From the Department of Biochemistry and Molecular
Biology and the § Molecular Medicine Division,
Department of Medicine, Oregon Health and Science University,
Portland, Oregon 97239
Received for publication, September 4, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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It has been shown that p300 binds to MDM2 and
leads to down-regulation of the p53 function. However, it remains
unclear whether the acetylase activity of p300 is necessary for
regulating MDM2 stability. In this study, we address this issue. First,
p300 did not acetylate MDM2 in solution and in cells. Second,
overexpression of p300 in cells increased the level of the MDM2 protein
but not its mRNA. Similarly, the acetylase-defective p300 AT2
mutant stabilized the MDM2 protein as well. Consistently, the
deacetylase inhibitor, trichostatin A, did not significantly affect the
half-life of the endogenous MDM2 protein, whereas p300 enhanced the
half-life of MDM2. Finally, both wild type and acetylase-defective
mutant p300 proteins associated with MDM2 in nuclear body-like
structures where MDM2 might be protected from proteasomal degradation.
Thus, these results suggest that p300 appears to stabilize MDM2 by
retaining this protein in a specific nuclear structure rather than by
acetylating it.
p300 and CBP1 are the
two acetylase-possessing transcriptional co-activators that have been
shown to mediate transcription by many different transcriptional
activators (1). They also regulate the function of the tumor suppressor
p53 protein and its homologs (2-8). It may be this regulation that
contributes to the role of p300/CBP in cell growth control and
neoplasia (Ref. 12 and references therein). Although p300 and CBP,
encoded by two different genes (9-11), share a significant homology in
their functional domains with similar biochemical functions, their
roles in development and neoplasia are not redundant (12, 13),
suggesting that each of them is essential for cell growth control.
Importantly, the intrinsic acetylase activity of these co-activators is
crucial for their regulatory function (14, 15). For instance, by
acetylating p53, p300/CBP stimulates its ability to bind to DNA in a
sequence-specific fashion in vitro (14), stabilizes its
protein (16), and enhances its transcription in cells (2-4, 17, 18).
Indeed, DNA damage signals stimulate p53 acetylation by p300 (15). This
effect can be reversed by deacetylases such as histone deacetylase 1 (HDAC1) and Sir2 p300 has also been shown to interact with the p53 negative regulator
MDM2 (5, 23), whose oncogenic activity is mainly attributed to its
ability to down-regulate p53 function (24). MDM2 possesses an E3-like
ubiquitin ligase activity and thus ubiquitinates p53 (25), leading to
its degradation (26, 27). MDM2 also binds to p53 when this
transcriptional activator resides at the promoter regions of its target
genes (28, 29), blocking p53-dependent transcription (24,
30). Because MDM2 itself is transcriptionally induced by p53, it serves
as a negative feedback regulator of this activator (31, 32).
Intriguingly, by binding to MDM2, p300 assists this protein in
degrading p53 (23). Though seemingly contradictory to the fact that p53
is activated by p300, as discussed above, the enhancement of
MDM2-mediated p53 degradation by p300 appears to be correlated with the
observation that MDM2 forms a ternary complex with p300 and p53 (33)
and inhibits p300-catalyzed p53 acetylation (16, 33). Reasonably,
inhibition of p300-catalyzed p53 acetylation would favor MDM2-mediated
p53 ubiquitination and, thus, degradation, because these enzymes target
the same set of lysine residues at the C-terminal domain of p53 (14,
15, 34). Hence, the MDM2-p300 interaction leads to down-regulation of
p53 function.
However, it is unclear whether p300 requires its acetylase activity to
assist MDM2 in degrading p53. We recently found that overexpression of
p300 increased the level of MDM2 (Fig. 2). This phenomenon was also
reported by others (35). Furthermore, our study as presented here shows
that p300 does not need its acetylase activity to stabilize MDM2.
First, p300 apparently did not acetylate MDM2 in solution and in cells.
