Physical and Functional Interactions between PML and MDM2*
Xiaolong Wei
,
Zhong Kang Yu
,
Arivudainambi Ramalingam
,
Steven R. Grossman
¶ 
,
Jiang H. Yu ||,
Donald B. Bloch || and
Carl G. Maki
**
From the
Department of Radiation Oncology, The
University of Chicago, Chicago, Illinois 60637, the
Departments of Adult Oncology and Cancer
Biology, Dana Farber Cancer Institute and the
¶Department of Medicine, Brigham and Women's
Hospital, Boston, Massachusetts 02115, and the
||Center for Immunology and Inflammatory Diseases,
Department of Medicine, Harvard Medical School and Massachusetts General
Hospital, Boston, Massachusetts 02114
Received for publication, December 2, 2002
, and in revised form, May 14, 2003.
 |
ABSTRACT
|
---|
The tumor suppressor protein PML and oncoprotein MDM2 have opposing effects
on p53. PML stimulates p53 activity by recruiting it to nuclear foci termed
PML nuclear bodies. In contrast, MDM2 inhibits p53 by promoting its
degradation. To date, neither a physical nor functional relationship between
PML and MDM2 has been described. In this study, we report an in vivo
and in vitro interaction between PML and MDM2 which is independent of
p53. Two separate regions of PML are recognized which can interact with MDM2.
The C-terminal half of PML, encoded by residues 300633, can interact
with the central region of MDM2 which includes the MDM2 acidic domain. In
addition, PML amino acids 1200, which encode the RING-finger and most
of the B box zinc binding motifs, can interact with the C-terminal,
RING-finger containing region of MDM2. Interestingly, PML mutants in which
sumoylation at lysine 160 was inhibited displayed an increased association
with MDM2, suggesting that sumoylation at this site may be a determinant of
PML-MDM2 binding. Coexpression with MDM2 caused a redistribution of PML from
the nucleus to the cytoplasm, and this required the PML N terminus and the
MDM2 RING-finger domain. These results suggest that interaction between the
PML N terminus and MDM2 C terminus can promote PML nuclear exclusion.
Wild-type MDM2 inhibited the ability of PML to stimulate the transcriptional
activity of a GAL4-CBP fusion protein. This inhibition required the central,
acidic region of MDM2, but did not require the MDM2 C terminus. Taken
together, these studies demonstrate that MDM2 and PML can interact through at
least two separate protein regions, and that these interactions can have
specific effects on the activity and/or localization of PML.
 |
INTRODUCTION
|
---|
Wild-type PML is a tumor suppressor and ubiquitously expressed nuclear
phosphoprotein. The PML gene was originally identified as a result of
a reciprocal translocation t(15:17) associated with acute promyelocytic
leukemia
(14).
The t(15:17) translocation disrupts the PML gene on chromosome 15 and
the retinoic acid receptor
(RAR)1
(RAR
) gene on chromosome 17 and is reciprocal in nature,
resulting in the generation of novel fusion proteins PML-RAR
and
RAR
-PML (5). The most
striking feature of wild-type PML is its localization to distinct nuclear foci
that have been termed PML nuclear bodies (PML-NBs), Kremer bodies, ND10 or
PODs (for PML oncogenic domains). These PML-NBs are multiprotein complexes
that are 0.10.2 µm in diameter, and cells typically contain
1030 PML-NBs/nucleus, although their number and size can vary during
the cell cycle (6,
7). In acute promyelocytic
leukemia cells, expression of PML-RAR
causes disruption of PML-NBs and
a redistribution of PML to a microspeckled nuclear localization pattern
(810).
PML-RAR
can form heterodimers with wild-type PML
(8,
10) as well as retinoid X
receptor, another retinoic acid receptor family member
(11). Current models suggest
that sequestration of normal PML by PML-RAR
inhibits the growth
suppressive activity of PML, whereas sequestration of retinoid X receptor
prevents the induction of differentiation
(5). Inhibition of both of
these pathways may be necessary for leukemogenesis.
The PML protein contains well characterized zinc binding domains in its
N-terminal half, including a RING-finger adjacent to two
cysteine/histidine-rich motifs known as B boxes. These domains, together with
an
-helical coiled-coil domain, comprise a conserved motif known as
RBCC (12). PML is covalently
modified by SUMO-1, a small ubiquitin-like polypeptide also known as
sentrin-1, UBL-1, or PIC-1
(13). Like ubiquitin, SUMO-1
is linked covalently to lysine residues on PML and other target proteins in an
ATP-dependent manner (14).
However, SUMO-1 modification seems to modulate the localization of its target
proteins rather than induce their degradation. Three major sites for SUMO-1
modification were identified in PML: Lys-65 in the RING-finger, Lys-160 in the
first B box, and Lys-490 in the nuclear localization signal
(15,
16). Sumoylation of PML is
essential for formation of PML-NBs and for its ability to recruit
NB-associated proteins (17,
18). Sumoylation was also
thought to be required for PML-dependent growth suppression. However, this
latter hypothesis was challenged by a recent study in which a mutant PML
deficient in sumoylation maintained its ability to inhibit growth when
overexpressed (19).
The mechanisms by which PML functions as a tumor suppressor and inhibits
growth have not been fully clarified. Mice with a targeted disruption of the
PML locus (PML/) display an increased incidence of carcinomas
after treatment with tumor-promoting agents
(20). Further, cells derived
from PML/ mice are resistant to apoptosis in response to various
apoptotic stimuli (21). These
results suggest that PML plays a central role in apoptosis signaling. A
relationship between PML and p53 was suggested by the finding that p53 could
interact directly with PML and was recruited by PML into PML-NBs
(22). Subsequent studies have
shown that PML corecruits both p53 and CBP/p300 to PML-NBs. CBP/p300 then
promotes the acetylation of p53, which increases p53 DNA binding affinity and
thus leads to an activation of p53-responsive genes
(23,
24). Perhaps the most
compelling evidence that links the PML and p53 growth-suppressive pathways
comes from studies of oncogene-induced senescence. In these studies, p53
activity was measured in PML+/+ and PML/ cells infected with
retroviruses expressing an activated ras oncogene (Ras V12). Ras V12
expression in PML+/+ cells resulted in the activation of p53, recruitment of
p53 into PML-NBs, and the induction of premature senescence. In contrast, p53
was neither activated nor recruited into PML-NBs in PML/ cells,
and the cells were resistant to ras-induced senescence
(23,
24). These studies provide
strong evidence that PML is required for the efficient activation of p53 in
response to aberrant oncogene signaling.
p53 levels and activity are controlled in large part by MDM2, the product
of a p53-inducible gene. MDM2 binds to the N terminus of p53 and inhibits the
activity of p53 as a transcription factor
(25,
26). Importantly, MDM2 binding
also promotes the ubiquitination of p53 and its subsequent degradation by the
proteasome, as well as the export of p53 from the nucleus to the cytoplasm
(2730).
Insofar as PML and MDM2 have opposing effects on p53 activity, it is of
interest to investigate the functional relationship between PML and MDM2.
