Haartman Institute and Molecular and Cancer Biology Program, Biomedicum Helsinki, University of Helsinki and Helsinki University Central Hospital, PO BOX 63, FIN-00014 Helsinki, Finland
* Author for correspondence (e-mail: marikki.laiho{at}helsinki.fi)
Accepted 16 June 2003
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Summary |
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Key words: Mdm2, p53, Nucleolus, PML, PML nuclear bodies, DNA damage, APL
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Introduction |
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Promyelocytic leukemia protein (PML) is a potential tumor suppressor
protein, and its overexpression induces either growth arrest or apoptosis
(Pearson and Pelicci, 2001).
Seven PML isoforms have been identified (PML I-VII according to the
nomenclature by Jensen et al.) (Jensen et
al., 2001
), and they differ only in their C-terminal sequence. PML
IV interacts with p53 and recruits it to the PML nuclear bodies (PML NB),
increasing p53 transcriptional activity upon
-radiation and by
oncogenic Ras (Pearson et al.,
2000
; Guo et al.,
2000
; Fogal et al.,
2000
). PML itself is essential for the formation of PML NBs and
the association of several regulatory and transcriptional factors, such as
SUMO-1, Sp100, Daxx, CBP, p53 and DNA damage repair proteins, such as Mre11,
to these bodies (Zhong et al.,
2000
; Lombard and Guarente,
2000
; Müller et al.,
2001
). The composition of NBs is cell-cycle regulated and also
dependent on cellular stress (Everett et
al., 1999
; Negorev and Maul,
2001
). Through its interactions, PML has been ascribed several
functions, including regulation of transcription, protein degradation,
cellular senescence and DNA repair
(Pearson et al., 2000
;
Ferbeyre et al., 2000
;
Pearson and Pelicci, 2001
;
Negorev and Maul, 2001
;
Bischof et al., 2002
;
Carbone et al., 2002
).
Sumoylation of PML is essential for the formation of the mature PML NBs
(Zhong et al., 2000
), and
interestingly, several other PML NB proteins are sumoylated as well,
suggesting that this modification directs their localization to the PML NBs or
that sumoylation takes place in the bodies. PML appears to affect the activity
of several proteins either by increasing (p53) or repressing (Daxx, Rb) their
activities (Zhong et al.,
2000
; Müller et al.,
2001
) possibly in the PML NBs. In acute promyelocytic leukemia
(APL) the function and localization of PML is altered because of the formation
of PML-RAR
fusion protein (de
Thé et al., 1990
; Dyck
et al., 1994
; Mu et al.,
1994
; Melnick and Licht,
1999
). The fusion protein blocks the expression of genes that are
essential for the normal myeloid differentiation and several PML-associated
apoptotic pathways. Treatment of APL cells with As2O3
induces the sumoylation and relocalization of PML-RAR
and PML into NBs
and leads to their degradation by the proteasome pathway
(Quignon et al., 1998
;
Zhong et al., 2000
;
Lallemand-Breitenbach et al.,
2001
; Miller et al.,
2002
).
Here we explore the relationship between the two regulators of the p53
pathway, Mdm2 and PML. Several studies have indicated the function of PML in
regulation of the p53 pathway upon -radiation and by oncogenic Ras.
Here we demonstrate that UV-radiation increases PML solubility and its
interaction with Mdm2. We find that Mdm2 and PML interact in vitro and that in
cellular stress, induced either by proteasome inhibition or UV-radiation, Mdm2
and PML form complexes in vivo and relocalize in a damage-specific manner.
Moreover, we show that p53 interacts with PML rapidly after UV damage prior to
its stabilization, and that this interaction preceeds p53-Mdm2 complex
formation. In vitro binding analyses demonstrated that p53, Mdm2 and PML form
trimeric complexes. In the trimeric complexes, the Mdm2-mediated degradation
of p53 is predicted to be obstructed because of PML binding to Mdm2 RING
domain. The results suggest that cellular stress, induced by UV, causes novel
associations between Mdm2, PML and p53 and that PML participates in the
activation of p53 through regulation of Mdm2.
