From the Lautenberg Center for General
and Tumor Immunology, The Hebrew University Hadassah Medical School,
Jerusalem 91120, Israel and § The Rockefeller University,
New York, New York 10021
Received for publication, September 28, 2000
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ABSTRACT |
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The p53 protein plays a key role in the cellular
response to stress by inducing cell growth arrest or apoptosis. The
polyproline region of p53 has been shown to be important for its growth
suppression activity. p53 protein lacking the polyproline region has
impaired apoptotic activity and altered specificity for certain
apoptotic target genes. Here we describe the role of this region in the regulation of p53 by its inhibitor Mdm2. p53 lacking the polyproline region was identified to be more susceptible to inhibition by Mdm2.
Furthermore, the absence of this region renders p53 more accessible to
ubiquitination, nuclear export, and Mdm2-mediated degradation. This
increased sensitivity to Mdm2 results from an enhanced affinity of Mdm2
toward p53 lacking the polyproline region. Our results provide a new
explanation for the impaired growth suppression activity of p53 lacking
this region. The polyproline region is proposed to be important in the
modulation of the inhibitory effects of Mdm2 on p53 activities and stability.
The p53 tumor suppressor has been implicated in the prevention of
many types of cancer (1). In response to various stress signals, such
as DNA damage, wild type
(wt)1 p53 promotes cell cycle
arrest (2). However, in response to certain oncogenic changes, or when
the stress is excessive, p53 can induce apoptotic cell death (3). The
apoptotic activity of p53 is mediated by both
transactivation-dependent and -independent pathways that
cooperate to mediate a full apoptotic response (3, 4). Several p53
apoptotic target genes have been identified, including bax,
Fas/Apo-1, KILLER/DR5, IGF-BP3,
PAG-608, PIG3, PERP, and MCG10 (Ref. 3
and references therein; Refs. 5 and 6). However, each of these target
genes contribute only partially to the overall apoptotic response
mediated by p53 (for reviews, see Refs. 3 and 4). Hence,
multiple apoptotic pathways presumably operate in parallel.
Little is known about the transactivation-independent apoptotic
activity of p53. New insights have been provided from studies defining
functional domains responsible for growth suppression. Initially,
Walker and Levine (8) revealed a polyproline region (amino acids
62-91) bearing five partially conserved PXXP motifs that are important for growth suppression. Deletion of these
motifs impairs the ability of p53 to induce apoptosis, without
affecting its ability to induce growth arrest (7-9). Intriguingly, p53 lacking the proline region has altered specificity for endogenous target promoters. There is a decrease in the induction of several apoptotic genes: PIG3, PIG6, PIG11,
p85, and BTG2 (9), most of which were implicated
in the cellular apoptotic response to oxidative stress (10, 11).
However, the induction of other apoptotic genes, such as bax
and KILLER/DR5, is unaffected (9). This proline-rich region
was also shown to be required for p53-dependent cell growth
arrest through Gas-1 (12), a plasma membrane protein highly expressed
during G0. The identifications of a germ line mutation
within the polyproline region (proline 82) in cancer patients with
Li-Fraumeni syndrome (13) and of somatic mutations in bladder tumors
(proline 85 and 89; Ref. 14) are consistent with this region playing an
important role in regulating p53 activity. Moreover, the phenotype
described for p53 lacking the polyproline region is similar to that
observed in tumor-derived p53 mutants (15).
The p53 protein is subject to tight regulation at multiple levels,
including protein stability, post-translational modifications, and
subcellular localization (for reviews, see Refs. 16 and 17). The key
player in p53 regulation is the proto-oncogene mdm2, which
is amplified in a variety of tumors (18). Mdm2 blocks the
transcriptional activity of p53 and its ability to induce growth arrest
and apoptosis (for reviews, see Refs. 18-20). In addition, Mdm2
promotes the degradation of p53 through the ubiquitin-proteasome pathway (21, 22) by acting as an E3-ligase (23). Negative regulation of
p53 by Mdm2 is modulated by specific modifications, such as
phosphorylation (20), or through the action of partner proteins, such
as p19ARF and c-Abl (for reviews, see Refs. 3 and 24).
