From the ¶ Department of Oncology-Pathology, Cancer Center
Karolinska, Karolinska Institutet and Hospital, S-171 76 Stockholm, Sweden and the Department of
Biochemistry and Molecular Biology, Wright State University, Dayton,
Ohio 45435
Received for publication, November 27, 2000, and in revised form, March 19, 2001
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ABSTRACT |
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Regulation of p53 involves a complex network of
protein interactions. The primary regulator of p53 protein stability is
the Mdm2 protein. ARF and MdmX are two proteins that have
recently been shown to inhibit Mdm2-mediated degradation of p53 via
distinct associations with Mdm2. We demonstrate here that ARF is
capable of interacting with MdmX and in a manner similar to its
association with Mdm2, sequestering MdmX within the nucleolus. The
sequestration of MdmX by ARF results in an increase in p53
transactivation. In addition, the redistribution of MdmX by ARF
requires that a nucleolar localization signal be present on
MdmX. Although expression of either MdmX or ARF leads to Mdm2
stabilization, coexpression of both MdmX and ARF results in a decrease
in Mdm2 protein levels. Similarly, increasing ARF protein levels in the
presence of constant MdmX and Mdm2 leads to a
dose-dependent decrease in Mdm2 levels. Under these
conditions, ARF can synergistically reverse the ability of Mdm2 and
MdmX to inhibit p53-dependent transactivation. Finally, the
association and redistribution of MdmX by ARF has no effect on the
protein stability of either ARF or MdmX. Taken together, these results
demonstrate that the interaction between MdmX and ARF represents a
novel pathway for regulating Mdm2 protein levels. Additionally, both
MdmX and Mdm2, either individually or together, are capable of
antagonizing the effects of the ARF tumor suppressor on p53 activity.
Cellular division is controlled predominantly by two distinct
tumor suppressors, p53 and
Rb.1 The p53 protein
regulates cell cycle arrest and apoptosis because of its ability to
transcriptionally activate specific target genes following various
forms of genotoxic stress (1). The Rb protein controls entry into
S-phase through its interaction with various members of the E2F family
of transcription factors (2). Signaling between these two pathways
involves an ever increasing number of proteins including p21WAF1, a
target of p53-dependent transactivation and inhibitor of
cyclin-dependent kinase activity (3, 4), and the Mdm2
protein, an antagonist of both p53 and Rb growth suppressive functions
(5-8). Additional overlap between p53 and Rb is provided by the
INK4A locus, which encodes both p16INK4a and p19ARF proteins
(9, 10). Although the coding transcripts of these proteins share common
exons, the primary amino acid sequences of both proteins are unique.
Overexpression of p16INK4A or p19ARF results in a cell cycle arrest
through distinct pathways involving either Rb or p53, respectively (9,
11-13). p16INK4A inhibits cyclin D-dependent kinases, thus
preventing the phosphorylation of Rb and the subsequent release of E2F
proteins (12, 13). p19ARF activates the p53 protein by binding to Mdm2
and sequestering it to the nucleolus (14, 15). The resulting
Mdm2·ARF complex is unable to mediate nucleocytoplasmic
shuttling of p53 (16) or act as an E3 ligase to ubiquitinate p53 (17).
In addition to interacting with Mdm2, ARF must also contain two
functional nucleolar localization signals (NrLS) (18-20). However,
recent evidence indicates that an additional NrLS within the C-terminal RING finger of Mdm2 is also required for nucleolar localization of the
ARF·Mdm2 complex. Deletion of the NrLS or a more subtle mutation of the basic residues of the NrLS to uncharged amino acids
results in failure of the two proteins to colocalize in the nucleolus
(19, 21).
Recently, we demonstrated that the MdmX protein, a p53-binding protein
with homology to Mdm2, could protect p53 from Mdm2-mediated degradation
while maintaining suppression of p53-dependent
transactivation (22). Moreover, additional studies have demonstrated
that Mdm2 is also stabilized through association with MdmX (23, 24). Given the homology between MdmX and Mdm2, we examined the localization of MdmX in the presence or absence of ARF. Our data indicate that coexpression of ARF with MdmX results in the mobilization of
full-length MdmX to the nucleoli, where it is not found in the absence
of ARF. Redistribution of MdmX by ARF reverses the MdmX-mediated inhibition of p53 transactivation. Interestingly, MdmX and ARF have an
antagonistic effect on each other with respect to their ability to
stabilize Mdm2, whereas MdmX and Mdm2 work synergistically to reverse
ARF induction of p53 transactivation.