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Materials and Methods |
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Cells and transfections
SaOS-2 osteosarcoma cells,
p53-/-mdm2-/- mouse embryo fibroblasts
(Montes de Oca Luna et al.,
1995) and WS1 human skin fibroblasts were cultured in a humidified
atmosphere containing 5% CO2 at 37°C. Cells were transfected by
electroporation (Gene Pulser II, Bio-Rad) and were treated with UVC (254 nm,
Stratalinker 2400, Stratagene), MG132 (10 µM, Affiniti Research Products)
or As2O3 (1 µM, Sigma) as indicated.
Immunofluorescence
Cells were fixed with 3.5% paraformaldehyde followed by permeabilization
with 0.5% NP-40. Alternatively, cells were permeabilized before fixation with
0.5% NP-40 for 5 minutes; 3% BSA was used for blocking. Primary antibodies
used for detection were as follows. Mdm2, IF-2 (Oncogene Sciences), SMP-14
(Santa Cruz Biotechnology) and 2A10; PML, PG-M3 and H-238 (Santa Cruz
Biotechnology), polyclonal PML IV antibody (G. del Sal); p53, DO-1, PAb421,
PAb1801 and FL393 (Santa Cruz Biotechnology). Mixes of the indicated
monoclonal antibodies were used for the detection of Mdm2 and p53.
In coimmunostainings, swine anti-rabbit or rabbit anti-goat FITC (DAKO), or goat anti-mouse conjugated Alexa594 or goat anti-rabbit conjugated Alexa488 (Molecular Probes) were used as fluorochromes. Absence of crossreactivity of the antibodies and conjugates was verified in separate experiments. The fluorochromes were visualized with Axioplan 2 Imaging MOT (Zeiss, Jena, Germany) equipped with appropriate filters (Chroma), and images were captured with Zeiss Axiocam CCD-video camera, followed by image processing and multilayer analysis with AxioVision program version 3.0. Confocal images were made with Bio-Rad MRC1024.
Immunoprecipitation and immunoblotting
Cellular lysates were prepared into EBC lysis buffer containing 25 mM
Tris-HCl pH 8.0, 120 mM NaCl, 0.5% NP-40, 4 mM NaF, 100 µM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 100 KIU/ml
aprotinin and 10 µg/ml leupeptin. Where indicated, an insoluble fraction
composed of EBC lysis buffer insoluble cellular pellet was collected. Protein
concentrations were determined by Bio-Rad Dc protein assay kit
(Bio-Rad), and after normalization of the protein concentrations, lysates were
immunoprecipitated with specific antibodies and collected on GammaBind-G
Sepharose (Pharmacia Biotech). Mixes of the indicated monoclonal antibodies
were used for the detection of Mdm2 and p53. Cellular lysates or
immunocomplexes were boiled in Laemmli sample buffer containing dithiothreitol
and were separated by 9% sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane
(Trans-Blot, Transfer Medium, Bio-Rad). Immunoblotting was performed by using
antibodies listed above followed by secondary antibodies conjugated with
horseradish peroxidase (HRP) and detection with enhanced chemiluminescence
(ECL) (Amersham Life Sciences). Total cell lysates were extracted in Laemmli
sample buffer, sonicated before boiling and analysed as above.
In vitro translation
In vitro translations of Mdm2, p53 and PML were performed with TNT Coupled
Reticulocyte Lysate System (Promega) from expression vectors containing T7
promoter. Translation products were precipitated with either Mdm2, p53 or PML
antibodies as above, in order to verify the direct interaction between these
two proteins.
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Results |
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In proteasome inhibitor-treated cells, however, PML has been found in the
nucleoli (Mattson et al., 2001). Given that we and others have recently shown
that Mdm2 is relocalized to the nucleoli in response to proteasome inhibitor
treatment (Klibanov et al.,
2001; Latonen et al.,
2003
), we wanted to address a possible association between Mdm2
and PML. To address the effect of downregulation of the proteasome on
localizations of PML and Mdm2, we treated SaOS-2 cells with the proteasomal
inhibitor MG132. Downregulation of the proteasome led to an increase in the
number of PML nuclear bodies and its translocation into the nucleoli that took
place within 6 hours (Fig. 1A)
(Everett et al., 1998
; Mattson
et al., 2001). Colocalization of PML and Mdm2 was detected both in the bodies
and in the nucleoli (Fig. 1A).
This was not exclusive however, as both were also present in separate
structures, Mdm2 in the nucleoplasm and PML in bodies devoid of Mdm2.