In this study we examined the role of the proline-rich region of p53 in
the regulation of p53 by Mdm2. We found that the apoptotic and
transcriptional activities of a p53 protein lacking the polyproline region were more susceptible to negative regulation by Mdm2 than its wt
counterpart. In the absence of this region, p53 is more accessible than
wt p53 to ubiquitination and nuclear export, and consequently more
susceptible to Mdm2-mediated degradation. These effects result from an
enhanced affinity of Mdm2 for p53 that lacks the proline region. Our
results support a role for the polyproline region of p53 in modulating
p53 regulation by Mdm2.
Cells and Transfection Assays--
Mouse embryo fibroblasts
(MEFs) were grown in Dulbecco's modified Eagle's medium, and H1299
and Saos-2 cells were grown in Roswell Park Memorial Institute medium
supplemented with 10% fetal calf serum at 37 °C. The Saos-2 cell
line is derived from an osteosarcoma, and the H1299 cell line is
derived from a lung carcinoma; both lines are devoid of any p53
expression. MEFs were derived from p53
The luciferase assay and Western blot analysis were carried out as
previously described (27). The apoptotic assay was carried out
essentially as previously described (26). Samples were analyzed in a
cell sorter (FACSCalibur) using the CellQuest software (Becton Dickinson). The apoptotic fraction was determined by measuring the
number of cells possessing a sub-G1 DNA content
(27).
For pulse-chase analysis, H1299 cells were transfected as indicated.
Twenty-four h post-transfection, cells were metabolically labeled with
100 µCi of [35S]Met plus Cys for 30 min. Cells were
then washed and chased in nonradioactive medium for the indicated
periods. Samples containing the same amount of radioactivity were
subjected to immunoprecipitation with the anti-p53 antibody PAb421 as
previously described (27). Immunocomplexes were resolved by
SDS-polyacrylamide gel electrophoresis and exposed to x-ray
film. The half-life of p53 proteins was quantified by scanning the
autoradiogram using a densitometer (Aida).
For immunofluorescent staining, H1299 cells were plated on glass
coverslips. Twenty-four h post-transfection cells were treated for
4 h with the proteasome inhibitor ALLN (150 µM;
Calbiochem) to prevent the degradation of p53 in the cytoplasm. Cells
were fixed in cold methanol and stained with anti-p53 antibodies
(PAb1801 and DO1) followed by Cy3-conjugated goat anti-mouse secondary antibody. Cells were stained simultaneously for DNA using
4',6-diamidino-2-phenylindole. Stained cells were observed under
the confocal microscope (Zeiss).
The antibodies used were as follows: anti-human p53 monoclonal
antibodies PAb1801, PAb421, and DO1, anti-Hdm2 SMP14 (28), and an
anti- Plasmids--
Expression plasmids were as follows: human wt p53
(pRC/CMV wtp53), human mutant p53 lacking the proline-rich region
(pRC/CMV p53 Ubiquitination Assay in Vivo--
The ubiquitination of p53
in vivo was detected by transfecting H1299 cells with 1.5 µg of hp53 The Inhibition of the Apoptotic Activity of p53
At the next step, we tested whether the impaired apoptotic activity of
p53 Mdm2 Inhibition of Transcriptional Activity Is Greater for
p53
Next we determined whether the differences in the transcriptional
activities between wt p53 and p53 Absence of the Polyproline Region Renders p53 More Susceptible to
Mdm2-mediated Degradation--
Because Mdm2 promotes p53 for
degradation, it was predicted from the above findings that p53
In the second assay, the half-life of p53 p53 The Lack of the Polyproline of p53 Enhances Its Nuclear
Export--
The nuclear export of p53 is essential for its degradation
by Mdm2 (32). Recently it has been shown that this export depends on
the ubiquitination of p53 by Mdm2 (33, 34). Because p53 p53 A p53 protein lacking the polyproline region of p53 has impaired
apoptotic activity (7, 9, 30). This impairment has been correlated with
altered specificity of p53 In this study we searched for an alternative explanation for the
impaired apoptotic activity of p53 The exact contribution of Mdm2-mediated degradation of p53 to the
overall inhibition of p53 by Mdm2 is difficult to assess. It appears
that low ratios of Mdm2 to p53 may be sufficient for inhibiting p53
activities, without promoting its degradation. This notion is
consistent with our findings that the mere presence of endogenous Mdm2
was sufficient to render p53
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
knockout mice
(KO) or from the
p53
/
/mdm2
/
double knockout mice (2KO) (25). Transfections were carried out as
outlined previously (26). The amounts of expression plasmids used in
each experiment are indicated in the corresponding figure legends. To
maintain a constant amount of plasmid DNA in each sample, an empty
vector was added.