Cell Lines and Antibodies--
U2OS cells are an osteosarcoma
cell line containing wild-type p53 and having no detectable ARF protein
(10). H1299 cells are a non-small cell lung carcinoma devoid of p53.
Cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. Antibodies include
monoclonal FLAG M2 (Sigma), monoclonal V5 (Invitrogen), monoclonal GFP
(Zymed Laboratories Inc.), and polyclonal ARF Ab-1
(Neomarkers). For immunofluorescence analysis, a Texas Red-conjugated
goat anti-mouse antibody (Jackson ImmunoResearch) was used. An
horseradish peroxidase-conjugated secondary antibody (Promega) was used
for chemiluminescent detection of proteins.
Plasmids and Transfections--
The p14ARF-GFP expression
plasmid has been described previously (25). The p19ARF-Myc/His
plasmid contains the murine ARF cDNA with a C-terminal Myc/His
epitope tag. Mdm2-FLAG is in the pFLAG-CMV-2 vector (Sigma), which
encodes a FLAG epitope tag onto the amino terminus of the Mdm2 protein.
Human MdmX and MdmX-(1-446) are in the pcDNA3.1/V5-His
vector, which encodes a V5 epitope tag and a six-histidine tag onto the
C terminus of the expressed protein. The human MdmX cDNA used as a
template for amplification was kindly provided by Dr. Aart
Jochemsen. The PG13-luc plasmid was constructed by cloning
13 copies of a synthetic p53 DNA binding site upstream of a SV40
promoter-luciferase gene. MG15-luc contains 15 copies of a
mutated p53 DNA binding site cloned upstream of the SV40
promoter-luciferase reporter gene. The pQE- Fluorescence Studies--
Immunofluorescence analysis was
performed 24 h after transfection. Transfected U2OS cells on
chamber slides (LabTech) were fixed in 3% paraformaldehyde,
permeabilized with 1% Triton X-100 in phosphate-buffered saline, and
blocked for 2 h with 10% goat serum, 0.01% Tween 20 in
phosphate-buffered saline. Primary antibody was added to a 1/10
dilution of blocking solution and incubated for 1 h. The cells
were washed five times and incubated with the secondary antibody for
1 h. Nuclei were stained with 25 µg/ml Hoechst Dye (Sigma) for 2 min. B23 immunofluorescence was performed as described previously
(25).
Immunoprecipitation, Western Analysis, and p53 Reporter
Assays--
For immunoprecipitation and Western blot experiments,
whole cell extracts were made 24 h following transfection by
incubating cell pellets for 30 min in lysis buffer (50 mM
Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) containing a
protease inhibitor mixture (Sigma). Immunoprecipitation analysis was
performed by adding 100 µg of pHOOK cell extracts with 1 µg of
monoclonal V5 antibody in a total volume of 300 µl of
phosphate-buffered saline containing 5 mM EDTA and 0.5%
Triton X-100. Following overnight incubation, 25 µl of protein
G-agarose was added and incubated an additional 1 h before
washing three times for 30 min each time. Pellets were resuspended in
25 µl of 2× SDS loading buffer and resolved using 10%
SDS-polyacrylamide gel electrophoresis followed by transfer of proteins
to a polyvinylidene difluoride membrane (Millipore) using a Transblot
system (Bio-Rad). Immunoblotting was performed as described previously
(26) using primary antibody dilutions of 1:1,000-1:2,500 and secondary
dilutions of 1:5,000-1:10,000. For p53 reporter assays, extracts were
made 24 h after transfection. p53 transactivation was determined
by quantifying luciferase activity in aliquots from whole cell extracts
using a luciferase assay system (Promega). Coexpression of ARF Localizes MdmX to the Nucleolus and Activates
p53 Transactivation--
Previous studies have demonstrated that ARF
is localized to the nucleolus (10, 15, 18, 25). The Mdm2 protein,
although not found in nucleoli in the absence of ARF, can be mobilized to nucleoli following either coexpression of ARF or induced ARF expression (14, 15). Based on the homology between Mdm2 and MdmX, we
first examined whether MdmX was found in nucleoli or could be
colocalized with ARF. U2OS cells, which do not express ARF yet do
possess wild-type p53 protein (10), were transiently transfected with
an expression plasmid encoding a V5-epitope tagged human MdmX protein
in the presence or absence of an expression plasmid encoding an
ARF·GFP fusion protein. In the absence of ARF (or in the presence of
GFP, data not shown), MdmX showed no nucleolar localization (Fig.