Arsenic trioxide recruits PML to NBs
(Müller et al., 1998;
Lallemand-Breitenbach et al.,
2001
). To test whether PML, located exclusively in the NBs after
arsenic treatment, affects Mdm2 localization, we treated SaOS-2 cells with
As2O3 for 16 hours. In
As2O3-treated cells Mdm2 was found relocalized to large
detergent-insoluble PML-bodies (Fig.
1A,C), and it also retained nucleoplasmic staining
(Fig. 1A). However, the
redistribution of Mdm2 following arsenic trioxide treatment was kinetically
slower than the MG132-induced changes but was clearly evident after 16 hours.
The results suggest that Mdm2 and PML can co-localize in response to cellular
stress and DNA damage in a spatially and temporally distinct manner.
To address whether PML can affect Mdm2 localization, we ectopically
expressed Mdm2 and PML III or PML IV in p53 and mdm2 null
fibroblasts (Montes de Oca Luna et al.,
1995). PML III and IV were expressed at high levels in the
transfected cells, and concentrated to large nuclear bodies
(Fig. 2). These structures did
not counterstain for DNA or RNA (SYTO Green) and they resembled larger
aggregates of natural PML bodies. Depending on the levels of the expressed
proteins, Mdm2 was found to co-localize with both PML forms, either
exclusively or to a large part, losing its even nucleoplasmic staining present
in untreated cells and concentrated to the PML bodies
(Fig. 2). The Mdm2 nucleolar
localization is determined by its nuclear localization signal (NoLS)
(Lohrum et al., 2000
). To
further address the capacity of PML to cause translocation of Mdm2, we tested
whether PML can affect the localization of a mutant Mdm2 lacking its NoLS
(
NoLS).
NoLS Mdm2 was transfected into
p53-/-mdm2-/- fibroblasts and the
cells were treated with MG132. Analysis of
NoLS Mdm2 transfected cells
(over 200 cells from at least three separate experiments) indicated that
NoLS Mdm2 and PML had an overlapping staining pattern and that
NoLS Mdm2 was detected only in nucleoli that contained nucleolar PML
(data not shown). This suggests that PML or other PML body proteins direct
Mdm2 independent of the Mdm2 NoLS. The results indicate that PML has the
capacity to sequester Mdm2 to the PML NBs as well as redirect Mdm2 into the
nucleoli.
|
Mdm2 and PML interact in vitro
Direct interaction of Mdm2 and PML was verified by in vitro translation and
co-immunoprecipitation analyses. The results showed that in vitro translated
full-length Mdm2 interacted with PML isoforms III and IV and that the
interaction was independent of PML sumoylation status
(Fig. 3A). Similar results were
obtained by coimmunoprecipitation analyses using an PML antibody (not shown).
However, Mdm2 interaction with PML III was weaker than with PML IV. There was
negligible interaction between Mdm2 and PML-RAR fusion protein,
suggesting that an intact C-terminus of PML, absent in the fusion protein, is
required for the interaction (Fig.
3A).
|
Similar analysis was performed by translating different Mdm2 deletion
constructs in vitro followed by coimmunoprecipitations with in vitro
translated PML IV (Fig. 3B).
Mdm2 C-terminal deletion mutants (6-339 and 1-440) had significantly weaker
interactions with PML IV than wild-type Mdm2 or Mdm2 deletion mutants
58-89,
89-222 and
222-437
(Fig. 3B). This suggests that
the Mdm2 C-terminus, including the RING finger domain responsible for p53
degradation, participates in the interaction. As only a fraction of PML
appears to interact with Mdm2, it is possible that additional modifications of
the proteins not present in the in vitro translation products increase the
efficiency of the interaction.
Mdm2 and PML interact in vivo in response to cellular stress
We further analysed the responses and interactions of endogenous Mdm2 and
PML in SaOS-2 cells following treatments with either MG132 or UV-radiation.
One major 92 kDa PML form was detected in the soluble and insoluble cellular
fractions. Based on its molecular weight, this form could represent either PML
III or IV. The PML levels increased in lysates of both UVC- and MG132-treated
cells, indicating its increase in the soluble nucleoplasmic fraction
(Fig. 4A). In addition, the
NP-40 insoluble fraction of PML decreased in the UV-treated cells
(Fig. 4A), whereas the total
levels of PML were unchanged at 6 hours (data not shown, see also
Fig. 5), suggesting that the
increase in the soluble fraction was because of the release of PML from the
PML bodies. Furthermore, PML derived from either MG132 or UV-treated SaOS-2
cells coimmunoprecipitated with endogenous Mdm2, demonstrating their
interaction and suggesting that both treatments increase the availability of
the interacting proteins (Fig.