-tubulin antibody (DM1A, Sigma).
proAE; Ref. 8), mouse wt mdm2
(pCOC-mdm2 X2; Ref. 27), and human Hdm2 (PCMV-Neo-Bam-Hdm2).
The reporter plasmid used was the bax luciferase
(29).
pro or hp53 expression plasmids alone or together with
the indicated amounts of expression plasmid for mdm2.
Twenty-two h post-transfection, cells were treated with 150 µM ALLN for 4 h. Following treatment, cells were
subjected to nuclear cytoplasmic fractionation. To prepare the
cytoplasmic fraction, the cell pellets were resuspended in cytoplasmic
buffer (10 mM Tris·HCl, pH 8.0, 10 mM KCl).
Cells were allowed to swell for 2 min, and then Nonidet P-40 was added
to 0.6% followed by centrifugation. The supernatant contained the
soluble cytoplasmic fraction. The pellets were washed once more with
the cytoplasmic buffer before proceeding to nuclear fractionation.
Preparation of the nuclear fraction from the remaining cell pellet was
undertaken by resuspending in high salt radioimmune precipitation
buffer (50 mM Tris, pH 8.0, 5 mM EDTA, 400 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, and 0.025%
SDS). The purity of the cytoplasmic fraction was verified by probing
with anti-
-tubulin antibody, whereas that of the nuclear fraction
was verified with anti-histone H2B antibody. Nuclear and cytoplasmic
extracts were subjected to Western blot analysis using the indicated antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pro by
Mdm2 Is Greater than That of wt p53--
Deletion of the
polyproline region of p53 has been shown to impair the apoptotic
activity of p53 (30). Because Mdm2 inhibits the apoptotic activity of
p53 (27, 31), we argued that one possible explanation for this
impairment is that the deletion of the polyproline region of p53
renders it more sensitive to inhibition by Mdm2. This conjecture was
tested by a transient apoptosis assay in Saos-2 cells. Cells were
transfected with expression plasmids for wt human p53 or mutant p53
lacking all five proline motifs (p53
pro; Ref. 8). Seventy-two h
post-transfection, cells were harvested, stained for p53, and subjected
to flow-cytometric analysis (27). Cells with background levels of
fluorescence represent the nontransfected subpopulation (NT
in Fig. 1A), whereas cells
with high fluorescent intensity represent the successfully transfected
subpopulation (T in Fig. 1A). The cell cycle
distribution of each subpopulation was analyzed separately, and the
proportion of cells with sub-G1 DNA content was
determined (Fig. 1B). Expression of wt p53 in these cells
was shown to induce apoptosis in 26% of the transfected subpopulation,
whereas expression of p53
pro induced only 14% apoptosis (Fig. 1,
C-E), which is consistent with previous reports (7, 30). It
should be noted that the expression level of p53
pro was similar, or
even slightly higher, than that of wt p53, as measured by the
fluorescent intensity of the two transfected subpopulations (Fig.
1F). This result demonstrates that under these experimental
conditions the impaired apoptotic activity of p53
pro is not due to
reduced levels of expression, consistent with previous findings (7,
30).
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Fig. 1.