However, to date neither a physical nor functional interaction between PML and
MDM2 has been described. The current report demonstrates an in vivo
and in vitro interaction between PML and MDM2 which can occur
independent of p53. Mapping studies indicate that PML and MDM2 can interact
through at least two separate protein regions and that these interactions can
affect both the localization and activity of PML.
 |
EXPERIMENTAL PROCEDURES
|
---|
Plasmid DNAsFLAG-tagged PML expression DNA was obtained
from Zhi-Min Yuan (Harvard School of Public Health). HA-tagged PML was
generated from this clone by PCR. The 3'-primer for PCR was the SP6
primer (Promega), and the 5'-primer was
5'-GCGAATTCACCATGTACCCATACGATGTTCCAGATTACGCTGAGCCTGCACCCGCCCG-3'.
The resulting PCR product was digested with EcoRI and XbaI
restriction enzymes and cloned into the corresponding sites of pCDNA-3.1. All
subsequent PML clones were generated by PCR using HA-PML wild-type as the
template. N-terminal deletions of PML were generated using the SP6 primer as
the 3'-primer, and the following 5'-primers: for HA-PML
100N,
5'-GCGAATTCACCATGTACCCATACGATGTTCCAGATTACGCTACACCCGCCCTGGATAACG-3';
for HA-PML
200N,
5'-GCGAATTCACCATGTACCCATACGATGTTCCAGATTACGCTAGCATCT ACTGCCGAGG-3';
for HA-PML
300N,
5'-GCGAATTCACCATGTACCCATACGATGTTCCAGATTACGCTGTGGACGCGCGGTACCAGC-3';
for HA-PML
400N,
5'-GCGAATTCACCATGTACCCATACGATGTTCCAGATTACGCTAAAGCCAGCCCAGAGGC-3';
PCR products were digested with EcoRI and XbaI restriction
enzymes and cloned into the corresponding sites of pCDNA-3.1. Sumoylation site
mutants of PML were generated using the QuikChange mutagenesis kit
(Stratagene) and HA-PML wild-type as a template. For HA-PML (K65R), the
following primer and its complement were used:
5'-GCGGAAGCCAGGTGCCCGAAGCTTCTGCCTTGTCTGC-3'. For HA-PML (K160R),
the following primer and its complement were used in the PCR:
5'-CAGTGGTTCCTCAGGCTCGAGGCCCGGC-3'. For HA-PML (K490R), the
following primer and its complement were used:
5'-GACCCAGTGCCCGCGGAAGGTCATCAGGATGGAGTCTGAGG-3'. C-terminal
deletions of HA-PML (K160R) were generated using
5'-GCGAATTCACCATGTACCCATACGATGTTCCAGATTACGCTGAGCCTGCACCCGCC CG-3'
as the 5'-primer and the following 3'-primers: for HA-PML K160R
(1500), 5'-CCGTCTAGATCACCTTGCCTCCTTCCCCTCC-3'; for HA-PML
K160R (1400), 5'-CCGTCTAGATCACTTGGATACAGCTGCATC-3'; for
HA-PML K160R (1300), 5'-CCGTCTAGATCAAGCCTCCAGCAGCTCGCGC-3';
for HA-PML K160R (1200),
5'-CCGTCTAGATCAGGTCAGCGTAGGGGTGCGG-3'. The resulting PCR products
were digested with EcoRI and XbaI and cloned into the
corresponding sites in pCDNA3.1. FLAG-tagged MDM2 DNAs were described
(31,
32) and were obtained from
Zhi-Min Yuan. FLAG-MDM2
NT lacks 120 amino acids from the N terminus.
DNAs encoding wild-type MDM2 and MDM2
217246 were obtained from
Steve Grossman (Dana Farber Cancer Institute). FLAG-tagged MDM2 (2202),
(100304) and (301488) were generated by PCR using wild-type MDM2
DNA as a template. For MDM2 (301488) the 5'-primer was
5'-CCGGGATCCCCAAAGAAGAAGAGGAAGGACTATTGGAAATGCACTTCATGC-3', and the
3'-primer was 5'-CCGTCTAGATCAAGTTAGCACAATCATTTGAATTGG-3'.
The resulting PCR products were digested with BamHI and XbaI
and cloned downstream and in-frame with the FLAG epitope in pCDNA3.1. MDM2
(301488) contains the SV40 large-T antigen NLS encoded within the
5'-primer. For MDM2 (2202) the 5'-primer was
5'-CCGGGATCCTGCAATACCAACATGTCTGTACC-3', and the 3'-primer
was 5'-CCGGAATTCTCATATTACACACAGAGCCAGGC-3'. For MDM2
(100304) the 5'-primer was
5'-CCGGGATCCTATACCATGATCTACAGGAACTTGG-3', and the 3'-primer
was 5'-CCGGAATTCTCATTTCCAATAGTCAGCTAAGG-3'. The resulting PCR
products were digested with BamHI and EcoRI and cloned
downstream and in-frame with the FLAG epitope in pCDNA3.1. MDM2 (6339)
was obtained from Arnold Levine.
Tissue Culture, Immunoblots, ImmunoprecipitationHuman
osteosarcoma cell lines Saos-2 cells (p53-null) and U2OS (p53 wild-type), and
35-2 cells (murine p53 and MDM2 double knockout) were grown in minimum
essential medium supplemented with 10% fetal bovine serum, 100 µg/ml
penicillin and streptomycin. Transfections in Saos-2 and U2OS cells were done
using the calcium phosphate method in 35-mm dishes when the cells were
80% confluent. 1620 h after addition of the DNA precipitate, cells
were washed twice with minimum essential medium minus serum and refed with
minimum essential medium plus 10% fetal bovine serum. Cell extracts were
prepared 810 h later. Transfections in 35-2 cells were done using
FuGENE-6 (Roche Applied Science), according to the manufacturer's
instructions. For immunoblot analysis and coimmunoprecipitations, cells were
rinsed with phosphate-buffered saline and scraped into 500 µl of lysis
buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM
NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2
µg/ml aprotinin, 5 µg/ml leupeptin). The scraped cells were lysed on ice
for 30 min with occasional light vortexing, followed by a 15-min
centrifugation to remove cellular debris. Protein extracts were then either
immunoprecipitated or resolved by SDS-PAGE, and transferred to a PolyScreen
polyvinylidene difluoride transfer membrane (PerkinElmer Life Sciences).
Antibodies used for immunoblotting were the anti-HA monoclonal antibody (HA.11
from Babco), an anti-MDM2 monoclonal antibody (SMP-14 from Santa Cruz), or an
anti-FLAG monoclonal antibody (M5 from Sigma). For immunoprecipitations, 300
µg of transfected cell extract was immunoprecipitated with 0.6 µg of the
anti-HA polyclonal antibody Y-11 (from Santa Cruz) or 0.2 µg of anti-MDM2
(SMP-14). To detect sumoylated PML species, cells were rinsed with
phosphate-buffered saline and scraped into 500 µl of
radioimmunoprecipitation assay buffer (20 mM Tris, pH 7.5, 2
mM EDTA, 150 mM NaCl, 0.25% SDS, 1% Nonidet P-40, 1%
deoxycholic acid), and then sonicated for 10 pulses (setting 5, 50% output)
using a Branson-450 sonifier. Lysates were centrifuged for 15 min prior to
analysis to remove cellular debris.