Second, overexpression of p300 in cells increased the level of the MDM2
protein, but not its mRNA, and led to elongation of the half-life
of MDM2. Similarly, the acetylase-defective p300 AT2 mutant stabilized
the MDM2 protein as well. Consistently, the deacetylase inhibitor,
trichostatin A (TSA), did not significantly affect the half-life of the
endogenous MDM2 protein. Moreover, both wild-type and
acetylase-defective mutant p300 proteins recruited MDM2 to nuclear
body-like structures where MDM2 might be protected from targeting by
nuclear proteosomes. Thus, these results demonstrate that p300
stabilizes MDM2 by retaining this protein in a specific nuclear
structure rather than by acetylating it.
Cell Culture--
Human lung small cell carcinoma H1299 cells,
human embryonic kidney epithelial 293 cells, human astrocytoma SJSA
cells (American Type Culture Collection) and mouse p53 null embryonic
fibroblast (MEF) cells were cultured as previously described (28,
52).
Buffers--
Lysis buffer consisted of 50 mM
Tris/HCl (pH 8.0), 0.5% Nonidet P-40, 1 mM EDTA, 150 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride. Buffer C 100 (BC100) included 20 mM
Tris/HCl (pH 7.9), 0.1 mM EDTA, 10% glycerol, 100 mM KCl, 4 mM MgCl2, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, and 0.25 µg/ml pepstatin A. 1× SSC consisted of 0.15 M NaCl and 15 mM sodium citrate (pH 7.0).
Antibodies and Reagents--
The monoclonal anti-FLAG antibody
was purchased from Sigma. The monoclonal anti-p53 antibody Pab421 and
the polyclonal or monoclonal anti-MDM2 antibody (4B11 and 2A10,
respectively) were described previously (5). Polyclonal anti-PCAF
antibodies (H-369 and FL-393) were purchased from Santa Cruz
Biotechnology. Monoclonal anti- Purification of Recombinant p300, p300-AT2, PCAF-HAT, MDM2,
GST-MDM2, and p53--
p300 and p300-AT2 were purified from
baculovirus-infected SF9 insect cells using immunoaffinity columns as
described (5, 6). His-p53 was purified from bacteria using a
nickel-nitrilotriacetic acid column as described (5, 6). MDM2 was
purified using an immunoaffinity column, and GST-MDM2 was purified
using the previously described method (5).
Transient Transfection and Western Blot Analysis--
H1299
cells (60% confluence in a 60-mm plate) were transfected with
pCDNA3-HA-MDM2 (0.5 µg) alone or together with pCDNA3-FLAG-p300 or
pCDNA3- FLAG-p300AT2 (see the legends for Figs. 1 and 2 for the
amount of p300 plasmids used). 36 h post transfection,
cells were harvested for preparation of whole cell lysates. Whole cell lysates containing 100 µg of protein were directly loaded onto an SDS
gel, and proteins were detected by ECL reagents (Bio-Rad) after Western
blotting using antibodies, as indicated in the legends for Figs.
1-4.
In Vitro p53 or MDM2 Acetylation Reaction--
p53 or MDM2
acetylation assays were carried out according to the published method
(7, 33). 20 µl of reaction mixture contained 50 mM
Tris-HCl (pH 8.0), 10% glycerol (v/v), 0.1 mM EDTA, 1 mM dithiothreitol, 10 mM sodium butyrate, 0.2 µCi [3H]acetyl-CoA or 500 nM acetyl-CoA
(Sigma), 50 ng p53, p300, p300AT2, PCAF-HAT, p53, MDM2 or GST-MDM2 (see
the legends for Figs. 1 and 2 for the amounts of the proteins, except
for p53, used in each reaction). The mixture was then incubated at
30°C for 60 min and analyzed on SDS-PAGE afterward. Acetylated p53 or
MDM2 was detected by autoradiography. MDM2 and p53 were detected by
Western blot using monoclonal anti-p53 antibody 421 and polyclonal
anti-MDM2 antibody.