The apoptotic activity of
p53 pro is more sensitive than wt p53 to
inhibition by Mdm2. Saos-2 cells were transfected with 1 µg of
either wt p53 or p53
pro expression plasmid alone or together with
expression plasmid for mdm2. Seventy-two h
post-transfection, cells were harvested, fixed, and stained for p53
using anti-p53 antibodies, DO1 and PAb1801, followed by fluorescein
isothiocyanate-conjugated goat anti-mouse secondary antibody.
Labeled cells were subjected to flow-cytometric analysis. Even numbers
of cells from each population were collected and analyzed separately.
A, fluorescent intensity of the nontransfected
(NT) subpopulation and of the p53-transfected subpopulation
(T). B, the cell cycle distribution of the
nontransfected subpopulation as determined by DNA content. The area of
the apoptotic cells is marked by Sub-G1. Also shown is the
DNA content of cells transfected with wt p53 (C) or
with p53
pro (D). The percentage of apoptotic
subpopulation is shown. E, a summary of triplicates from one
of five independent experiments. F, histogram showing the
fluorescent intensity of wt p53 (dark line) and p53
pro
(light line). Also shown is the DNA content of cells
transfected with wt p53 and mdm2 expression plasmids
(G) or p53
pro and mdm2 (H).
I, relative apoptotic activity of wt p53 and p53
pro alone
or together with mdm2. The extent of apoptosis obtained for
each p53 plasmid alone was taken as 100% relative apoptosis
(black bars), and the residual activity in the presence of 2 µg (gray bars) or 4 µg (white bars) of Mdm2
was calculated relative to this value. Standard errors from three
independent experiments are indicated. J, histogram showing
the fluorescent intensity of wt p53+Mdm2 (dark line) or
p53
pro+Mdm2 (light line).
pro results from an increased sensitivity to Mdm2-mediated inhibition. For this purpose, the apoptotic activities of wt p53 and
p53
pro were compared in the presence of increasing amounts of
mdm2 expression plasmid. Saos-2 cells were transfected with 1 µg of expression plasmid for wt p53 or p53
pro, together with an
expression plasmid for mdm2. In the presence of 2 and 4 µg of mdm2 expression plasmid, the apoptotic activity of wt p53
was reduced by 25 and 50%, respectively (Fig. 1, G and
I), whereas that of p53
pro was reduced by 50 and 80%,
respectively (Fig. 1, H and I). Here too the p53
fluorescent intensities of cells transfected with wt p53 or p53
pro
were identical, even in the presence of Mdm2 (Fig. 1J).
Taken together, these results strongly implicate the involvement of
Mdm2 in the impaired apoptotic activity of p53 lacking the polyproline region.
pro than for wt p53--
The inhibition of p53-mediated
apoptosis by Mdm2 was shown to be largely due to inhibition of the
transcriptional activity of p53 (27, 31). Based on the findings above,
we predicted that the impaired apoptotic activity of p53
pro results
from enhanced sensitivity of its transcriptional activity to inhibition
by Mdm2. To test this prediction, the transcriptional activity of wt
p53 and of p53
pro was compared in fibroblasts that lack p53 and
either express or lack endogenous Mdm2. MEFs derived from
p53
/
KO or from
p53
/
/mdm2
/
2KO were used (25; a generous gift from Dr. G. Lozano). Transcriptional activity was measured using the luciferase reporter plasmid driven under the bax promoter. The induction of the bax
reporter by low levels of p53
pro was previously shown to be impaired
(7). The KO and 2KO MEFs were transfected with the bax
luciferase reporter plasmid together with expression plasmids for wt
p53 or p53
pro. In MEFs expressing endogenous mdm2, the
transcriptional activity of p53
pro was 50% lower than that induced
by wt p53 (Fig. 2A), consistent with previous reports using similar conditions (7). However,
this difference in activity was diminished in MEFs lacking mdm2 expression, where the transcriptional activity
of p53
pro was even slightly higher than that of wt p53 (Fig.