4B). Although the interaction was detected in
As2O3-treated cells as well, it did not increase as
compared to the controls (not shown). The results suggest that Mdm2 and PML
interact at least in the nucleoplasmic fraction. Finally, as SaOS-2 cells are
null for p53, the Mdm2-PML interaction was clearly independent of PML-p53
interaction.
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|
Mdm2, PML and p53 form temporal complexes in response to DNA
damage
To test the timing and relationship of the complexes between Mdm2, PML and
p53, we treated human fibroblasts expressing wild-type p53 with UVC and
incubated the cells for different periods of time as indicated in
Fig. 5. Cellular lysates were
precipitated either with an p53 antibody mix or antibody against PML followed
by immunoblotting. As amply demonstrated previously
(Latonen et al., 2001), p53
levels increased at 3 hours after the damage during which its interaction with
Mdm2 was low to negligible. p53-Mdm2 interaction increased at later timepoints
coinciding with an increase in Mdm2 levels by p53
(Fig. 5A). Following UV
radiation, p53 formed a complex with PML, but only at a kinetically narrow
window between 1-3 hours after the damage
(Fig. 5A and data not shown).
Mdm2 complex formation with PML was also transient, peaking at 3 hours
(Fig. 5A). Thus, p53-PML and
Mdm2-PML complexes were present in UV-radiated cells early after the damage
and these interactions preceeded p53 stabilization and Mdm2-p53 complex
formation. Although these interactions took place in the nucleoplasmic
fraction, it is possible that the interactions may take place in the PML
bodies as well.
To verify the possible translocation of Mdm2, p53 and PML to the NP-40
insoluble fraction of the cells (including NBs, nucleoli, DNA bound fraction)
we also performed immunoblotting analyses of the insoluble fractions. Shortly
after the UV-radiation (1 hour) p53 was found in the NP-40 insoluble pellets
in a slower migrating 65 kd form (Fig.
5B). As this corresponds to the size of sumoylated p53
(Rodriguez et al., 1999) (data
not shown), it is possible this form represents p53 undergoing
SUMO-modification. In response to UV, PML levels in the insoluble fraction
initially decreased, whereas a slower migrating PML form appeared at later
timepoints (Fig. 5B). There
were no major changes in the insoluble fraction of Mdm2.
To correlate the changes of the respective proteins in the insoluble and soluble fractions of the cells, we also analysed total cellular lysates. This indicated, as expected from previous analyses, that total levels of p53 and Mdm2 increased at later timepoints starting from 3 and 12 hours after the UV-damage, respectively (Fig. 5C). Also, total levels of PML showed an increase 12 hours after the damage (Fig. 5C).
To address whether kinetics of p53-PML interaction correlate rapid p53 localizations to the PML bodies, we performed immunostaining of UVC-treated WS1 cells. p53 shows gradual nucleoplasmic accumulation without specific localization to the PML NBs at any given timepoint (Fig. 6). PML present in the NBs, however, was rapidly dissociated from the NBs and was increasingly found in the nucleoplasm and in the perinucleolar regions (Fig. 6).
PML forms trimeric complexes with Mdm2 and p53 in vitro
Considering the in vitro results on the interaction domains of Mdm2, PML
and p53 and the in vivo results on their temporal complex formation, we tested
whether the proteins form mutually exclusive complexes. p53, Mdm2 and PML IV
were translated in vitro and mixed with increasing amounts of either Mdm2 or
PML IV and were tested for their capacity for complex formation
(Fig. 7). Addition of
increasing amounts of Mdm2 to the in vitro reactions led to an increase in
p53-bound PML and vice versa, presence of increasing amounts of PML led to
increased complex formation between p53 and Mdm2
(Fig. 7). This suggests that
the interactions of PML with Mdm2 and p53 are not mutually exclusive, whereas
PML-Mdm2 interaction promotes p53 binding. This further suggests that PML has
separate binding sites for Mdm2 and p53.