In Vivo Acetylation Labeling--
The acetylation in cells (60%
confluence in a 10-cm plate) was performed according to the published
method with minor modifications (53). Human H1299 cells were
transfected with the MDM2 expression plasmid alone or together with the
p300 or p300-AT2 plasmid. 30 h after transfection, the cells were
pretreated with cycloheximide (CHX) (5 µg/ml, Sigma) for 1 h and
then incubated in Dulbecco's modified Eagle's medium containing 0.5 mCi/ml [3H]sodium acetate (Amersham Biosciences) and 5 µg/ml CHX for 1 h at 37°C before cell lysates were prepared.
Immunoprecipitation with the lysates and antibodies against MDM2 was
carried out, and immunoprecipitated proteins were analyzed by SDS-PAGE
and detected by autoradiography for 2 months. MDM2 was detected by Western blotting.
Northern Blot Analysis--
Northern blot analysis was conducted
as described (54). Total RNA was isolated from transfected H1299 cells
using the Trizol reagent (Invitrogen). 15 µg of RNA were
loaded onto a 1.5% agarose gel and transferred to a
nitrocellulose membrane. The membrane was exposed to UV light in a UV
cross-linker (Fisher) and incubated with 32P-labeled
cDNA probes encoding human MDM2 at 42°C overnight. After washing
with 4× SSC once and 1× SSC twice, the blot was exposed to x-ray film overnight.
Analysis of MDM2 Half-life in Cells--
H1299 cells were
transfected with the MDM2 plasmid alone or together with the p300 or
p300-AT2 expression plasmid as described above. 30 h after
transfection, the cells were treated with TSA or
Me2SO for 6 h. Separately, mouse p53 null
embryonic fibroblast cells, human HEK, and H1299 cells were also
treated with TSA or Me2SO for 6 h. Then, all the cells
were treated with 50 µg/ml CHX and harvested at different time points
afterward for the preparation of cell lysates. Lysates containing 100 µg of proteins were loaded directly onto an SDS gel for Western blot
analysis with antibodies against MDM2. The level of MDM2 from the
transfected cells was quantified by scanning the blot and using the
Adobe Photoshop program and plotted with the Cricket Graph (Fig.
3A). These experiments were repeated twice.
Construction and Use of p300 Adenoviruses--
A wild-type human
p300 cDNA and the AT2 mutant lacking histone acetyltransferase
activity (7, 37) were used to generate recombinant adenoviruses with
the adenase Vector Kit (Quantum Biotechnologies). Each p300 cDNA
was first cloned into a modified shuttle plasmid containing a
tetracycline-inducible promoter using standard molecular biological
manipulations. Each pShuttle:TetR:p300 recombinant plasmid was
linearized and recombined with the pedally plasmid in Escherichia
coli strain BJ5183 as described by the manufacturer. Five
micrograms of purified pAdEasy:TetR:p300 and pAdEasy:TetR:p300AT2
plasmids were then digested with PacI endonuclease and
transfected into low passage HEK293 cells using FuGENE-6 at a ratio of
1 µg of plasmid DNA per 5 µl of FuGENE 6. After 2 days, the
transfected cells were replated in 6-well dishes, and growth medium
containing 1.25% Seaplaque Agarose was added 1 day later to promote
the formation of recombinant viral plaques, as described in the
manufacturer's protocol. Approximately 17 days later, individual plaques were picked, amplified in the HEK293 cells, and purified over a
discontinuous CsCl gradient as outlined in the supplier's protocol.
Cells were co-infected with approximately equal amounts of two
recombinant adenoviruses, one encoding a tetracycline-inhibited transactivator (Ad:tTA), and the other either Ad:GFP, Ad:FLAG-p300, or
Ad:FLAG-p300-AT2. Cells at ~50% confluent density were washed once
with PBS and then incubated for 3 h at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and
an equal mixture of two viruses at a multiplicity of infection (m.o.i.)
that depends upon cell type. The medium was then replaced with fresh
growth medium, and the cells were incubated until confluence before
being used for experiments.