2A). These results support the notion that the impaired
transcriptional activity of p53
pro, at least for some promoters such
as bax, is due to increased sensitivity to inhibition by
Mdm2. The complete absence of transcriptional activity for certain
genes, such as PIG3, involve a loss of DNA binding ability
to the corresponding promoters (7, 9, 15).
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Fig. 2.
Enhanced sensitivity of
p53 pro transcriptional activity to inhibition
by Mdm2. A, Single
(p53
/
) and double knockout
(p53
/
/mdm2
/
)
fibroblasts were transfected with 20 ng of expression plasmid for
either wt p53 or p53
pro together with 500 ng of the bax
luciferase reporter plasmid. Twenty four h post-transfection, cells
were harvested, and the luciferase activity was determined. The
luciferase activity is shown in arbitrary units; the average and S.D.
from three independent experiments are shown. B, one dish
from each type (as indicated) was used to visualize the p53 protein by
Western blot analysis using a mixture of anti-p53 antibodies, DO1 and
PAb1801.
pro in the 2KO line were due to
altered expression of p53
pro. The steady state level of each form of
p53 was measured by Western blot analysis using anti-p53 antibodies.
For this purpose a sample was taken from one of multiple dishes that
were used in the luciferase assay. This analysis revealed that within
each cell line p53
pro was expressed at a similar level, or even
slightly higher than wt p53 (Fig. 2B). The expression levels
of both proteins were reduced in the KO cells due to endogenous mdm2 expression (Fig. 2B). These results support
the notion that the impaired transcriptional activity of p53
pro
results from an increased sensitivity to inhibition by Mdm2 as compared
with wt p53.
pro
would be more sensitive than wt p53 to degradation by Mdm2. In the
assays described above the steady state levels of wt p53 and p53
pro
were found to be equivalent (Figs. 1, F and J,
and 2B). To examine the possibility that p53
pro is more
susceptible to Mdm2-mediated degradation, we used two assays. First, we
compared the steady state levels of wt p53 and p53
pro by using very
low amounts of expression plasmids for each p53 form in the presence or
absence of Mdm2. For this purpose lung adenocarcinoma cells, H1299,
lacking p53 expression were transfected with 50 ng of each p53
expression plasmid alone or together with increasing amounts of
mdm2 expression plasmids (100, 200, or 300 ng). Twenty-four
h post-transfection, cells were harvested and subjected to Western blot
analysis using anti-p53 antibody. When expressed alone, the level of
the p53
pro protein was higher than that of wt p53 (Fig.
3, lanes 1 and 5).
However, in the presence of mdm2 (100 ng) the expression
level of wt p53 was reduced by 52%, whereas that of p53
pro was
reduced by 82% (Fig. 3, lanes 2 and 6). This
reduction was normalized to the total amounts of protein extracts
loaded in the relevant lanes by re-probing the same blot with
anti-
-tubulin.
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Fig. 3.
p53 pro is more
sensitive than wt p53 to Mdm2-mediated degradation. H1299 cells
were transfected with 50 ng of either wt p53 (lane 1) or
p53
pro (lane 5) expression plasmid alone or together with
100 ng (lanes 2 and 6), 200 ng (lanes
3 and 7), or 300 ng (lanes 4 and
8) of expression plasmid for mdm2.
Twenty-four h post-transfection, cells were harvested, and cell
extracts were subjected to Western blot analysis as in Fig. 2. The
amount of p53 in each lane was quantified using a densitometer (Aida).
The amount of protein loaded was monitored by reactivity of the same
blot with anti-
-tubulin. The positions of wt p53, p53
pro, and
-tubulin are marked by arrows.
pro and wt p53 was
compared. H1299 cells were transfected with expression plasmids for wt
p53 or p53
pro. Twenty-four h post-transfection, cells were
metabolically labeled with [35S]Met/Cys and then chased
in nonradioactive medium for 0, 1, 3, or 6 h. The p53 proteins
were precipitated from each cell extract and subjected to
SDS-polyacrylamide gel electrophoresis analysis. At 0 h chase
time, the amount of p53
pro was similar to, or even higher than, that
of wt p53, hence excluding the possibility that the p53
pro protein
is synthesized at a reduced rate. However, with increasing chase time,
the amount of radioactively labeled wt p53 was elevated (Fig.