1A, panel l). However, upon
coexpression of ARF, MdmX became localized to the nucleolus (Fig.
1A, panels j and k). When MdmX and ARF
signals were overlaid they demonstrated clear colocalization (Fig.
1A, panels m and n). ARF localization to the
nucleolus (Fig. 1A, panel e) was confirmed in transfected U2OS cells by demonstrating that ARF colocalized with the nucleolus protein B23 (Fig. 1A, panels p and q).
To determine the effect of ARF expression on MdmX regulation of p53
transactivation, U2OS cells were transfected with the p53-responsive
promoter PG13-luc. As expected, transfection of the
p53-responsive promoter (PG13-luc) into cells possessing
wild-type p53 protein led to a 6-fold increase in p53 transactivation
when compared with transfection of a p53 reporter plasmid containing mutant p53 DNA binding sites (MG15-luc; Fig.
1B). Coexpression of a mdmX expression vector led to a
modest 20% decrease in the activity of the p53 responsive promoter
(Fig. 1B). With increasing levels of mdmX expression vector,
p53 transactivation levels do continue to decrease in U2OS cells (data
not shown). Interestingly, the addition of a 1:1 or 1:2 ratio of
mdmX:ARF expression vectors led to a
dose-dependent increase in p53 transactivation, surpassing levels of p53 transactivation seen in the absence of exogenous MdmX or
ARF. The 2.7-fold increase in p53 transactivation reporter activity
seen on comparing the 1:2 mdmX:ARF transfections with the
PG13-luc only transfected U2OS cells most likely means
that the elevated ARF protein levels effectively sequestered
both the exogenous MdmX and endogenous Mdm2, thereby stabilizing and
maximizing the nuclear pools of free p53 protein (Fig.
1B).
The Requirement of a Nucleolar Localization Signal in
MdmX--
Based on the requirement for a conserved stretch of basic
amino acids, or NrLS, in the RING domain of Mdm2 (19, 21), we examined
whether MdmX contains a similar NrLS. Fig.
2A compares the NrLS of Mdm2
with the putative NrLS of both human and murine MdmX. Both human and
murine MdmX sequences have a conserved
(R/K)(R/K)X(R/K) motif previously shown to be
required for the nucleolar localization of the ARF·Mdm2 complex (19,
21). To examine the importance of the putative NrLS in MdmX, a deletion
mutant of MdmX lacking the RING finger domain, which contains the NrLS,
was constructed, and its ability to colocalize with ARF was tested. The
deletion matched a similar deletion made in Mdm2, which demonstrated
the requirement for a NrLS in the Mdm2 RING finger for proper
Mdm2·ARF nucleoli localization (19, 21). As seen with Mdm2,
MdmX-(1-446), which lacked the MdmX RING domain, was unable to
colocalize to the nucleolus with ARF (Fig. 2B, panel h).
Surprisingly, ARF was not completely prohibited from entering the
nucleolus when coexpressed with MdmX-(1-446) (Fig. 2B, panel
e), contrasting the results reported with NrLS-deficient Mdm2
proteins (19, 21). In any event, expression of MdmX-(1-446) did lead
to a greater accumulation of ARF in the nucleoplasm when compared with
coexpression with full-length MdmX (Fig. 2B, compare
panels d and e).
The inability of MdmX-(1-446) to maintain ARF within the nucleoplasm
may simply represent differences in ARF binding to MdmX and
MdmX-(1-446). To test this possibility, H1299 cells were
transfected as described in Fig. 2B, MdmX protein was
immunoprecipitated, and the interaction with ARF was confirmed by
Western analysis. Based on immunoprecipitation results (Fig.