Immunofluorescent Staining and Fluorescent Microscopic
Analysis--
Human astrocytoma SJSA cells containing detectable MDM2
were co-infected with the recombinant adenovirus encoding a
tetracycline-inhibited transactivator (Ad:tTA) and the recombinant
adenovirus harboring GFP, FLAG-p300, or FLAG-p300AT2 at an m.o.i. of
1000. 48 h after infection, cells were fixed for immunofluorescent
staining with monoclonal anti-FLAG antibodies and polyclonal anti-MDM2
antibodies as well as DNA staining with 4',6-diamidino-2-phenylindole
(DAPI). The Alexa Fluor 488 (green) goat anti-mouse antibody and the
Alexa Fluor 546 (red) goat anti-rabbit antibody (Molecular Probes,
Eugene, Oregon) were used for FLAG and MDM2, respectively. Stained
cells were analyzed under the Zeiss Axiovert 25 fluorescent microscope. The infected cells were also harvested for Western blot analysis using
antibodies against p300, MDM2, and p300 Does Not Acetylate MDM2 in Vitro and in Cells--
The
interaction between p300 and MDM2 (33) prompted us to examine whether
MDM2 is also a substrate for this acetylase. To this end, purified
p300, MDM2, and GST-MDM2 were used in a set of in vitro
acetylation assays with purified PCAF-HAT fragment and p53 as controls.
[3H]acetyl-CoA was used as a substrate, and acetylated
proteins were detected by autoradiography. As shown in Fig.
1A, both p300 and the PCAF-HAT
fragment acetylated p53 (lanes 2 and 3) as
expected (33, 36). However, none of these acetylases were able to
acetylate either MDM2 or GST-MDM2 (lanes 4-9). To further
confirm this result, MDM2 was titrated in the same acetylation reaction
setting with increasing amounts of p53 as positive controls. Again, p53
was acetylated by p300, but not by the p300 AT2 mutant (7), in a
dose-dependent fashion (lanes 1-5 of Fig.
1B). Although a significant amount of the MDM2 protein was
used in the reaction, no acetylated MDM2 was detected even after an
extended exposure (lanes 6-10 of Fig. 1B). Thus,
these results demonstrate that p300, like PCAF (28), does not acetylate
MDM2 in solution.
To determine whether this observation is also true in cells, we
performed an in vivo acetylation assay by co-introducing
p300 with MDM2 into human small cell lung carcinoma H1299 cells that are free of p53 proteins. Cells were labeled with
[3H]acetic acid 36 h after transfection and
harvested 2 h after labeling in the presence of the deacetylase
inhibitor TSA. Although both MDM2 and p300 were well expressed, we were
unable to detect acetylated MDM2 in the presence of the exogenous p300
(Fig. 1C). Taken together, these data demonstrate that p300
is unable to acetylate MDM2 in solution and in cells.
Wild Type as Well as Acetylase-defective Mutant p300 Stabilize MDM2
in Cells--
We next tested whether p300 affects the level of MDM2 in
cells. To this end, H1299 cells were transfected with plasmids encoding HA-MDM2 and p300 alone or together. Protein levels were detected by
Western blot analysis using antibodies against p300 and HA, respectively. As shown in Fig.
2A, the MDM2 level increased
dramatically when MDM2 was co-expressed with p300. This increase was in
a p300 dose-dependent manner, but it was not due to the
increase of the mdm2 gene transcription by p300, as the
mRNA level of the mdm2 gene did not change significantly
(two bottom panels of Fig.
2A) no matter whether the p300 level was high or low
(top panel). Surprisingly, the
acetylase-defective p300AT2 mutant (37) was also able to induce the
level of MDM2 in a dose-dependent manner (Fig.
