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INTRODUCTION |
The p53 tumor suppressor plays a key role in the cellular response
to stress. The p53 protein exists in a latent inactive form with a
short half-life. However, in response to stress the p53 protein
undergoes post-translational modifications and accumulates (1, 2).
Activation of p53 induces cell growth arrest or apoptosis. These
activities are mediated by its target genes, such as
p21waf/cip1 and bax (1, 2).
The activities and stability of the p53 protein are tightly regulated.
A key negative regulator of p53 is mdm2, a cellular proto-oncogene amplified in sarcomas (reviewed in Ref. 3). Mdm2 blocks
the transcriptional activity of p53 and its ability to induce growth
arrest and apoptosis (reviewed in Ref. 3). Importantly, Mdm2 also
promotes the rapid degradation of p53 through the ubiquitin-proteasome
system (4-6). Since mdm2 is a direct target gene of p53, it
shuts down its own expression through a negative autoregulatory
feedback loop (reviewed in Ref. 3).
The activity of p53 is also positively regulated. A physical and
functional link has been demonstrated between p53 and c-Abl, a
nonreceptor tyrosine kinase. Both genes are activated in response to
similar genotoxic stresses (7) and undergo phosphorylation and
activation by DNA-dependent protein kinase or ATM (Ref. 8; reviewed in Ref. 9). This link suggests that the two proteins may act
in a common pathway during the cellular response to DNA damage. c-Abl
binds p53 in vitro (10) and in vivo (11), and consequently the transcriptional activity of p53 is enhanced (10, 12).
p53 is required by active c-Abl to induce cell growth arrest and
apoptosis (10, 11, 13, 14).
This study focuses on the mode of cooperation between c-Abl and p53. We
demonstrate that c-Abl enhances p53 activity through inhibition of
Mdm2-mediated p53 degradation. As a consequence of this neutralization
of Mdm2, the p53 protein is stabilized in an active form. Therefore, we
suggest that by relieving the inhibitory effect of Mdm2, c-Abl can act
as a positive regulator of p53.
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EXPERIMENTAL PROCEDURES |
Cells and Transfection Assays--
Mouse embryo fibroblasts were
grown in Dulbecco's modified Eagle's medium, H1299, and Saos-2 cells
were maintained in RPMI with 10% fetal calf serum. Transfections,
luciferase assay, and Western blot analysis were carried out as
described previously (15). The antibodies used were: anti-human p53
monoclonal antibodies PAb1801, PAb421, and DO1, anti-human Mdm2 SMP14,
anti-c-Abl monoclonal antibody (8E9), and anti-
-tubulin antibody (DM
1A Sigma).
Immunoprecipitation was carried out essentially as described previously
(15). p53 complexes were immunoprecipitated with PAb421, resolved by
SDS-polyacrylamide gel electrophoresis, and subjected to Western
blotting using anti-Mdm2. Flow cytometric analysis was carried out
essentially as described in Haupt et al. (15). Samples were
analyzed in a cell sorter (FACSCalibur, Becton Dickinson).
Plasmids--
Expression plasmids were: human
wt1 p53 (pCMV-Neo-Bam-p53),
mutant p53 (pRCp53Gln14,Ser19 and pRCp53Gln22,Ser23); human wt mdm2 (pCMV-Neo-Bam-mdm2) and human mutant
mdm2 (pCMV-Neo-Bam-hdm2
ring); mouse
mdm2 (pCOC-mdm2-X2; Ref. 15); and mouse wt
c-abl (pCMV-c-abl IV) and kinase defective
c-abl (pCMV-c-abl K290R). All of these cDNAs
were driven under the CMV promoter. The reporter plasmids used were:
cyclin G-luciferase, mdm2 luciferase,
p21 luciferase, and bax luciferase (16).
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RESULTS |
c-Abl Enhances the Transcriptional Activity of p53--
c-Abl can
enhance the ability of p53 to induce the mdm2 promoter and a
synthetic responsive element (10, 11). Here we show that this
cooperative effect holds true for other promoters. H1299 lung carcinoma
cells lacking p53 were transfected with p53 alone or together with
c-abl, and the luciferase reporter gene was driven by either
the cyclin G or the mdm2 promoter (16). Activation of both promoters was increased by c-Abl more than 2-fold
(Fig. 1A). Similar results
were obtained using the p21 and bax promoters
(data not shown). This enhancement of p53 transcriptional activity was
independent of the kinase activity of c-Abl (data not shown),
consistent with previous findings (10, 11).