Immunofluorescence StainingFor immunofluorescence staining,
cells were plated on glass coverslips and transfected, washed, and refed as
described above. 24 h after transfection, cells were rinsed with
phosphate-buffered saline plus 0.1 mM CaCl2
and1mM MgCl2 and fixed with 4% paraformaldehyde for 30
min at 4 °C. Paraformaldehyde was then replaced with 50 mM
NH4Cl for 5 min, and cells were permeabilized with 0.1% Triton
X-100 plus 0.2% bovine serum albumin. PML staining was carried out using the
anti-HA monoclonal antibody HA.11 (Babco) as the primary antibody and
rhodamine red-conjugated anti-mouse antibody (Jackson Labs) as the secondary
antibody. MDM2 staining was carried out using the anti-MDM2 polyclonal
antibody N-20 (Santa Cruz) as the primary antibody, and either
7-amino-4-methylcoumarin-3-acetic acid-conjugated anti-rabbit antibody, or
fluorescein isothiocyanate-conjugated anti-rabbit antibody (Jackson Labs) as
the secondary antibody. Specimens were then examined under a fluorescent
microscope.
GST Fusion Protein ProductionGST-tagged PML wild-type DNA
was generated by PCR using HA-PML wild-type as a template. The 3'-primer
for PCR was 5'-GGCGCGGCCGCCTCACCAGGAGAACCCACTTTCATTG-3', and the
5'-primer was 5'-CCGGGATCCGAGCCTGCACCCGCCCGATCTCCG-3'. The
resulting PCR product was digested with BamHI and NotI
restriction enzymes and cloned into the corresponding sites of pGEX-4T-3.
GST-tagged PML K160R DNA was generated by PCR using the same primers but
HA-PML K160R as a template. DNAs encoding GST alone or GST-PML were used to
transform BL-21 bacterial cells, and transformed cells were grown at 37 °C
until reaching log phase. GST protein expression was induced by incubation in
0.2 mM isopropyl-1-thio-
-galactopyranoside for 3 h. To purify
the GST proteins, cells were lysed by sonication in lysis buffer (10
mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% Triton X-100, 150
mM NaCl), and the resulting lysate was incubated for 12 h at 4
°C with glutathione-Sepharose beads. The beads were pelleted by
centrifugation and washed with lysis buffer. For MDM2 binding, the GST-tagged
proteins bound to beads were incubated with MDM2 protein in 300 µl of lysis
buffer for 56 h. Unbound MDM2 protein was removed by five washes (1 ml
each) with lysis buffer. Bound proteins were eluted by boiling for 10 min in 1
x loading buffer, resolved by SDS-PAGE, and examined by immunoblot
analysis with the anti-MDM2 antibody SMP-14.
Expression and Isolation of MDM2 from Insect CellsDNA
encoding FLAG-tagged MDM2 that was either wild-type, lacked amino acids
217246 (MDM2
p300BD), or lacked the C-terminal RING domain
between residues 443 and 491 (MDM2
CT) was cloned into a baculovirus
expression system and used for expression in insect cells. MDM2 protein was
isolated by passing insect cell lysate over a column of Sepharose beads
conjugated to an anti-FLAG antibody. Bound proteins were washed several times
and eluted with a peptide encoding the FLAG epitope.
 |
RESULTS
|
---|
PML protein levels increase upon exposure of cells to interferons (IFNs)
through direct transcriptional activation of the PML gene. To test for an
in vivo interaction between PML and MDM2, H1299 cells (p53-null) were
exposed to IFN-
for 48 h to induce endogenous PML expression, and the
cells were then examined by immunofluorescence staining and confocal
microscopy with antibodies against MDM2 and PML. These studies revealed
colocalization between the endogenous PML and MDM2 proteins in
IFN-
treated H1299 cells (Fig.
1A). Because H1299 cells lack p53 expression, these
results suggest that MDM2 and PML can form an endogenous complex, either
directly or indirectly, in the absence of p53. We also attempted to
coimmunoprecipitate PML and MDM2 from IFN-
-treated H1299 cells (not
shown). However, we have been unable to immunoprecipitate large amounts of
endogenous PML using our normal cell lysis conditions because the PML protein
is insoluble under these conditions. We can solubilize PML under relatively
harsh lysis conditions (high detergent), but these conditions are denaturing
and therefore destroy any putative PML-MDM2 interactions. To examine the
PML-MDM2 interaction further, Saos-2 cells (p53-null) were transfected with
DNAs encoding MDM2 and epitope-tagged (HA-tagged) PML. Cell lysates were then
immunoprecipitated with an anti-HA antibody and examined by immunoblotting
with an antibody against MDM2. As shown in
Fig. 1B, MDM2
coimmunoprecipitated with HA-PML in Saos-2 cells, again demonstrating that
MDM2 and PML can interact in the absence of p53. To test the possibility that
MDM2 and PML may interact directly, wild-type PML protein fused to GST was
generated in bacteria, purified on glutathione-agarose beads, and mixed with
partially purified, recombinant MDM2 protein produced in insect cells.
Association between PML and MDM2 was then assessed in GST pull-down assays. As
shown in Fig. 1C,
wild-type (wt) MDM2 formed a complex with GST-PML wt, whereas no complex was
observed between MDM2 and the GST protein alone. These results indicate that
MDM2 and PML can interact with each other and suggest that this may be a
direct interaction. An MDM2 protein lacking the C-terminal amino acids
443491 (MDM2
CT) also formed a complex with GST-PML wt but not
with the GST protein control, indicating that this in vitro binding
between PML and MDM2 does not require the MDM2 C terminus.
We next wished to identify the regions of PML and MDM2 required for their
interaction. PML contains several well characterized protein domains,
including a RING-finger, two B boxes, and an
-helical coiled-coil
(Fig. 2A) (for review,
see Ref. 12). To map the
regions of PML required for MDM2 binding, N-terminal deletion mutants of
HA-PML were expressed with MDM2 in transiently transfected cells. Cell lysates
were then immunoprecipitated with an anti-HA antibody and examined by
immunoblotting with an antibody against MDM2. As shown in
Fig. 2B, MDM2
coimmunoprecipitated with HA-tagged wild-type PML, consistent with the results
of Fig. 1. Interestingly,
deletion mutants of PML lacking 100 or 200 amino acids from the N terminus
displayed a much stronger interaction with MDM2 compared with wild-type PML,
whereas a PML deletion mutant lacking 400 amino acids from the N terminus was
unable to bind MDM2. Similar binding results were obtained in the reciprocal
coimmunoprecipitation, which involved first immunoprecipitating with an
anti-MDM2 antibody, followed by immunoblotting with an anti-HA antibody
(Fig. 2C). In these
studies, wild-type PML again displayed relatively weak binding to MDM2,
whereas mutants lacking 100, 200, or 300 amino acids from the PML N terminus
displayed stronger association with MDM2, and a mutant lacking 400 amino acids
from the N terminus failed to bind MDM2. These results indicate that the
C-terminal region of PML between residues 300 and 633 can form a complex with
MDM2 and suggest that sequences within the N terminus of PML may be inhibitory
to this PML-MDM2 complex formation.