2B). Consistent with this result, the deacetylase inhibitor TSA did not significantly affect the half-life of either exogenous (Figs. 2C or 3) or endogenous MDM2 in H1299 cells
(Figs. 2D). This finding was also true in HEK293, p53
null MEF, and Saos2 cells (data not shown). In addition, the
NAD-dependent deacetylase inhibitor vitamin B3 (nicotinic
acid, 5 mM) was without effect on the half-life of MDM2
either (data not shown). Although MDM2 level increased in the presence
of TSA, this increase was inconsistent, as it was not reproducible in
H1299 and several other cell lines including HEK293 and p53 null MEF
cells (Fig. 2D, and data not shown). The stabilization of
MDM2 by p300 appeared to require a direct association between these
proteins, as the MDM2 level did not significantly increase when two
p300 deletion mutants that lack the MDM2-binding domain (amino acids
350-450) (5, 23) were independently co-introduced with MDM2 into H1299
cells (Fig. 2E). These results indicate that p300 does not
need its acetylase activity to increase the level of MDM2.
P300 Extends the Half-life of MDM2, Which Is Not Affected by a
Deacetylase Inhibitor--
To test whether the increase of MDM2 level
is due to its stabilization by p300, the turnover rate of exogenous
MDM2 proteins was determined. H1299 cells were transfected with the
MDM2 expression plasmid alone or together with the p300 expression
vector. 30 h post transfection, cells were treated with TSA or
50% ethanol (used for preparation of TSA). 3 h after TSA
treatment, cells were treated with the translation inhibitor
cycloheximide to stop the synthesis of nascent MDM2 proteins and
harvested at different time points immediately after treatment. As
shown in Fig. 3, the half-life of MDM2
was about 15 min in the absence of exogenous p300, whereas it extended
to ~25 min in the presence of p300. Again, acetylation may not be
involved in this extension, as the deacetylase inhibitor TSA did not
extend the turnover rate of MDM2 further (Fig. 3). Although the
half-life of the endogenous MDM2 was a little bit longer than that of
the exogenous MDM2 (compare Fig. 2, panel C with panel
D), overall it was no longer than 20 min, as tested in three
different cell lines, including H1299, HEK293, and MEF cells (Fig. 2,
C and
D).2 These results
suggest that p300 increases the MDM2 level by stabilizing this protein
and that this stabilization is not through acetylation modification of
MDM2.
Both Wild Type and Mutant p300s Can Associate with MDM2 in Nuclear
Body-like Structures--
Because p300 does not acetylate MDM2, how
may p300 stabilize MDM2? There are two possibilities. First, p300 may
retain MDM2 in certain subcellular structures in order to prevent this
protein from being targeted by the proteosome machinery.
Alternatively, p300 may inhibit MDM2 auto-ubiquitination by binding to
this protein. We tested the first hypothesis by infecting human
astrocytoma SJSA cells, which contain a high level of MDM2 (Fig.
4), with the recombinant adenovirus
encoding GFP, FLAG-p300, or FLAG-p300AT2. 24 h after infection,
cells were either harvested for Western blot to detect p300 and MDM2 or
analyzed by immunofluorescent staining with antibodies against MDM2 or
FLAG. Again, overexpression of both the wild type and mutant p300
proteins, but not GFP, induced the level of the endogenous MDM2 (Fig.
4A). Immunofluorescent staining revealed that p300 and
p300-AT2 proteins co-localized with endogenous MDM2 in the nucleus,
whereas GFP defused throughout the whole cell (Fig. 4B).
Interestingly, when examining individual cells under a higher
magnification lens (×32), we found that MDM2 co-localized exactly with
p300 in nuclear body-like structures, forming focus-like spots (Fig.