4, lanes 3 and 5),
consistent with previous findings (21), suggesting that the half-life
of exogenous wt p53 was longer than 6 h. By contrast, the amount
of radioactively labeled p53
pro increased after a 1-hour chase, and
thereafter the levels decreased. Thus, the half-life of p53
pro is
significantly shorter than that of wt p53 (Fig. 4). Taken together,
these two assays demonstrate that p53
pro is less stable than wt p53
and is more sensitive than wt p53 to degradation by Mdm2.
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Fig. 4.
The p53 pro protein
is less stable than wt p53 protein. H1299 cells were transfected
with 1 µg of expression plasmid for either wt p53 or p53
pro.
Twenty-four h post-transfection, cells were labeled for 30 min
and then chased in nonradioactive medium for the indicated periods.
Cell extracts containing equal amounts of radioactivity were used for
immunoprecipitation of p53 using the anti-p53 antibody PAb421.
Immunocomplexes were subjected to SDS-polyacrylamide gel
electrophoresis and then exposed to film. The amount of radioactively
labeled p53 was measured using a densitometer.
pro Is More Susceptible to Ubiquitination than Is wt
p53--
The findings that p53
pro is less stable than wt p53 (Fig.
4) raised the possibility that p53
pro is more susceptible to
ubiquitination than is wt p53. This possibility was tested in an
in vivo ubiquitination assay. It has recently being reported
that p53 undergoes ubiquitination in the nucleus (33). Therefore, the
extent of ubiquitination of wt p53 and p53
pro was examined in the
nuclear and cytoplasmic fractions. To test the effect in
vivo, H1299 cells were transfected with expression plasmids for wt
p53 or p53
pro. Twenty-four h post transfection, cells were treated
with ALLN for 4 h, to prevent p53 degradation, prior to harvest.
Extracts from nuclear and cytoplasmic fractions were subjected to
Western blot analysis using anti-p53 antibody (PAb421). p53 bands of
molecular weight larger than p53 and p53
pro represent p53-ubiquitin
conjugates (Fig. 5A,
lane 3). These bands do not appear in the absence of ALLN
and were previously shown to contain ubiquitin molecules by
coimmunoprecipitation assay using ubiquitin-hemagglutinin tag
(data not shown). Effective ubiquitination of p53
pro but not of wt
p53 was observed (Fig. 5A, lanes 1 and
3). This ubiquitination is believed to be mediated by Mdm2.
Attempts to measure the effect of exogenous Mdm2 on the ubiquitination
of p53
pro failed because of its high sensitivity to Mdm2-mediated
degradation (data not shown) even in the presence of ALLN, as observed
above (e.g. Figs. 3 and 6).
Interestingly, the ubiquitination was confined almost exclusively to
the nuclear fraction. This result supports the notion that p53
pro is
more susceptible to ubiquitination than is wt p53 and that the
ubiquitination of p53
pro occurs largely in the nucleus.
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Fig. 5.
Enhanced ubiquitination of p53 lacking the
polyproline region. H1299 cells were transfected with 1.5 µg of
expression plasmid for either wt p53 or p53 pro. Twenty-two h after
transfection, cells were treated with ALLN for 4 h before nuclear
(N) and cytoplasmic (C) fractions were prepared
and subjected to Western blot analysis using anti-p53 antibody. The
positions of wt p53 (lanes 1 and 2) and p53
pro
(lanes 3 and 4) are indicated by
arrows, and the position of p53-ubiquitin conjugates is also
indicated (Ub-p53). The positions of the molecular mass
markers in kDa are shown.