3A), both full-length MdmX and
MdmX-(1-446) were capable of interacting with ARF. Comparing the
immunoprecipiations to a parallel immunoblot (Fig. 3A, lower panel), there appears to be a slight but reproducible decrease in
the amount of ARF bound to MdmX-(1-446) relative to full-length MdmX.
Perhaps the interaction between ARF and MdmX is not as stable in the
nucleoplasm as it is in the nucleolus. The nucleolar localization of
ARF in transfected H1299 cells (Fig. 3B) confirmed that ARF localization was comparable with that seen in U2OS cells. We also examined a more truncated version of MdmX, amino acids 1-363, which
also binds to ARF but is unable to colocalize to the nucleolus with ARF
(data not shown). Taken together, the data in Figs. 2 and 3 are
consistent with the similar interpretation made for Mdm2, namely that
MdmX must contain a functional RING finger domain in order for ARF to
mobilize it to the nucleolus but not for association with ARF.
Regulating Mdm2 Stability and p53 Transactivation--
Because ARF
and MdmX have both been reported to individually stabilize the Mdm2
protein (10, 23, 24), we next examined how MdmX affects the stability
of Mdm2 either alone or in the presence of increasing levels of ARF
protein. Although studies examining mdmX gene
expression have failed to uncover any significant modulation (27, 28),
one group has reported substantially higher levels of mdmX mRNA in
the thymus of both human and mouse (29) suggesting that elevated MdmX
protein may occur in specific tissues. In agreement with previously
reported data, MdmX was able to stabilize the Mdm2 protein (Fig.
4A, lane 3). In
contrast, coexpression of MdmX and ARF together had a
dose-dependent antagonistic effect on the ability of both
proteins to stabilize Mdm2 protein (Fig. 4A, lanes
4-6). Interestingly, there was also a slight, but reproducible,
decrease in MdmX protein levels as ARF protein levels increased and
Mdm2 protein levels decreased (Fig. 4A, lanes 3-6). It is possible that the decrease in MdmX protein levels results from a decrease in MdmX·Mdm2 complex formation. This would be
in agreement with one recent study in which MdmX mutants lacking the
RING finger domain showed decreased protein stability relative to
full-length MdmX (30).
As expected, overexpression of MdmX-(1-446) protein was not able to
stabilize Mdm2 protein when expressed in U2OS cells (Fig. 4B, lane 5), nor was it able to antagonize the
ability of ARF protein to stabilize Mdm2 protein (Fig. 4B,
lane 4). The inability of MdmX-(1-446) to reverse the
ARF-induced stability of Mdm2 implies that the association of MdmX and
ARF may not be sufficient to reverse the ARF stability of Mdm2 and that
nucleolar localization of ARF and MdmX is required to destabilize Mdm2.
The results shown in Fig. 4A demonstrated that the stability
of both MdmX and Mdm2 decreased as ARF protein levels increased. To
determine whether the decrease in MdmX stability was dependent upon
Mdm2 or ARF, MdmX was expressed in U2OS cells with increasing ARF in
the absence of Mdm2. Under these transfection conditions, increasing
ARF protein levels did not affect MdmX protein levels (Fig.
5A), consistent with the
stability of MdmX in Fig. 4A being more dependent on the
presence of elevated Mdm2 than ARF levels. Finally, there was no
alteration in ARF levels as in transfections containing increasing
levels of MdmX expression vector (Fig. 5B). Taken together,
the results shown in Fig. 5 suggest that the association between MdmX
and ARF has no dramatic effect on the stability of either protein.
Finally, to examine the effects of coexpressing Mdm2, MdmX, and ARF on
endogenous p53 transactivation, U2OS cells were transfected with the
indicated plasmids and the p53-responsive promoter,
PG13-luc (Fig. 6). As expected, transfection of Mdm2
and MdmX expression vectors together, led to a >90% decrease in p53
transactivation (Fig. 6A). The
inhibition of p53 transactivation by transfection with mdm2 and mdmX
expression vectors was reversed, in a dose-dependent manner, by coexpression with ARF expression vectors (Fig.