4C). By striking contrast, MDM2 in the absence of exogenous
p300 apparently did not display such patterns and was instead evenly
distributed in the nucleus (Fig. 4C, see arrows
and also see the GFP-MDM2 image). Also, the focus-like spots of MDM2
were not seen when SJSA cells were exposed to
The study we described here demonstrates that p300 can stabilize MDM2
by binding directly to this protein and confining it in the focus-like
structure, thus protecting it from targeting by proteosomes. This
protection does not appear to require acetylation, as the
acetylase-defective p300AT2 mutant displayed the same function as the
wild type p300 to stabilize MDM2. The latter observation is not in
favor of the idea that p300 might inhibit MDM2 auto-ubiquitination by
chemically modifying the lysines of MDM2 that are potential target
residues for auto-ubiquitination. Consistently, others have shown that
p300 still enhances MDM2-mediated p53 ubiquitination (35) and
degradation (23, 35), suggesting that p300 may not inhibit the E3-like
ubiquitin ligase activity of MDM2. Nevertheless, it still remains to be
clarified whether p300 may prevent MDM2 auto-ubiquitination by
associating with this protein and causing a steric hindrance without
affecting ubiquitination of other substrates. Noticeably, p300 only
extended the turnover rate of MDM2 by ~10 min. Two possible
interpretations of this observation are either that the association of
p300 with MDM2 may be weak and dynamic so that the latter undergoes
active self-destruction in cells, or that p300 may require other
proteins to further stabilize MDM2.
There are two other mammalian cellular proteins that have been shown to
stabilize MDM2 by direct association. They are p14arf (p19arf for
mouse) (38-41) and MDMx (42, 43). Similar to p300, they all are stable
proteins and not directly regulated by p53. However, the outcomes of
the associations of these proteins with MDM2 and the mechanisms by
which they regulate MDM2 are distinct. P14arf binds to MDM2, retains it
in the nucleolus (39, 40), and inhibits its ubiquitin ligase activity
(44). Thus, p14arf prevents MDM2-mediated p53 degradation and
subsequently activates p53, although it also stabilizes MDM2. In
contrast, MDMx binds to MDM2 through their Ring finger domains (45, 46)
and inhibits its auto-ubiquitination without affecting its ubiquitin
ligase activity on p53, leading to p53 inactivation partially by
stabilizing MDM2 (38, 42, 45). Normally, MDMx locates in the cytoplasm and is transported to the nucleus with the help of MDM2 (47, 48). Thus,
these studies suggest that MDM2 and MDMx act cooperatively to
inactivate p53. This notion is substantially supported by the MDM2-p53
and the recent MDMx-p53 double knockout studies (49-51). Similarly to
MDMx, but unlike p19arf, p300 appears to enhance p53 degradation when
co-expressed with MDM2 (23, 48). However, p300 alone can also stabilize
p53 by acetylating this protein and possibly by preventing
MDM2-mediated p53 ubiquitination, as p300 and MDM2 target the similar
lysine residues of p53 (14, 16, 34). How p300 regulates p53 in two
opposite ways may depend upon the status or existence of MDM2. It is
possible that p300 activates p53 at the very early stage of cellular
response to DNA damage when MDM2 level is low, whereas at the late
stage of this response it may stabilize newly synthesized MDM2 that is transcriptionally activated by p53 and thus assists this ubiquitin ligase to fulfill its negative feedback regulation of p53. This hypothesis is in agreement with the fact that the half-life (10-15 min) of MDM2 is shorter than that (30 min) of p53; thus the former needs to be stabilized, such as by p300, in order to control the latter. Whether and how p14arf and MDMx may be involved in these aspects by interplaying with p300 to modulate p53 stability and function would be an interesting and important question to address for
a better understanding of the p53 regulatory network. Also, whether
CBP, similarly to p300, functions to stabilize MDM2 remains to be
investigated
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(19-22). Thus, up-regulating p53 activity is an
important function for p300 and CBP.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-tubulin antibodies were purchased
from Sigma. The baculovirus harboring human PCAF, p300, or MDM2 was
described previously (5, 6, 28). pCDNA3-HA-MDM2 and pCMV-p53 were
as described (30). PCAF-HAT was generously provided by John Denu
(Oregon Health and Science University, Portland, OR). TSA was purchased
from Calbiochem. pCDNA3-FLAG-p300 and pCDNA3- FLAG-p300AT2
were described previously (7, 37).