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Fig. 6.
p53 pro is more
cytoplasmic than wt p53. H1299 cells were transfected with
p53
pro or wt p53. Twenty four h after transfection, cells were
treated with ALLN before being stained for p53 (A,
right-hand side) using anti-p53 followed by Cy3-conjugated
secondary antibody. The nuclei were visualized by staining the DNA
using 4',6-diamidino-2-phenylindole (A,
left-hand side). Stained cells were examined under the
confocal microscope and photographed at × 800 magnification.
B, a summary of p53 localization in stained cells (over 300 cells for each p53). The staining phenotype was categorized into two
groups: nuclear staining (dark bars) or nuclear plus
cytoplasmic staining (light bars).
pro is more
sensitive than wt p53 to ubiquitination, the possibility that p53
pro
is being shuttled to the cytoplasm more efficiently than wt p53 was
raised. This notion was tested by immunofluorescent staining of H1299
cells transfected with expression plasmids for p53
pro or wt p53. To
prevent p53 degradation, cells were treated with ALLN for 4 h.
Fixed cells were stained for p53 using anti-p53 antibodies (PAb1801 and
DO1), followed by Cy3-conjugated secondary antibody. The nuclei were
visualized by staining the DNA with 4',6-diamidino-2-phenylindole. In the great majority of cells the wt p53 protein expression was confined to the nucleus (Fig. 6). By contrast, in a significant
proportion of cells the p53
pro was expressed both in the cytoplasm
and nucleus (Fig. 6). A summary of p53 localization in several hundred
transfected cells is presented in Fig. 6B. Presumably, this
difference is underestimated because of the impaired induction of
endogenous Mdm2 by p53
pro (9). The effect of exogenous Mdm2 on this
distribution was difficult to assess because of the high sensitivity of
p53
pro to degradation by Mdm2, even in the presence of ALLN (data
not shown). This result suggests that the lack of the polyproline of
p53 increases its nuclear export.
pro Has Enhanced Binding Affinity to Hdm2--
The overall
increased sensitivity of p53
pro to Mdm2 suggested that p53 lacking
the polyproline region may be more accessible to binding by Mdm2. This
notion was tested by comparing the binding of human Mdm2 (Hdm2) to wt
p53 or p53
pro using a coimmunoprecipitation assay. H1299 cells were
transfected with each expression plasmid alone or with Hdm2 together
with each p53 expression plasmid. Twenty-two h post-transfection, cells
were incubated with ALLN (150 µM) for 2 h to protect
p53 from degradation, before cell extracts were prepared.
Immunocomplexes were identified by immunoprecipitation of p53 using
anti-p53 antibody (PAb421), followed by Western blot analysis using
anti-Hdm2 antibody (SMP14). As shown in Fig. 6A, Hdm2 bound
stronger to p53
pro than to wt p53 despite the higher expression of
the latter. When the binding was normalized to the amount of p53
proteins expressed, Hdm2 bound p53
pro 10-fold stronger than wt p53.
These results suggest that the absence of the proline-rich region of
p53 enhances the binding affinity of p53 to Hdm2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pro for certain promoters (7, 9). Notably,
Zhu et al. (9) have shown that p53
pro has reduced ability
to induce certain endogenous apoptotic target genes, including
BTG2, p85, PIG3, PIG6, and
PIG11 (10, 11, 35). However, several lines of evidence argue
for an additional explanation for the impaired apoptotic activity of
p53
pro. First, a direct contribution of these apoptotic genes to the
apoptotic response of p53 has not yet been demonstrated. Second, the
induction of several other p53 apoptotic target genes, such as
KILLER/DR5, BAX, PIG2, and
PIG7, is not affected by the deletion of the proline-rich
region (9). Third, deletion of the corresponding region in p73
did
not affect the induction of PIG3 (15). Fourth,
despite the phenotypic similarity between tumor-derived p53 mutants and
p53
pro, very few mutants have been identified in this region.