View larger version (44K):
[in this window]
[in a new window]
|
FIG. 2. Binding of PML deletion mutants to MDM2. A, schematic of
the PML protein showing the positions of the RING-finger domain (R),
the two B box zinc binding domains (B1, B2), the predicted
-helical coiled-coil, and the three sites of sumoylation. B,
U2OS cells were transfected with DNAs encoding wild-type MDM2 and either
wild-type PML or the indicated deletion mutant of PML. PML protein was
precipitated with an anti-HA antibody, and the immunoprecipitates
(IP) were examined with an antibody against MDM2 (top
panel). Steady-state levels of HA-PML and MDM2 were monitored by
immunobloting (IB) of lysates without prior immunoprecipitation
(middle and lower panels). The molecular mass (mw)
in kDa of protein standard is indicated next to each blot. C, U2OS
cells were transfected with DNAs encoding either wild-type MDM2 or an MDM2
mutant that localizes in the cytoplasm (MDM2 NLS-), and the indicated form of
HA-PML. MDM2 protein was precipitated with an anti-MDM2 antibody, and the
immunoprecipitates were examined with an antibody against HA (top
panel). Steady-state levels of HA-PML and MDM2 were monitored by
immunobloting of lysates without prior immunoprecipitation (middle
and lower panels).
|
|
Wild-type PML is post-translationally modified by the covalent attachment
of SUMO, a small ubiquitin-like molecule, at three separate lysine residues in
the PML sequence (Lys-65, Lys-160, and Lys-490;
Fig. 2A)
(13,
15,
16). Sumoylation is mediated
by the sumo-conjugating enzyme Ubc9 and requires interaction between Ubc9 and
the RING-finger domain within the first 100 amino acids of PML
(16). The finding that PML
mutants lacking the first 100 amino acids displayed an increased association
with MDM2 suggested that sumoylation of PML may be inhibitory to PML-MDM2
binding. To investigate this possibility, PML mutants were generated in which
each sumo site lysine was converted to arginine either singly or in
combination, and association between these mutants and MDM2 was monitored by
coimmunoprecipitation. As shown in Fig.
3A, MDM2 coimmunoprecipitated with wild-type PML to a
relatively small extent in cells transiently expressing both proteins. MDM2
also coimmunoprecipitated with PML K65R and PML K490R to a comparable extent.
In contrast, the PML K160R and PML 3KR mutants displayed a much stronger
association with MDM2 compared with either wild-type PML or the other sumo
site mutants. These findings suggested that PML sumoylation at lysine 160 may
be an important regulator of MDM2 binding. In light of these results, we
wished to confirm the sumoylation status of PML that coimmunoprecipitates with
MDM2. Sumoylated PML is largely insoluble under gentle conditions of cell
lysis. Therefore, to observe sumoylated PML, transfected cells were lysed in
buffer containing a relatively high detergent concentration
(radioimmunoprecipitation buffer) and subsequently examined by immunoblotting
with an anti-HA antibody. These studies revealed a series of high molecular
mass PML species that migrated above wild-type HA-PML and each of the single
sumo site mutants (Fig.
3B). Comparing the pattern of these species allowed us to
identify each as PML that is sumoylated at Lys-65, Lys-160, or Lys-490. In
contrast, these high molecular mass bands were not detected with expression of
the PML 3KR mutant. To characterize the sumoylation state of PML bound to
MDM2, wild-type PML and each of the sumo site mutants were expressed either
alone or with MDM2. Transfected cells were then immunoprecipitated with either
an anti-HA or anti-MDM2 antibody and examined by immunoblotting with an
anti-HA antibody (Fig.
3C). In these studies the K160R and 3KR PML mutants again
showed a much stronger association with MDM2 than the other forms of PML, and
only the nonsumoylated PML species could be coimmunoprecipitated with MDM2.
Taken together, these results indicate that nonsumoylated PML is the primary
form of PML that coimmunoprecipitates with MDM2 and suggest that sumoylation
at lysine 160 may be inhibitory to PML-MDM2 binding.

View larger version (50K):
[in this window]
[in a new window]
|
FIG. 3. Binding of MDM2 to PML sumoylation site mutants. A, U2OS
cells were transfected with DNAs encoding wild-type MDM2 and either wild-type
PML or the indicated sumoylation site mutants of PML. PML protein was
precipitated with an anti-HA antibody (IP), and the
immunoprecipitates were examined with an antibody against MDM2 (top
panel). Steady-state levels of HA-PML and MDM2 were monitored by
immunobloting (IB) of lysates without prior immunoprecipitation
(middle and lower panels). B, cells were
transfected with DNAs encoding the indicated forms of HA-PML. The cells were
then lysed in radioimmunoprecipitation buffer with sonication, as described
under "Experimental Procedures," and examined by immunoblotting
with an anti-HA antibody. The position of nonsumoylated PML, as well as PML
sumoylated at lysine 65, 160, and 490, are indicated. The asterisks
(*) indicate higher molecular mass bands that are most likely forms of HA-PML
which are sumoylated at multiple sites. C, cells were transfected
with DNAs encoding MDM2 and the indicated form of HA-PML. Cell lysates were
immunoprecipitated with either an anti-HA antibody or an anti-MDM2 antibody,
and the immunoprecipitates were examined with an anti-HA antibody (upper
panels). The nonsumoylated and sumoylated forms of PML are indicated. The
asterisk indicates the position of the antibody heavy chain used in
the immunoprecipitation. Steady-state levels of HA-PML were monitored by
immunobloting of lysates without prior immunoprecipitation (lower
panels).
|
|
The strong interaction between HA-PML K160R and MDM2 allowed us to use this
mutant in mapping other PML regions that may potentially interact with MDM2.