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Discussion |
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Here we demonstrate that cellular UV-damage leads to rearrangements of the
PML NBs. UVC-insult led to an apparent increase in the soluble PML and small
PML aggregates in the nucleoplasmic fraction, suggesting that PML NBs undergo
rearrangements and more PML is found in a nucleoplasmic diffusible form. This
finding is corroborated by a recent study by Seker et al.
(Seker et al., 2003). A
physiological release of PML from the bodies occurs during mitosis
(Ascoli and Maul, 1991
;
Everett et al., 1999
). Based
on the observation that the transcriptional inhibitor actinomycin D causes
scattering of the PML body components throughout the nucleoplasm
(Kießlich et al., 2002
),
it has been suggested that transcriptional activity is required for the
integrity of PML NBs. Even though the sumoylation of PML appears essential for
the formation of mature PML bodies and its release from these sites is
believed to involve desumoylation, other mechanisms resulting in free PML may
also exist. In fact, Cd2+ exposure releases PML from the bodies to
the nucleoplasm utilizing p38 MAPK and ERK1/2 signalling pathways
(Nefkens et al., 2003
). These
pathways are also activated by cellular stress, including UV radiation.
However, the mechanism of the UV-induced release still needs to be examined in
more detail. The UV-damage-induced dispersion of PML is a novel finding,
demonstrating its regulation by other types of stress besides viral infections
(Zhong et al., 2000
), oncogene
activation (Pearson et al.,
2000
) or gamma-radiation (Guo
et al., 2000
).
In UV-treated cells, PML and Mdm2 also relocalized to perinucleolar areas.
These dotted structures around the nucleoli resemble the nucleolar necklaces
(Granick, 1975), which are
formed in cells treated with a transcription inhibitor, DRB, and are involved
in the transcription of rRNA (Panse et
al., 1999
). The units are composed of a small fibrillar centre
surrounded by dense fibrillar component in a reorganized nucleolus, and are
believed to correspond to active single gene (rRNA) transcription units
(Panse et al., 1999
).
Nucleolar necklace also contains various transcription factors, including RNA
pol I and TFIIH (Hoogstraten et al.,
2002
). DRB does not inhibit rRNA transcription by RNA pol I,
although it causes alterations in rRNA processing and RNA pol II functions
(Granick, 1975
). PML also
associates with RNA pol II at sites of active transcription
(Kießlich et al., 2002
),
although the bodies themselves do not contain RNA or DNA
(Grande et al., 1996
). DNA
helicase II (NDH II) is a component of the PML bodies in a manner dependent on
active transcription. Inhibition of RNA pol II leads to its relocalization to
the perinucleolar area (Fuchsová et
al., 2002
). The cause or purpose of the relocalization of Mdm2 and
PML to these structures is presently not clear, although inhibition of RNA pol
II by UV-radiation is suggestive that this could be an initiating factor for
the translocation (Ljungman et al.,
1999
). Our findings imply a role for these proteins in the rRNA
transcription unit of the nucleolus, either activatory or inhibitory.
UV-damage promotes the interaction of PML and Mdm2 despite the fact that
levels of Mdm2 are slightly decreased early on after the damage
(Latonen et al., 2001).
Although the stress response caused by downregulation of the proteasome leads
to PML-Mdm2 interaction, the distinct localizations of the proteins in
response to UV-treatment as compared with proteasome dysfunction (nucleoplasm
and perinucleolar dots versus nucleoli) distinguish the nature of their
response. Possible bridging factors between these two proteins were excluded
by in vitro translation assays in which the interaction was shown to be
direct, although we cannot absolutely exclude the presence of a connecting
factor in the reticulosyte lysate. In vitro analyses showed that Mdm2 binds
both PML III and IV, although the in vitro interaction with PML III was much
weaker. Mdm2 is subject to DNA damage-provoked phosphorylation and
dephosphorylation (Khosravi et al.,
1999
; Blattner et al.,
2002
). Further studies should demonstrate whether the
modifications (phosphorylation, sumoylation) of Mdm2 or alternatively, PML,
affect their interactions and whether there is a preference for a certain PML
isoform to bind Mdm2.
The Mdm2 binding site in PML was similar to the p53 interaction domain, as
shown by their absence of binding with the PML-RAR fusion protein.