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Fig. 1.
Cooperation between p53 and c-Abl in the
activation of p53-responsive promoters. A, H1299 cells
were transfected transiently with a reporter plasmid containing the
luciferase gene driven by the cyclin G promoter (panel
I) or mdm2 promoter (Na-Luc; Ref. 16) (panel
II), together with wt p53 alone or p53 plus c-abl. The
amount of plasmid DNA per transfection was 25 ng for p53, 3 µg for
c-abl, and 1 µg for the reporter plasmids. A similar ratio
of these plasmids was used in the experiments described in Figs. 2, 3,
and 5. Luciferase activity is shown in arbitrary units, along with the
standard deviation of triplicates. B, transcriptional
activity of endogenous p53 in c-abl null lines stably
transduced with lacZ (abl / + LacZ) or c-abl (abl / + abl). Cell extracts from equal numbers of
abl / + LacZ,
abl / + abl, and NIH3T3 cells
(positive control for endogenous c-Abl) were subjected to Western blot
analysis using anti-c-Abl antibody (8E9) followed by an
anti- -tubulin antibody (panel I).
abl / + LacZ and
abl / + abl cells were transfected
with the luciferase reporter driven by the p21 promoter
(light shaded columns) or the CMV promoter (dark
shaded columns), and the luciferase activity was determined
(panel II). Standard deviations are indicated.
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We next tested whether this cooperation occurs also at physiological
levels of p53 and c-Abl. This was tested in mouse embryonic fibroblast
cell lines, derived from c-abl null mice, which express either lacZ (abl
/
+ lacZ) or wild type c-abl
(abl
/
+ abl). The expression of
c-Abl protein in the c-abl reconstituted lines was found to
be lower than that of endogenous c-Abl in NIH3T3 control cell line
(Fig. 1B, panel I), confirming that it is within the physiological range. When assayed with a
p21waf1/cip1 luciferase reporter gene, p53 activity
was 2.5-fold higher in the c-Abl reconstituted line than in the
lacZ expressing cells (Fig. 1B). This effect was
independent of c-Abl kinase activity (data not shown). It should be
noted that the luciferase activities of CMV-driven reporter control
were similar in both lines (Fig. 1B, panel II).
These results imply that c-Abl can positively modulate the
transcriptional activity of p53 at physiological levels of both proteins.
c-Abl Stabilizes the p53 Protein--
The mechanism by which p53
transcriptional activation is enhanced by c-Abl (Fig. 1 and Refs. 10
and 11) is yet to be elucidated. Because p53 is regulated largely at
the protein level (reviewed in Refs. 9 and 17), we investigated whether
c-Abl can affect p53 expression level. H1299 cells were transfected
with a low amount of a p53 expression vector either alone or together
with increasing amounts of a c-abl expression vector, and
the steady state expression levels of p53 were determined. The
expression of p53 was elevated by c-abl in a
dose-dependent manner (Fig. 2, lanes 1-3). This effect
was observed at different inputs of p53 plasmids (data not shown). A
similar elevation of p53 by c-Abl was observed in Saos-2, an
osteosarcoma cell line (data not shown), supporting the generality of
this effect.

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Fig. 2.
Elevation of p53 steady state level by
c-Abl. H1299 cells were transfected with human wt p53 expression
plasmid (50 ng) alone (lanes 1 and 4) or together
with 2 µg (lane 2) or 4 µg (lane 3) of
c-abl expression plasmid or with 2 µg (lane 5)
or 4 µg (lane 6) of the kinase-defective mutant
c-abl K290R (abl k). Cell extracts were
subjected to Western blot analysis with a mixture of anti-p53
antibodies (PAb1801 + DO-1) (A). The same blot was reprobed
with anti-c-Abl antibody (8E9) (B) and with an
anti- -tubulin antibody (C).
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The kinase defective c-Abl, c-abl K290R, was also able to
elevate p53 levels in a dose-dependent manner (Fig. 2,
lanes 4-6), although to a lesser extent than that observed
with wt c-Abl (e.g. compare lane 2 versus 5). Thus, c-Abl kinase activity is not essential for
the elevation of p53 expression level, although it may have a
contributory effect, suggesting that c-Abl may elevate p53 expression through more than one mechanism.
c-Abl Blocks Mdm2-mediated Degradation of p53--
Our finding
raised the intriguing possibility that c-Abl elevates p53 level by
overcoming its destabilization by Mdm2. This assumption was tested in
H1299 cells by determining the effect of c-Abl on the ability of Mdm2
to promote p53 degradation. Although the level of the p53 protein was
markedly reduced by Mdm2 (Fig. 3A, panel I,
lane 1 versus lane 2), in the presence of c-Abl the p53
protein was largely protected from degradation (Fig. 3A,
panel I, lane 3). The same results were obtained
in Saos-2 cells (data not shown). Like wt c-Abl, the kinase defective
c-Abl K290R was able to neutralize the promotion of p53 degradation by
Mdm2 (Fig. 3B). Thus, c-Abl protects p53 from
destabilization by Mdm2 in a kinase-independent manner.