To this end, C-terminal deletion mutants of HA-PML K160R were tested for MDM2
binding by coimmunoprecipitation. Because the nuclear localization signal
(NLS) of PML is located in the C terminus between residues 487 and 493,
C-terminal deletion mutants that lack the NLS may localize in the cytoplasm
rather than the nucleus. Therefore, a cytoplasmic, NLS mutant of MDM2 was
included in these studies to ensure colocalization of the transfected
proteins. As shown in Fig. 4,
full-length HA-PML (K160R) formed a strong complex with MDM2 in cotransfected
cells. Similarly, C-terminal deletion mutants of HA-PML (K160R) also formed a
strong complex with either wild-type MDM2 or the MDM2 NLS mutant, including a
deletion mutant encoding only residues 1200. In these studies, the
C-terminal deletion mutants of HA-PML (K160R) displayed varying degrees of
binding to either wild-type MDM2 or the MDM2 NLS mutant. Forms of PML (K160R)
that are 1633 and 1500 contain the NLS and localize to the
nucleus of transfected cells (not shown). These forms of HA-PML (K160R) bind
most strongly to the nuclear localized wild-type MDM2 protein. In contrast,
HA-PML (K160R) 1400 lacks the NLS and localizes to the cytoplasm,
unable to enter the nucleus without being actively imported. Not surprisingly,
HA-PML (K160R) 1400 associates strongly with the cytoplasmic MDM2 NLS
mutant and only weakly with wild-type MDM2. The smaller HA-PML (K160R) mutants
(1300 and 1200) also lack the NLS but are small enough to
diffuse freely in and out of the nucleus, at least to some extent. These forms
of HA-PML (K160R) display a diffuse localization pattern throughout the cell
(not shown) and can bind both nuclear wild-type MDM2 and the cytoplasmic MDM2
NLS mutant. These results demonstrate that residues 1200 in PML-K160R
encode a separate MDM2 binding region. It should be noted that PML residues
1200 (wild-type) displayed little to no binding to MDM2 when tested in
a coimmunoprecipitation study similar to that shown in
Fig. 4 (data not shown),
consistent with the hypothesis that the K160R mutation uncovers an MDM2
binding site in this region of PML which would otherwise not be evident.

View larger version (74K):
[in this window]
[in a new window]
|
FIG. 4. Recognition of an MDM2 binding region within the PML N terminus.
Cells were transfected with DNAs encoding wild-type MDM2 or MDM2 (NLS-) either
alone or cotransfected with the indicated deletion mutants of HA-PML (K160R).
Transfected cell lysates were immunoprecipitated (IP) with an anti-HA
antibody and examined by immunoblotting (IB) with an antibody against
MDM2 (upper panel). The asterisk indicates the position of
the antibody heavy chain used in the immunoprecipitation. Reprobing of the
blot with an anti-HA antibody shows the expression level of the transfected
PML proteins (lower panel).
|
|
Our results indicate the presence of multiple MDM2 binding domains in PML,
one located in the C terminus between residues 300 and 633, and a second that
is revealed by the K160R mutation and is located between residues 1 and 200.
We wished to define the regions of MDM2 required for interaction with either
of these two regions of PML. To this end, MDM2 deletion mutants lacking
various portions of the N terminus, C terminus, or central region were tested
for their ability to coimmunoprecipitate with a form of PML which contained
both binding domains (PML 1633 (K160R)), or with PML mutants that
expressed either MDM2 binding region alone. The results of these studies are
summarized in Fig. 5. PML
1633 (K160R), which contains both MDM2 binding regions, formed a strong
complex with all of the MDM2 deletion mutants tested. This suggests the
presence of multiple PML binding regions in MDM2, the deletion of any one of
which may have little effect on PML binding. PML
200N also formed a
complex with all of the tested MDM2 mutants, although lesser binding was
observed between this form of PML and an MDM2 mutant that lacked the acidic
domain between residues 222 and 272. This suggests that MDM2 residues between
222 and 272 may be required for strong interaction between MDM2 and the
C-terminal half of PML. PML 1200 (K160R) formed a complex with each
MDM2 mutant except one that lacked the C terminus (MDM2 6339),
suggesting that MDM2 residues between 340 and 491 are required for interaction
with HA-PML 1200 (K160R). To map the PML interacting regions further,
MDM2 mutants that expressed only the N terminus, C terminus, or central
regions were tested in coimmunoprecipitation studies for binding to either PML
1200 (K160R) or PML
200N
(Fig. 5). In these studies, PML
1200 (K160R) formed a strong complex with the C terminus of MDM2
(residues 300488), but not with the MDM2 N terminus (residues
2202) or central region (residues 100304), demonstrating that
the MDM2 C terminus can form a specific complex with residues 1200 in
PML-K160R. In contrast, PML
200N could be coimmunoprecipitated with
MDM2 mutants encoding the central region between residues 100 and 304, but not
with MDM2 mutants that encoded only the N (residues 2202) or C
(residues 300488) terminus. These results are consistent with the
observation that PML
200N displayed weak binding to an MDM2 mutant that
lacked the central acidic domain (residues 222272). In summary, these
studies indicate that the C terminus of MDM2 can form a complex with PML
residues 1200 in the K160R mutant, whereas the MDM2 central region,
which includes the acidic domain between residues 222 and 272, can interact
with the C-terminal half of PML (Fig.
5).
Having identified multiple interactions between PML and MDM2, we next
wished to determine the consequence of these interactions on the localization
and/or activity of PML. The most striking feature of PML is its localization
into multiprotein, nuclear foci structures, PML-NBs
(33). Although the functions
of PML are not fully understood, their proper localization and ability to
recruit and interact with other NB components are considered essential for PML
to act as a tumor suppressor
(33,
34). To assess the effect of
MDM2 on PML localization, cells were transfected with DNAs encoding HA-tagged
PML either alone or cotransfected with various forms of MDM2. Localization of
the transfected PML and MDM2 proteins was then monitored by immunofluorescence
staining. As illustrated in Fig.
6A, HA-PML (wild-type) and MDM2 localized almost
exclusively to the nucleus when expressed alone. In contrast, coexpression
with wild-type MDM2 caused a marked redistribution of PML to the cytoplasm,
whereas the MDM2 protein remained nuclear
(Fig. 6B). These
results indicated that nuclear MDM2 could trigger a redistribution of PML from
the nucleus to the cytoplasm. We scored the extent to which PML was localized
to the cytoplasm when expressed alone or with MDM2, and the results are
graphed in Fig. 7. We next
wished to investigate the region of MDM2 which may be required to promote this
nuclear exclusion of PML. As shown in Figs.
6 and
7, MDM2 deletion mutants
lacking either the central acidic domain (
222272) or the
N-terminal p53 binding domain (
120N) could efficiently promote the
nuclear exclusion of PML, indicating that neither the central domain nor p53
binding domain is required for this effect. Similar results were obtained in
Saos-2 cells (p53-null; data not shown), further demonstrating that MDM2 can
promote PML nuclear exclusion in the absence of p53. An MDM2 mutant lacking
the C terminus (MDM2 6339) failed to promote PML nuclear exclusion
(Figs. 6 and
7), suggesting that PML binding
to the MDM2 C terminus may be required for this effect. Consistent with this
hypothesis, an N-terminal deletion mutant of PML (HA-PML (
200N)) was
completely resistant to MDM2-mediated nuclear exclusion. Given that the MDM2 C
terminus can interact with the PML N terminus, these results suggest that
binding between the N terminus of PML and the C terminus of MDM2 is required
to promote the nuclear exclusion of PML.

View larger version (17K):
[in this window]
[in a new window]
|
FIG. 6. MDM2 promotes nuclear exclusion of PML in transiently transfected
cells. U2OS cells were transfected with DNAs encoding HA-PML (wild-type)
or HA-PML ( 200N), either alone or with the indicated forms of MDM2, and
examined by immunofluorescence staining with anti-HA and anti-MDM2 antibodies.