These binding domains could be indicative of competitive interactions.
However, although Mdm2 bound both PML III and IV, p53 interacts only with PML
IV (Fogal et al., 2000
).
Moreover, the in vitro interaction analyses showed that increased levels of
Mdm2 promoted PML-p53 interaction and vice versa, an increase in PML enhanced
p53-Mdm2 complex formation. The results strongly favor the formation of a
trimeric PML-p53-Mdm2 complex in which PML can accommodate the binding of both
p53 and Mdm2. Mdm2, through its separate binding sites for p53 and PML, can
increase the number of p53 molecules bound to PML. Furthermore, because Mdm2
interacts with PML through its RING-domain, it is unlikely that the trimeric
complex would promote p53 degradation.
p53, in the nucleoplasmic fractions, interacted transiently with PML in the
UV-treated cells. At the same time, insoluble p53 was found in a
slower-migrating form, corresponding to its SUMO modification
(Rodriguez et al., 1999).
Similarly, PML present in the insoluble fraction migrated as a higher
molecular weight form in UV-damaged cells whereas the PML form present in
control cells decreased, suggesting that both proteins undergo modifications
following UV-damage (Fig. 5C).
The sumoylation of p53 affects its transcriptional activity in UV-treated
cells (Rodriguez et al., 1999
;
Gostissa et al., 1999
). The
localization of p53 to NBs is not dependent on this modification
(Fogal et al., 2000
;
Kwek et al., 2001
), but the
SUMO-1-conjugating enzyme, Ubc9, exists in NBs
(Duprez et al., 1999
). It is
thus possible that sumoylation takes place in PML NBs and is a prerequisite
for the activation of p53. In addition, p53 is modified in a PML-dependent
manner through acetylation by CBP
(Ferbeyre et al., 2000
;
Pearson et al., 2000
), and
conversely, can be deacetylated through SIRT1 deacetylase, which localizes to
PML NBs (Langley et al.,
2002
). Following UV-damage, p53 is phosphorylated on Ser46 by
HIPK2 (D'Orazi et al., 2002
).
Both events are presumed to increase p53 stability and enhance its
transactivation function and may be mediated through events taking place in
the PML NBs. Alternatively, acetylation of p53 prevents its degradation by
Mdm2 because of overlapping lysines targeted by acetylation and ubiquitination
(Li et al., 2002b
). Mdm2 can
promote p53 deacetylation by recruiting histone deacetylase-1 (HDAC1), and the
deacetylation is suggested to lead to enhanced p53 degradation
(Ito et al., 2002
). Finally,
herpesvirus-associated ubiquitin-specific protease (HAUSP) stabilizes p53 by
deubiquitinating it both in vitro and in vivo
(Li et al., 2002a
), and
interestingly, this protein also exists in PML bodies, suggesting that p53
stabilization could take place in NBs. The PML and PML NBs thus appear to be
modifiers of several processes affecting p53 and its activity. Yet, evidence
by Bishof et al. indicates that several p53 modifications (Ser46
phosphorylation, Lys382 acetylation) induced by PML IV-mediated senescence do
not require the integrity of PML bodies (Bishof et al., 2002). We find no
evidence that p53, following UV-damage, is found in the PML NBs
(Fig. 6). However, the clearly
evident p53-PML interactions in the nucleoplasmic fractions, and their changes
in the insoluble fractions suggest that they undergo rapid dynamic
interactions and possibly, modifications. We therefore propose that the
capacity of PML to directly complex with Mdm2, and the formation of trimeric
PML-Mdm2-p53 complexes, allow for p53 stabilization following UV-damage. In
fact, PML IV overexpression has been shown to induce both p53 stabilization
and acetylation (Bischof et al.,
2002
). The mechanism for p53 stabilization has, however, remained
unknown. Our results show that one stabilizing factor, in addition to
acetylation of p53, may be interference of Mdm2 E3 ligase activity by PML. We
suggest that PML inhibits Mdm2-mediated degradation of p53 by binding to the
Mdm2 C-terminus, hindering its ubiquitin ligase activity in DNA-damaged cells.
In addition, both PML and Mdm2 are relocalized to the perinucleolar
structures. The presented results unravel complex interactions and regulation
of p53, Mdm2 and PML following diverse types of cellular stress and DNA
damage.
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Acknowledgments |
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References |
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