-tubulin.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
p300 does not acetylate MDM2.
A, the in vitro acetylation assay. 50 ng of p53
and 300 ng of MDM2 and GST-MDM2 or 200 ng of p300 and PCAF-HAT as well
as 0.2 µCi of [3H]acetyl-CoA were used in this assay.
This autoradiogram was exposed for 1 week. B, titration of
substrates in acetylation reaction. The same acetylation as that
described above was conducted except that different amounts of p53 (10, 30, and 60 ng) and MDM2 (50, 100, and 200 ng) were used here as
substrates. Acetylated proteins were detected by autoradiography
(top panel), and p53 and MDM2 were detected by Western blot
(WB; middle and bottom panels).
C, in vivo acetylation labeling. In
vivo acetylation labeling was carried out as described under
"Experimental Procedures." MDM2 was immunoprecipitated with the
anti-MDM2 antibody and analyzed by SDS-PAGE. The bottom
panel presents the result of autoradiography after 2 months
of exposure of the gel to x-ray film, and the middle
panel shows the MDM2 level. The top
panel shows the levels of FLAG-p300 and FLAG-p300AT2.
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Fig. 2.
Both wild type and mutant p300 proteins
induce MDM2 in cells. A, p300 induces the protein but
not the mRNA level of MDM2 in cells. Transfection followed by
Western blot (WB) and Northern blot (NB) analyses
was performed as described under "Experimental Procedures." The
top three panels show the result of
Western blotting for FLAG-p300, HA-MDM2, and -tubulin, and the
two bottom panels show the result of a
Northern blot for HA-MDM2. B, both wild type and
acetylase-defective mutant p300 proteins induce MDM2 in cells.
Co-transfection of H1299 cells with plasmids encoding HA-MDM2 alone or
together with FLAG-p300 or FLAG-p300AT2 followed by a Western blot was
carried out as described under "Experimental Procedures." 100 µg
of protein were loaded directly onto an SDS gel. The p300 level is
shown on the top panel, the MDM2 level is shown
in the middle panel, and the tubulin level is
shown in the bottom panel. C, TSA does not affect
the half-life of the exogenous MDM2. H1299 cells were transfected with
the HA-MDM2-encoding plasmid and, 48 h post transfection, treated
with 5 µM TSA (bottom panel) or vehicle (50%
ethanol, top panel) for 6 h. Then, cells were treated
with 50 ng of CHX and harvested at different time points as indicated
at the top. Cell lysates containing 100 µg of proteins
were loaded directly onto an SDS gel for Western blot analysis of MDM2
with anti-HA antibodies. D, TSA does not increase the
half-life of the endogenous MDM2. HEK293, H1299, and the p53 null MEF
cells were treated with 5 µM TSA followed by CHX
treatment and harvested at different time points as described above.
Cell lysates containing 100 µg of proteins were loaded onto an SDS gel for
Western blot analysis of MDM2 with anti-MDM2 antibodies. A
representative result with H1299 cells is shown here.
Asterisk indicates a nonspecific band cross-reacting with
the MDM2 antibody. Top panel shows the result
without TSA; bottom panel shows the result with TSA.
E, wild type p300 but not the MDM2 binding-defective
deletion mutant p300 induces MDM2 level. H1299 cells were transfected
with the MDM2 expression vector (0.5 µg) alone or together with
pCDNA3 plasmids encoding p300 (2 µg), Gal-
242-1737 (1× = 1 µg; 2× = 2 µg) or Gal-1945-2424 (1× = 1 µg; 2× = 2 µg). Lysates containing 100 µg of proteins were loaded onto
an SDS gel, and WB was carried out to detect MDM2. Bottom
panel shows a protein loading control.
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Fig. 3.