Therefore, it appears that the differential regulation of certain p53
apoptotic target genes provides only a partial explanation for the
impaired apoptotic activity of p53
pro.
pro. The high sensitivity of p53
to its negative regulator Mdm2 prompted us to ask whether the
polyproline region of p53 affects its regulation by Mdm2. Indeed, the
Mdm2 inhibition of the apoptotic activity is greater for p53
pro than
for wt p53 (Fig. 1). Similarly, Mdm2 inhibition of the transcriptional
activity is greater for p53
pro than for wt p53 (Fig. 2). These
differences are likely to have been underestimated because the
induction of endogenous Mdm2 by p53
pro is ~30% that of wt p53
(9). This differential induction of mdm2 is consistent with
the findings that the steady state level of the p53
pro protein appears to be higher than that of the wt p53 protein (Figs. 2 and 3).
These findings provide an additional explanation for the impaired
growth suppression activity of p53
pro observed in previous studies
(7-9, 30). Furthermore, our findings help to explain the low frequency
of mutations within the polyproline region of p53 in tumors. Such
mutations are expected to have increased sensitivity to Mdm2-mediated
degradation, and consequently are unlikely to be selected during tumor development.
pro less active than wt p53 (Fig. 2) and
more susceptible to nuclear export and ubiquitination than wt p53 (Fig.
5), although its presence was insufficient for promoting
p53
pro for degradation (Figs. 1-4). Interestingly, almost all the
detected ubiquitination of p53 was observed in the nucleus. The
requirement for the ubiquitination of p53 in the nucleus for its
nuclear export (33, 34) is consistent with the enhanced shuttle of
p53
pro to the cytoplasm (Fig. 6), providing further support for the
link between the ubiquitination and nuclear export of p53 (32, 33). On
the other hand, in the presence of high ratios of Mdm2 to p53, as
achieved by the addition of exogenous Mdm2, p53
pro was more
sensitive than wt p53 to Mdm2-mediated degradation (Fig. 4).This
increased sensitivity to Mdm2 inhibitory effects is explained by the
enhanced binding of p53
pro to Mdm2, as compared with wt p53 (Fig.
7).
View larger version (60K):
[in a new window]
Fig. 7.
Enhanced binding of Mdm2 to
p53 pro. H1299 cells were transfected with
the combination of expression plasmids as indicated. Twenty-two h
post-transfection, cells were treated with 150 µM ALLN
for 2 h. Cell extracts were prepared, and the extent of p53-Mdm2
binding was monitored by immunoprecipitation (IP) of p53
using the anti-p53 antibody PAb421, followed by immunoblot analysis
(IB) using the anti-Mdm2 monoclonal antibody SMP14
(A). Aliquots of cell extracts were taken prior to
immunoprecipitation and were subjected to immunoblot using the anti-p53
antibodies DO1 and PAb1801 (B) or the anti-Hdm2 antibody
SMP14 (C). The positions of Hdm2, p53, and p53
pro are
shown by arrows.
It is not clear at this stage why p53pro binds Mdm2 with higher
affinity than does wt p53. The polyproline region may be involved
directly or indirectly in Mdm2 binding. This region may serve as an
anchor for a third partner, presumably an SH3-containing protein, which
may modulate the interaction between p53 and Mdm2. In the absence of
the polyproline region the putative protein would be unable to bind
p53, and consequently would be unable to modulate Mdm2 binding to p53.
Further studies aimed at identifying proteins interacting with the
polyproline region of p53 are required to test this conjecture.
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ACKNOWLEDGEMENTS |
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We thank M. Oren for the generous gift of plasmids, D. Lane for the generous gift of antibody, and G. Lozano for the generous gift of MEFs. We are grateful to S. Moody-Haupt for critical comment and to S. Coen for technical help.
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FOOTNOTES |
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* This work was supported by the Center for Excellence Grant of the Israel Science Foundation and by the Research Career Development Award from the Israel Cancer Research Fund awarded to Y. H.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.
¶ To whom correspondence should be addressed. Tel.: 972-2-6757103; Fax: 972-2-6424653; E-mail: haupt@md.huji.ac.il.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M008879200
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ABBREVIATIONS |
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The abbreviations used are: wt, wild type; MEF, mouse embryo fibroblast; KO, knockout mice; 2KO, double knockout mice; Hdm2, human Mdm2.
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