6A). The increase in p53 transactivation seen in
transfections of increasing ARF in the presence of constant Mdm2 and
MdmX most likely results from with ARF nucleolar sequestration of Mdm2
(14, 15) and MdmX (Fig. 1) or the decreased protein stability of Mdm2
and MdmX (Fig. 4A). Finally, consistent with the antagonist
effects of ARF and MdmX functioning through nucleolar sequestration, a
5-fold increase in p53 transactivation detected when ARF was
overexpressed in U2OS cells was inhibited in a
dose-dependent manner with transfection of increasing
levels of mdmX expression plasmid (Fig.
6B). Taken together with the results in Fig. 1B,
these data demonstrate that MdmX regulation of p53 transactivation is
not limited to its direct association with p53 (29) or with Mdm2 (22)
but can also be elicited through MdmX association with ARF.
Both ARF and MdmX are capable of regulating p53 protein levels
through binding to and inhibiting Mdm2 function. However, MdmX also
binds directly to p53, resulting in the nucleoplasmic sequestration of
p53 protein and inhibition of p53 transactivation (22). We demonstrate
in this report that in addition to mobilizing Mdm2 to the nucleolus,
ARF can also interact with and direct the nucleolar localization of
MdmX (Fig. 1A). Redistribution of MdmX requires the
C-terminal RING domain of MdmX that contains a conserved stretch of
basic amino acids recently identified as an NrLS in Mdm2 (Fig. 2).
Mobilization of MdmX from the nucleoplasm to the nucleoli segregates
MdmX from p53, resulting in a decrease in the ability of MdmX to
interact with and inhibit p53 transactivation (Fig. 1B).
Furthermore, we show that coexpression of MdmX with ARF and Mdm2
results in a decrease in Mdm2 stability, indicating that interaction of
MdmX and ARF prohibits the stabilizing interaction between Mdm2 and ARF
and likely blocks MdmX interactions with Mdm2. Interestingly, although
the coexpression of MdmX, Mdm2, and ARF results in decreased Mdm2
protein levels relative to MdmX and Mdm2 alone, it also resulted in a
lower level of p53 transactivation relative to ARF alone (Fig.
6A). These data are consistent with a model in which the
nucleolar sequestration of MdmX through MdmX·ARF complexes
releases Mdm2, leading to its eventual for degradation (Fig.
7).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal plasmid was used to
normalize transfection efficiency. In the p53 transactivation studies,
ARF, mdm2, and mdmX expression plasmids were used. Transfections were
performed using LipofectAMINE (Life Technologies, Inc.). In Fig. 3, a
pHOOK plasmid (Invitrogen) was included with each transfection to allow
immunoselection of transfected cells with Capture-Tec beads
(Invitrogen). For Western analysis in Figs. 4 and 5, transfection
efficiencies were normalized by the inclusion of a pGL3-luciferase
reporter plasmid (Promega) in each transfection.
-Galactosidase activity
was determined by incubating protein extracts in 100 mM
sodium phosphate, pH 7.3, 1 mM MgCl2, 50 mM
-mercaptoethanol, and 650 µ g/ml
O-nitrophenyl-
-D-galactopyranoside at
37 °C for 0.5-2 h.
-Galactosidase activity was calculated by
converting the absorbance read at 420 nm into milliunits of
-galactosidase activity per microliter of extract. All luciferase and
-galactosidase assays were performed in duplicate. Error bars of
relative p53 transactivation represent the average deviation for the
relative luciferase units divided by the milliunits of
-galactosidase.
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ABSTRACT
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Fig. 1.