Both wild type and acetylase-defective mutant
p300 proteins induce the half-life (t1/2) of
MDM2 in cells. Transfection of H1299 cells followed by TSA and CHX
treatments was conducted as described in the Fig. 2 legend. The level
of MDM2 was analyzed by using Western blot with anti-HA antibodies,
quantified by measuring the density of MDM2 signals, and calculated and
plotted using the CA Cricket Graph program. The x-axis
indicates the time after CHX treatment, whereas the y-axis
indicates the arbitrary level of MDM2. Values were also shown in linear
forms, and the half-life of MDM2 under three different conditions is
marked. Formulas for calculating t1/2 are Ymdm2 = 0.009X + 0.302; Ymdm2/p300 = 0.021X + 0891; and Ymdm2/AT2 = 0.021X + 0.883.
-irradiation (Fig.
4D), suggesting that the stabilization of MDM2 by p300 and the co-localization of MDM2 with p300 were not due to a nonspecific and
stressful effect caused by overexpressing p300. Although we observed a
marginal increase of MDM2 3 h after
-irradiation of SJSA cells,
this increase was caused by endogenous and activated p53 in response to
the physical stress (Fig. 4D, top). Thus, in combination with the above data, these results suggest that p300 may
protect MDM2 from proteosome-mediated degradation by keeping MDM2 in
nuclear body-like subnuclear structures.
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Fig. 4.
p300 associates with MDM2 in nuclear
focus-like structures. A, induction of
endogenous MDM2 by p300 and p300AT2 but not by GFP. SJSA cells were
infected with recombinant adenovirus encoding GFP, p300, or p300AT2.
48 h after infection, cells (100 µg protein) were harvested for
Western blot (WB) analysis of p300 and MDM2 with -tubulin
as a control. B and C, immunofluorescent staining
analyses. SJSA cells were infected as described for panel
A. Cells were fixed for immunofluorescent staining as
described under "Experimental Procedures." Viruses encoding GFP,
p300, and p300AT2 used for infection are indicated at the
top. Antibodies used for staining are indicated at the
left. Images were taken under a fluorescent microscope with
a lens of either 10× (B) or 32× magnification.
C, arrows indicate a comparison between the cells
that expressed high and low levels of exogenous p300 proteins.
D, SJSA cells were irradiated with 7 gray of
-ray and
harvested for Western blot analysis with antibodies against MDM2, p53,
and
-tubulin (top) or fixed for immunofluorescent
staining with anti-MDM2 antibodies and 4',6-diamidino-2-phenylindole
(DAPI; bottom) at 0, 1, and 3 h intervals,
respectively. No single focus-like spot of MDM2 was identified in the
nucleus after irradiation. Representative images taken under the 32×
magnification lens is shown in this figure.
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ACKNOWLEDGEMENTS |
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We thank Hunjoo Lee and Yeon-Jin Veach for technique assistance for this study, David M. Keller and Jayme Gallegos for critically reading this manuscript, members of the Lu lab for stimulating discussion, and Drs. John Denu, Zhiming Yuan, and TsoPang Yao for generously providing us with reagents used in this study.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA095441 and CA93614 (to H. L.).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 National Institutes of Health Grant DK42748 and a National Institutes of Health postdoctoral fellowship.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Oregon Health and Science
University, 3181 S. W. Sam Jackson Park Rd. L224, Portland, OR 97239. Tel.: 503-494-7414; Fax: 503-494-8393; E-mail:
luh@ohsu.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M209030200
2 S. X. Zeng, Y. Jin, and H. Lu, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CBP, cAMP-response element-binding protein binding protein; CHX, cycloheximide; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; HAT, histone acetyl transferase; HEK, human embryonic kidney; MEF, mouse3 p53 null embryonic fibroblast; m.o.i., multiplicity of infection; PCAF, p300/CBP-associated factor; PMSF, phenylmethylsulfonyl fluoride; TSA, trichostatin A.
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