazia, 1984
). Therefore, it
plays a crucial role during mitosis in the equal and correct segregation of
chromosomes as well as in the exit of cytokinesis
(Piel et al., 2000
). Indeed,
many human tumor cells, including those lacking the tumor supressor p53
(Fukasawa et al., 1996
), have
abnormally high number of centrosomes
(Pihan et al., 1998
;
Winey, 1996
), and it has long
been proposed that such aberrations may cause aneuploidy and contribute to
cancer development (Brinkley,
2001
; Doxsey,
1998
). More recently, Nigg and collaborators
(Meraldi et al., 2002
;
Meraldi and Nigg, 2001
) have
put forward a different mechanism where centrosome anomalies arise through
failures in cell division, which lead to tetraploidization and subsequent
propagation in tumor tissues, especially when the p53 pathway is abrogated or
deregulated. Whatever the molecular scenario for the origin of centrosome
aberrations may be, a strong correlation between centrosome amplification and
aneuploidy exists and probably contributes to the selection of rare survival
daughter cells that have acquired a mutator phenotype
(Salisbury, 2001
;
Salisbury et al., 1999
).
Overexpression of hPARP-3 or its N-terminal domain in HeLa cells interfered
with the G1/S cell cycle transition, perhaps by titrating out a key regulator
normally required at this critical stage when the decision to divide is taken.
The lack of effect on HU-induced centrosome overduplication in CHO cells
suggests that hPARP-3 does not interfere directly with centrosome duplication.
Our results rather point to a possible role for hPARP-3 in cell cycle
progression at the G1/S transition. What is the connection between the
influence of hPARP-3 at the G1/S transition and its preferential localization
to the daughter centriole? The respective role of the two centrioles, mother
and daughter, has been recently documented
(Piel et al., 2000); they
separate and function as independent structures at two stages of the cell
division: (i) after the formation of the cleavage furrow, the mother centriole
nucleates a microtubule aster, whereas the daughter centriole exhibits a
considerable mobility, which progressively slows down from the onset of
centrosome duplication at the G1/S border, up to late G2. (ii) At the end of
telophase, just before abscission, the mother centriole moves to the
intercellular bridge. Its repositioning back to the cell centre seems to
provoke the completion of the cell division characterized by the narrowing of
the intracellular bridge and abscission. On the basis of these observations,
which clearly indicate that each centriole plays a specific role, it is
tempting to speculate that hPARP-3, associated to the daughter centriole, may
control its maturation until the G1/S restriction point is past. The
regulating function of centrosome in the G1 to S transition has been recently
studied using microsurgery and laser ablation. Indeed, when centrosomes were
removed from somatic vertebrate cells, a proportion of cells completed cell
division but failed to undergo the next round of DNA synthesis, suggesting a
critical role of centrosomes in cell cycle progression
(Hinchcliffe et al., 2001
;
Khodjakov and Rieder, 2001
;
Piel et al., 2000
).
Alternatively, the presence of both hPARP-1 and hPARP-3 at the centrosome may be a part of a detection/signalling pathway aimed at monitoring the eventual presence, in the midbody, of broken DNA that originate from tension forces between two daughter cells experiencing unbalanced chromosome segregation. Indeed, both DNA and poly(ADP-ribose) can be easily detected in some arrested daughter cells as a long thin filament in the midbody (C.S. and G.dM., unpublished). Thus, DNA-binding enyzmes PARP-1 and PARP-3 might contribute to an ultimate cell division checkpoint linking the mitotic fidelity to the DNA damage surveillance network.
In conclusion, hPARP-3 and hPARP-1 add to the growing number of proteins
that have been recently found, transiently or constitutively, associated with
the centrosome. Conversely, the centrosomal protein centrin-2 has been
recently identified as a constituent of the XPC (xeroderma pigmentosum)
complex, a key component of global genome nucleotide excision repair acting as
the initial damage detector (Araki et al.,
2001), which points to an even more general link between DNA
damage and repair and cell division (Su
and Vidwans, 2000
).
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Acknowledgments |
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References |
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