MdmX colocalizes with ARF and affects
p53 transactivation. A, U2OS cells were transfected
with cDNAs encoding GFP-tagged ARF (1.0 µg) and/or V5-tagged
human MdmX (1.0 µg) as indicated. Cells were fixed 24 h after
transfection and analyzed for GFP and MdmX (left panel) or
B23 (right panel) fluorescence. Expression of MdmX alone
resulted in no significant localization within the nucleolus
(panel l). In contrast, coexpression of ARF with MdmX
resulted in the mobilization of MdmX to the nucleolus (panels
m and n). The immunofluorescence of the nucleolar
protein B23 demonstrated that ARF is localized to the nucleolus
(panels o-q). B, U2OS cells were transfected
with either MG15-luc or PG13-luc, pQE- gal,
and cDNAs encoding the indicated plasmids. Whole cell extracts were
produced 24 h after transfection. Relative p53 transactivation
represents the p53 induction of luciferase activity normalized to the
transfection efficiency (
-galactosidase assays). The expression of
ARF reverses MdmX inhibition of p53 transactivation and increases p53
transactivation in a dose-dependent manner. The pattern has
been observed in three separate transfection experiments.
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Fig. 2.
The C terminus of MdmX is required for
colocalization with ARF. A, amino acid alignment of
human Mdm2, human MdmX, Mdm2, and MdmX demonstrating the conservation
of a nucleolar localization sequence,
(R/K)(R/K)X(R/K). B, cellular localization
of MdmX and ARF·GFP proteins in transfected U2OS cells. Cells were
examined for nuclear DNA (top panels), GFP (middle
panels), and MdmX (bottom panels). Although
coexpression of full-length MdmX with ARF resulted in the mobilization
of MdmX to the nucleolus (panel g), deletion of the NrLS of
MdmX abrogated the ability of ARF to sequester MdmX to the nucleolus
(panel k).
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Fig. 3.
Direct interaction of ARF and MdmX.
A, H1299 cells were transfected with ARF (1.5 µg) and a
V5-tagged human MdmX or MdmX-(1-446) (4.0 µg) as indicated. A
pHook plasmid was also included (1.5 µg), and the transfected
population was selected using Capture Tec beads. Western blot analysis
of MdmX immunoprecipitated cellular extracts (top
panel). Both full-length MdmX and MdmX-(1-446) are able to
interact with ARF. The bottom panels are Western blots
containing 10% of each whole cell extract probed for GFP-tagged ARF or
MdmX. B, H1299 transfected with ARF and examined by
immunofluorescence for ARF and B23 localization. Like U2OS cells (Fig.
1), ARF colocalizes with B23 in the nucleolus of H1299 cells.
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Fig. 4.
Interaction between MdmX and ARF destabilizes
Mdm2 and inhibits p53 transactivation. A, Western blot
analysis of cellular extracts from transfected U2OS cells was used to
determine Mdm2, ARF, and MdmX protein levels. Coexpression of MdmX with
Mdm2 resulted in the stabilization of Mdm2. However, expression of
increasing amounts of ARF in the presence of MdmX resulted in a
dose-dependent decrease in the stabilization of Mdm2
protein by MdmX. B, extracts from transfected U2OS cells
were used in Western blot analysis to monitor MdmX-(1-466) and ARF
proteins levels. The stabilization of Mdm2 observed following the
coexpression of ARF is not affected by an NrLS-deficient MdmX.
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Fig. 5.
Expression of ARF and MdmX does not alter
protein stability. U2OS cells were transfected with cDNAs
encoding: A, V5-tagged human MdmX and increasing amounts of
ARF·GFP; or B, ARF·GFP and increasing amounts of
V5-tagged human MdmX. After 24 h, cell extracts were made and
Western analysis was performed to determine ARF and MdmX protein
levels. The protein stability levels of MdmX and ARF are not
altered by one another.
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Fig. 6.
p53 transactivation represents a balance of
MdmX/Mdm2 and ARF. p53 transactivation levels in U2OS cells
transfected with mdm2, mdmX, and ARF
expression vectors. p53 transactivation was monitored by transfecting
the PG13-luc (PG) luciferase reporter plasmid.
A, cotransfection of mdmX and mdm2
expressed results in a decrease in p53-dependent
transactivation. The addition of ARF expression vector increases p53
transactivation in the presence of constant MdmX and Mdm2.
B, increasing amounts of mdmX expression vector
results in a dose-dependent reversal of the increase in p53
transactivation induced by ARF. MG15-luc reporter activity
was not affected by overexpression of ARF (data not shown). Relative
p53 transactivation represents the p53 induction of luciferase activity
normalized to the transfection efficiency ( -galactosidase
assays).