A, representative immunofluorescence staining patterns are shown for
HA-PML (wild-type) and MDM2 (wild-type) when expressed alone. B,
representative immunofluorescence staining patterns are shown for each
transfection condition.
|
|

View larger version (26K):
[in this window]
[in a new window]
|
FIG. 7. Quantification of MDM2-mediated nuclear exclusion of PML. Cells were
transfected with the indicated combinations of HA-PML and MDM2 and examined by
immunofluorescence staining. The localization pattern for HA-PML in each case
was scored for 150 cells in three separate experiments. The graph illustrates
the average results from three separate experiments and shows the percentage
of cells with the indicated HA-PML staining patterns. MDM2 exhibited complete
nuclear staining in >90% of transfected cells when expressed alone or when
coexpressed with HA-PML. Cells in which MDM2 did not exhibit complete nuclear
staining were excluded from the analysis.
|
|
One NB-associated protein with which PML interacts is the transcriptional
coactivator CBP. PML binding has been reported to stimulate the ability of a
GAL4-CBP fusion protein to activate transcription from a gene promoter
containing GAL4 DNA binding sites
(35). We wished to test
whether MDM2 could block the ability of PML to activate CBP. As shown in
Fig. 8, HA-PML activated
GAL4-CBP-dependent transcription, consistent with previous studies
(35). Coexpression of
wild-type MDM2 decreased the ability of PML to activate GAL4-CBP, although
having relatively little effect on the activity of GAL4-CBP alone. These
results indicate that wild-type MDM2 can inhibit the ability of PML to
activate the GAL4-CBP fusion protein. To address the region of MDM2 required
for this effect, we tested various MDM2 deletion mutants for their ability to
inhibit PML in this assay. As shown in Fig.
8, the C-terminal deletion mutant MDM2 (6339) efficiently
inhibited the ability of PML to activate the GAL4-CBP fusion protein. These
results indicate that binding between the MDM2 C terminus and PML is not
required to inhibit PML in this assay. Given that MDM2 (6339) is unable
to promote PML nuclear exclusion, these results also indicate that nuclear
exclusion is not required for the MDM2-mediated inhibition of PML. An MDM2
mutant that lacks the N terminus (MDM2
120N) was also able to inhibit
the ability of PML to activate the GAL4-CBP fusion protein. In contrast, an
MDM2 deletion mutant lacking residues 222272 had little to no effect on
the ability of PML to activate the GAL4-CBP fusion protein. These results
indicate that the ability of MDM2 to inhibit PML in this assay requires the
central, acidic region of MDM2 and therefore likely involves binding between
the central region of MDM2 and PML.

View larger version (19K):
[in this window]
[in a new window]
|
FIG. 8. MDM2 inhibits PML-dependent activation of GAL4-CBP. A, 35-2
cells (p53 and MDM2 double knockout) were transfected with a GAL4-responsive
luciferase promoter and DNAs encoding GAL4-CBP (125 ng), HA-PML (200 ng), and
the indicated forms of MDM2 (500 ng). Relative luciferase activity mediated by
the GAL4-CBP fusion protein was measured. The graph shows the average results
from three to seven independent experiments (±S.E.).
|
|
 |
DISCUSSION
|
---|
The tumor suppressor protein PML has received considerable attention
recently due, at least in part, to its involvement in apoptosis signaling and
its direct interaction with p53. PML binding recruits p53 to PML-NBs,
multiprotein complexes that localize to discreet foci within the nucleus. The
effect of p53-PML binding is to increase p53 activity as a transcription
factor (22). Current models
suggest that recruitment to PML-NBs brings p53 in close proximity with
CBP/p300 (36). Acetylation of
p53 by CBP/p300 then increases p53 DNA binding affinity, leading to an
activation of p53-responsive genes. This activation of p53 most likely
contributes to the tumor suppressor function of PML. In addition to p53, PML
can interact with a variety of other proteins and colocalize with them in
PML-NBs. Included among these are proteins involved in recombination (BLM,
RAD51, RAD52), transcriptional activation and repression (CBP, Daxx, Sp100),
protein degradation (HAUSP, PA28), and the regulation of cell growth (pRB)
(for review, see Refs. 37 and
38). However, in most cases
the physiologic relevance of these interactions and their effect on cell
proliferation have not been clarified fully.
The current report identifies MDM2 as a novel PML-interacting protein.
Confocal microscopy revealed colocalization between the endogenous PML and
MDM2 proteins in IFN-
treated H1299 cells (p53-null), suggesting
that they can complex one another in cells. Further, MDM2 immunoprecipitated
with PML in cells transiently expressing both proteins, and recombinant MDM2
produced in insect cells formed a strong complex with a bacterially expressed
GST-PML fusion protein. These results indicate that MDM2 and PML can interact
with each other in a manner independent of p53 and suggest that this may be a
direct interaction. Mapping studies revealed at least two separate regions of
MDM2 and PML involved in their interaction. First, the central region of MDM2,
which includes the MDM2 acidic domain, could form a complex with the
C-terminal portion (residues 200633) of PML. The C terminus of PML is
of particular interest when considering interactions between PML and other
proteins. At least seven different PML isoforms have been described, all of
which vary in their C termini and are generated by alternative splicing
(12). It will be of interest
in the future to determine whether interaction with the central region of MDM2
is an activity shared by all PML isoforms or occurs in an isoform-specific
manner. The C terminus of MDM2 (residues 300488) could also form a
complex with residues 1200 in the PML K160R mutant. The N terminus of
PML and the C terminus of MDM2 both contain a RING-finger domain within their
sequences, and we considered that the interaction between these two regions
might be mediated through their respective RING-fingers. However, preliminary
results from our laboratory suggest that the PML RING-finger is not required
for residues 1200 to interact with MDM2 (not shown), suggesting that
this interaction with MDM2 is not through their corresponding RINGs.
Interestingly, the extent to which MDM2 could bind PML in
coimmunoprecipitation experiments was increased markedly when sumoylation of
PML at lysine 160 was inhibited, either through deletion of PML protein
regions required for Lys-160 sumoylation or through mutation of the Lys-160
site. This finding raises the possibility that PML sumoylation at Lys-160 may
in some way be inhibitory to PML-MDM2 binding. Lys-160 is located within the
N-terminal region of PML which can interact with the MDM2 C terminus. One
possibility, therefore, is that sumoylation at this site blocks access of this
region of PML to MDM2. However, it should be noted that sumoylation at Lys-160
is required for the proper localization of PML into PML-NBs
(39,
40). A second possibility,
therefore, is that sumoylation at Lys-160 may indirectly affect PML-MDM2
coimmunoprecipitation by directing PML into PML-NBs.