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ABSTRACT
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Fig. 7.
Model of MdmX, Mdm2, and ARF nuclear
interactions. Based on results from these studies and previous
reports (22-24), the effects of ARF on Mdm2 and MdmX nuclear
localization and effects on p53 stability are described. Additionally,
the observations concerning Mdm2 and MdmX with respect to p53 stability
and transactivation (TA) are detailed.
Because the ability of MdmX to stabilize Mdm2 (Fig. 7A) is lost upon coexpression of ARF, we predict that the existence of an equilibrium between MdmX·Mdm2, Mdm2·ARF, and MdmX·ARF complexes may ultimately balance the levels of both Mdm2 and p53 (Fig. 7, B and C). Clearly a key factor in this effect is the ratio of MdmX and Mdm2 protein. It is worth noting that the MdmX protein has a much greater half-life than Mdm2; therefore, it is likely that in these present studies when cotransfecting equal molar amounts of mdm2 and mdmX plasmids, the MdmX protein concentration is significantly higher than that for Mdm2 protein. Studies comparing the various binding affinities between the Mdm2, MdmX, and ARF heterodimeric complexes are currently ongoing. At the cellular level we speculate that in tissues such as the thymus, brain, and testis, where higher levels of mdmX transcripts have been reported relative to other tissues (28, 29), that ARF-MdmX interactions (Fig. 7C) may play a role consistent with those observed in these overexpression studies.
Taken together, the observations detailed here support a model whereby MdmX is responsible for maintaining the nucleoplasmic localization of Mdm2 and p53 in actively dividing cells. During a DNA-damaging event in which p53 and Mdm2 would become induced, MdmX would likely be of little consequence because reports from our laboratory and others have shown that MdmX proteins are not induced following DNA damage (27, 28). However, induction of ARF by oncogenic stimuli, such as deregulated Myc expression (31), and its subsequent regulation of Mdm2 may be affected differentially in tissues expressing high levels of MdmX. The resulting antagonistic effect of MdmX on ARF may weaken the ability of ARF to sequester Mdm2, resulting in more rapid Mdm2 and p53 turnover and an inactivation of p53 function. Consistent with this hypothesis, a small percentage of human gliomas contain an amplified mdmX gene without p53 mutation or mdm2 gene amplification. Furthermore, gliomas containing amplified mdmX showed no accumulation of p53 or Mdm2 protein (32). We are presently exploring the possibility that ARF-MdmX interactions in these tumors produce this malignant phenotype.
Although our original model outlined MdmX as an inhibitor of
Mdm2-mediated degradation (22), the data presented here argue that MdmX
together with ARF may act antagonistically to allow more rapid Mdm2
turnover (Fig. 7). Most likely, both mechanisms are utilized to
maintain steady state levels of Mdm2 and p53 in normal cells. It is
also possible that the interaction between MdmX and ARF affects
pathways other than those relating to p53 and Mdm2. In fact,
exogenously expressed ARF can induce a cell cycle arrest in triple
knock-out cells lacking p53, mdm2, and ARF (33). Interestingly, the
Mdm2 binding domain of ARF, amino acids 1-14, is required for ARF to
induce an arrest in a p53/Mdm2 null background. Although it is
tempting to speculate that ARF interacts with MdmX or similar Mdm2-like
proteins to reverse the inhibition of a yet unidentified protein,
further studies into the effects of ARF-MdmX interactions in the
absence of Mdm2 remain to be completed.
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ACKNOWLEDGEMENTS |
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We thank Dr. Madhavi Kadakia for reviewing the manuscript and providing us with the V5-MdmX expression vector.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA64430 (to S. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the Biomedical Sciences Ph.D. program. Present address: Dept. of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., NB20 Cleveland, OH 44195.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Wright State University, 3640 Colonel Glenn Hwy., Dayton, OH 45435. Tel.: 937-775-4494; Fax:
937-775-3730; E-mail: steven.berberich@wright.edu.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M010685200
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ABBREVIATIONS |
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The abbreviations used are: Rb, retinoblastoma protein; NrLS, functional nucleolar localization signal; GFP, green fluorescent protein.
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