PML is the major component of multiprotein, nuclear foci structures,
PML-NBs (33,
34). PML appears to be
absolutely required for NB formation, as other NB-associated proteins fail to
localize in NBs when PML is not present
(39,
40). However, the biochemical
and molecular functions of PML-NBs are still unknown. Most current models
propose that PML-NBs are nuclear scaffolds that serve as either temporary
storage sites for catalytically active proteins or as catalytic surfaces where
specific biochemistries can occur
(34). In either case, proper
localization of PML, as well as its ability bind and recruit other
NB-associated proteins, is considered essential for PMLs normal function. We
observe that transient overexpression of MDM2 disrupts PML-NBs and promotes a
redistribution of PML to the cytoplasm. Given the importance of proper
localization in PML function, it is possible that MDM2 may inhibit one or more
activities associated with PML through this disruption of its normal
localization. The C terminus of MDM2 (residues 340488) is necessary to
alter PML localization, and the N terminus of PML is also required for PML to
be redistributed to the cytoplasm by MDM2 (Figs.
6 and
7). Given the interaction
between the PML N terminus and MDM2 C terminus, these results suggest that
binding between these two regions is responsible for the cytoplasmic
redistribution of PML. It is interesting to note that the disruption of
PML-NBs and cytoplasmic redistribution of PML observed here with MDM2
overexpression is strikingly similar to the effect of certain viral proteins
on PML. During a variety of viral infections, including those of the herpes,
adeno, and retro families, PML-NBs become disrupted, and, in some cases, PML
is relocalized to the cytoplasm
(4145).
This disruption of PML-NBs has been linked in some cases to host cell
shut-off, inhibition of apoptosis, and establishment of a chronic viral
infection (45). In contrast,
PML and other proteins associated with PML-NBs are induced after exposure to
antiviral IFNs, and the number of PML-NBs increases
(46). Based on these findings
it has been proposed that PML-NBs may represent preferential targets for viral
infections and that PML could play a role in the mechanism of the antiviral
action of IFNs. Viral proteins that disrupt PML-NBs and/or cause a
relocalization of PML to the cytoplasm include the herpes simplex virus type-1
ICP0 protein, the adenovirus E4-ORF-3 protein, the human cytomegalovirus IE1
protein, the lymphocytic choriomeningitis virus Z protein, and the rabies
virus P protein (45; for
review, see Ref. 46). Like
MDM2, the herpes simplex virus type-1 ICP0 and lymphocytic choriomeningitis
virus Z proteins both contain a RING-finger domain. Further, lymphocytic
choriomeningitis virus Z and rabies P protein cause a cytoplasmic
relocalization of PML, and herpes simplex virus type-1 ICP0 promotes the
proteasome-dependent degradation of PML. It remains to be seen whether the
relocalization of PML observed here with MDM2 expression occurs through a
similar or distinct mechanism as in cells expressing these viral proteins.
CBP is a transcriptional activator with histone acetyltransferase activity
and is a component of PML-NBs
(47). PML forms a direct
complex with CBP, and this complex formation stimulates the ability of a
GAL4-CBP fusion protein to activate gene transcription
(35). In the current report,
wild-type MDM2 inhibited the ability of PML to activate GAL4-CBP-mediated
transcription. This inhibition required the central, acidic region of MDM2,
suggesting that binding between the central region of MDM2 and PML is required
to inhibit PML in this assay. An MDM2 mutant that lacked the C terminus, and
was therefore unable to redistribute PML to the cytoplasm, also inhibited the
ability of PML to activate CBP. These results indicate that the MDM2-mediated
nuclear exclusion of PML is not required for MDM2 to inhibit PML, at least in
this assay system. Functional interactions among PML, MDM2, and CBP are of
particular interest with regard to the PML-mediated activation of p53. Current
models suggest that PML corecruits p53 and CBP to PML-NBs, bringing them into
close proximity (for review, see Ref.
36). CBP can then acetylate
p53, resulting in an increased p53 DNA binding activity, thus leading to an
activation of p53-responsive genes. This activation of p53 most likely
contributes to the tumor suppressor function of PML. Conceivably, MDM2 could
inhibit this activation of p53 by either disrupting PML-NBs or blocking the
ability of PML to activate CBP. Such a mechanism could contribute to the
MDM2-mediated inhibition of p53.
 |
FOOTNOTES
|
---|
* This work was supported by NCI, National Institutes of Health Grant
1RO1CA8091801 and American Cancer Society Grant RSG-01-042 (to C. G.
M.). The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 

Present address: Dept. of Cancer Biology, Cancer Research Center,
University of Massachusetts Medical School, Worcester, MA 01605. 
**
To whom correspondence should be addressed: Dept. of Radiation Oncology, the
University of Chicago, 5841 S. Maryland Ave., MC1105 Room G-06, Chicago, IL
60637. E-mail:
cmaki{at}rover.uchicago.edu.
1 The abbreviations used are: RAR, retinoic acid receptor; CBP, CREB-binding
protein; CREB, cAMP enhancer-binding protein; GST, glutathione
S-transferase; HA, hemagglutinin; NB, nuclear body; NLS, nuclear
localization signal; wt, wild-type. 
 |
REFERENCES
|
---|
- de The, H., Lavau, C., Marchio, A., Chomienne, C., Degos, L., and
Dejean, A. (1991) Cell
66,
675684[Medline]
[Order article via Infotrieve]
- Goddard, A. D., Borrow, J., Freemont, P. S., and Solomon, E.
(1991) Science
254,
13711374[Medline]
[Order article via Infotrieve]
- Kakizuka, A., Miller, W. H., Jr., Umesono, K., Warrell, R. P., Jr.,
Frankel, S. R., Murty, V. V., Dmitrovsky, E., and Evans, R. M.
(1991) Cell
66,
663674[Medline]
[Order article via Infotrieve]
- Pandolfi, P. P., Grignani, F., Alcalay, M., Mencarelli, A., Biondi,
A., LoCoco, F., Grignani, F., and Pelicci, P. G. (1991)
Oncogene 6,
12851292[Medline]
[Order article via Infotrieve]
- Pandolfi, P. P. (2001) Hum. Mol.
Genet. 10,
769775[Abstract/Free Full Text]
- Koken, M. H., Linares-Cruz, G., Quignon, F., Viron, A.,
Chelbi-Alix, M. K., Sobczak-Thepot, J., Juhlin, L., Degos, L., Calvo, F., and
de The, H. (1995) Oncogene
10,
13151324[Medline]
[Order article via Infotrieve]
- Everett, R. D., Lomonte, P., Sternsdorf, T., van Driel, R., and
Orr, A. (1999) J. Cell Sci.
112,
45814588[Abstract/Free Full Text]
- Dyck, J. A., Maul, G. G., Miller, W. H., Jr., Chen, J. D.,
Kakizuka, A., and Evans, R. M. (1994)
Cell 76,
333343[Medline]
[Order article via Infotrieve]
- Koken, M. H., Puvion-Dutilleul, F., Guillemin, M. C., Viron, A.,
Linares-Cruz, G., Stuurman, N., de Jong, L., Szostecki, C., Calvo, F., and
Chomienne, C. (1994) EMBO J.
13,
10731083[Abstract]
- Weis, K., Rambaud, S., Lavau, C., Jansen, J., Carvalho, T.,
Carmo-Fonseca, M., Lamond, A., and Dejean, A. (1994)
Cell 76,
345356[Medline]
[Order article via Infotrieve]
- Perez, A., Kastner, P., Sethi, S., Lutz, Y., Reibel, C., and
Chambon, P. (1993) EMBO J.
8,
31713182
- Jensen, K., Shiels, C., and Freemont, P. S. (2001)
Oncogene 49,
72237233[CrossRef]
- Boddy, M. N., Howe, K., Etkin, L. D., Solomon, E., and Freemont, P.
S. (1996) Oncogene
13,
971982[Medline]
[Order article via Infotrieve]
- Seeler, J. S., and Dejean, A. (2001)
Oncogene 20,
72437249[CrossRef][Medline]
[Order article via Infotrieve]
- Kamitani, T., Kito, K., Nguyen, H. P., Wada, H., Fukuda-Kamitani,
T., and Yeh, E. T. (1998) J. Biol. Chem.
273,
2667526682[Abstract/Free Full Text]
- Duprez, E., Saurin, A. J., Desterro, J. M., Lallemand-Breitenbach,
V., Howe, K., Boddy, M. N., Solomon, E., de The, H., Hay, R. T., and Freemont,
P. S. (1999) J. Cell Sci.
112,
381393[Abstract/Free Full Text]
- Muller, S., Matunis, M. J., and Dejean, A. (1998)
EMBO J. 17,
6170[Abstract/Free Full Text]
- Zhong, S., Hu, P., Ye, T. Z., Stan, R., Ellis, N. A., and Pandolfi,
P. P. (1999) Oncogene
56,
79417947
- Bischof, O., Kirsh, O., Pearson, M., Itahana, K., Pelicci, P. G.,
and Dejean, A. (2002) EMBO J.
13,
33583369[CrossRef]
- Wang, Z. G., Delva, L., Gaboli, M., Rivi, R., Giorgio, M.,
Cordon-Cardo, C., Grosveld, F., and Pandolfi, P. P. (1998)
Science 279,
15471551[Abstract/Free Full Text]
- Wang, Z. G., Ruggero, D., Ronchetti, S., Zhong, S., Gaboli, M.,
Rivi, R., and Pandolfi, P. P. (1998) Nat.
Genet. 20,
266272[CrossRef][Medline]
[Order article via Infotrieve]
- Fogal, V., Gostissa, M., Sandy, P., Zacchi, P., Sternsdorf, T.,
Jensen, K., Pandolfi, P. P., Will, H., Schneider, C., and Del Sal, G.
(2000) EMBO J.
19,
61856195[Abstract/Free Full Text]
- Pearson, M., Carbone, R., Sebastiani, C., Cioce, M., Fagioli, M.,
Saito, S., Higashimoto, Y., Appella, E., Minucci, S., Pandolfi, P. P., and
Pelicci, P. G. (2000) Nature
406,
207210[CrossRef][Medline]
[Order article via Infotrieve]
- Ferbeyre, G., de Stanchina, E., Querido, E., Baptiste, N., Prives,
C., and Lowe, S. W. (2000) Genes Dev.
14,
20152027[Abstract/Free Full Text]
- Momand, J., Zambetti, G. P., Olson, D., George, D., and Levine, A.
J. (1992) Cell
69,
12371245[Medline]
[Order article via Infotrieve]
- Oliner, J. D., Kinzler, K. W., Meltzer, P. S., George, D., and
Vogelstein, B. (1992) Nature
358,
8083[CrossRef][Medline]
[Order article via Infotrieve]
- Haupt, Y., Maya, R., Kazaz, A., and Oren, M. (1997)
Nature 387,
296299[CrossRef][Medline]
[Order article via Infotrieve]
- Kubbutat, M. H., Jones, S. N., and Vousden, K. H.
(1997) Nature
387,
299303[CrossRef][Medline]
[Order article via Infotrieve]
- Boyd, S. D., Tsai, K. Y., and Jacks, T. (2000)
Nat. Cell Biol. 9,
563568[CrossRef]
- Geyer, R. K., Yu, Z. K., and Maki, C. G. (2000)
Nat. Cell Biol. 9,
569573[CrossRef]
- Gu, J., Nie, L., Kawai, H., and Yuan, Z. M. (2001)
Cancer Res. 18,
67036707
- Kawai, H., Nie, L., Wiederschain, D., and Yuan, Z. M.
(2001) J. Biol. Chem.
276,
4592845932[Abstract/Free Full Text]
- Maul, G. G., Negorev, D., Bell, P., and Ishov, A. M.
(2000) J. Struct. Biol.
129,
278287[CrossRef][Medline]
[Order article via Infotrieve]
- Borden, K. L. B. (2002) Mol. Cell.
Biol. 22,
52595269[Free Full Text]
- Doucas, V., Tini, M., Egan, D. A., and Evans, R. M.
(1999) Proc. Natl. Acad. Sci. U. S. A.
6,
26272632[CrossRef]
- Pearson, M., and Pelicci, P. G. (2001)
Oncogene 49,
72507256[CrossRef]
- Zhong, S., Salomoni, P., and Pandolfi, P. P. (2000)
Nat. Cell Biol. 5,
E85E90[CrossRef]
- Negorev, D., and Maul, G. G. (2001)
Oncogene 49,
72347242[CrossRef]
- Zhong, S., Muller, S., Ronchetti, S., Freemont, P. S., Dejean, A.,
and Pandolfi, P. P. (2000) Blood
95,
27482752[Abstract/Free Full Text]
- Lallemand-Breitenbach, V., Zhu, J., Puvion, F., Koken, M., Honore,
N., Doubeikovsky, A., Duprez, E., Pandolfi, P. P., Puvion, E., Freemont, P.,
and de The, H. (2001) J. Exp. Med.
193,
13611371[Abstract/Free Full Text]
- Borden, K. L., Campbell Dwyer, E. J., and Salvato, M. S.
(1998) J. Virol.
72,
758766[Abstract/Free Full Text]
- Desbois, C., Rousset, R., Bantignies, F., and Jalinot, P.
(1996) Science
273,
951953[Abstract]
- Maul, G. G., and Everett, R. D. (1994) J.
Gen. Virol. 75,
12231233[Abstract]
- Yu, E., Joo, Y. K., and Lee, I. (1999) Int.
J. Mol. Med. 3,
591596[Medline]
[Order article via Infotrieve]
- Blondel, D., Regad, T., Poisson, N., Pavie, B., Harper, F.,
Pandolfi, P. P., de The, H., and Chelbi-Alix, M. K. (2002)
Oncogene 21,
79577970[CrossRef][Medline]
[Order article via Infotrieve]
- Regad, T., and Chelbi-Alix, M. K. (2001)
Oncogene 20,
72747286[CrossRef][Medline]
[Order article via Infotrieve]
- Chan, H. M., and La Thangue, N. B. (2001)
J. Cell Sci. 114,
23632373[Abstract/Free Full Text]