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Fig. 3.
c-Abl protects p53 from Mdm2-mediated
degradation in a kinase-independent manner. H1299 cells were
transfected with p53 alone (100 ng) or with various combinations of
mdm2 (500 ng) and c-abl (2 or 4 µg)
(A) as indicated or with the kinase-defective mutant K290R
(abl k) (B). Cell extracts were subjected to
Western blot analysis using antibodies against p53 (panel I)
and then reprobed with anti- -tubulin (panel II).
C, H1299 cells were transfected with p53Gln22,Ser23 alone
(100 ng) (lane 1) or together with c-abl at 2 µg (lane 2) or 4 µg (lane 3). Cell extracts
were analyzed for p53 (panel I) and -tubulin (panel
II).
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The involvement of Mdm2 in the stabilization of p53 by c-Abl was
further examined by using two p53 mutants, p53Gln14,Ser19 and
p53Gln22,Ser23, which do not bind Mdm2 (18) and are resistant to its
destabilizing effect (4, 5). If c-Abl stabilizes p53 by neutralizing
Mdm2, the expression level of these mutants should not be elevated by
c-Abl. Indeed, unlike wt p53, p53Gln22,Ser23 was not stabilized by
c-Abl (Fig. 3C). Similar results were obtained with
p53Gln14,Ser19 (data not shown). These results further support the
notion that c-Abl stabilizes p53 by preventing its degradation by Mdm2.
p53 Binds Mdm2 in the Presence of c-Abl--
The finding that
c-Abl neutralizes Mdm2-promoted p53 degradation raised the possibility
that c-Abl may prevent the interaction between p53 and Mdm2, which is
essential for p53 degradation (4, 5). This conjecture was tested by a
co-immunoprecipitation assay. To increase the sensitivity of this assay
a mutant form of human Mdm2 (Hdm2), Hdm2
RING, lacking the RING
finger domain, was used. This mutant binds p53 without promoting its
degradation (Fig. 4B and Ref.
5), thereby allowing the detection of stable p53-Hdm2 complexes. H1299
cells were transfected with p53 and Hdm2
RING in the presence or
absence of c-Abl. Larger amounts of expression plasmids were used to
obtain amounts of p53 and Hdm2 sufficient for detection. With such
amounts of DNA the p53 protein is stable (4), explaining why further
stabilization by c-Abl is only marginal (Fig. 4B). The
amount of p53-Hdm2 complex was determined by immunoprecipitation using
the anti-p53 antibody PAb421, followed by Western blot analysis with
the anti-Mdm2 monoclonal antibody SMP14. Fig. 4A shows that
Hdm2
RING co-immunoprecipitated with p53 irrespective of the presence
of c-Abl (lane 3 versus lane 4). In fact, the
amount of co-immunoprecipitated Hdm2
RING was even larger in the
presence of c-Abl (Fig. 4A, lane 4), correlating with the elevation in Hdm2
RING expression (Fig. 4D).
Thus, c-Abl does not interfere with Mdm2-p53 complex formation.

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Fig. 4.
Effect of c-Abl on p53-Mdm2 interaction.
A, H1299 cells were transfected with wt p53 alone (1 µg)
(lane 2) or together with Hdm2 RING
(hdm2 R; 2 µg) (lane 3) and c-abl
(4 µg) (lane 4). Cell extracts were subjected to
co-immunoprecipitation using the anti-p53 monoclonal antibody PAb421.
Immunoprecipitated proteins were resolved by 10% SDS-polyacrylamide
gel electrophoresis and subjected to Western blotting using anti-Mdm2
antibody, SMP14. An aliquot of each cell extract before
immunoprecipitation was subjected to Western blot analysis using
anti-p53 (B), anti-c-Abl (C), or anti-Mdm2
(D) antibodies. IP, immunoprecipitation;
IB, immunoblot.
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c-Abl Relieves Mdm2-mediated Inhibition of p53 Activity--
The
significant effect of c-Abl on Mdm2-mediated degradation of p53
prompted us to evaluate its biological consequences. Since c-Abl
prevents the degradation of p53 by Mdm2, it is conceivable that c-Abl
may promote p53 activities even in the presence of Mdm2. This
prediction was tested in two functional assays. First, we determined
whether c-Abl can relieve the inhibitory effect of Mdm2 on the
transcriptional activity of p53. H1299 cells were transfected with a
luciferase reporter gene driven by the cyclin G promoter
together with p53 alone or p53 plus mdm2, and the effect of
c-abl on each plasmid combination was tested. Fig.
5 shows partial inhibition of the
transcriptional activity of p53 by Mdm2, consistent with previous
studies (4). However, this inhibition was entirely alleviated by c-Abl;
in fact, transcriptional activation by p53 was even increased despite
the presence of Mdm2. Thus, the neutralization of the inhibitory effect
of Mdm2 by c-Abl renders p53 transcriptionally active.

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Fig. 5.
c-Abl overcomes the inhibitory effect of Mdm2
on the transcriptional activity of p53. H1299 cells were
transfected with p53 alone (20 ng) or together with the indicated
combinations of mdm2 (70 ng) and c-abl (4 µg),
along with the cyclin G luciferase reporter plasmid (1 µg). Luciferase activity is shown in arbitrary units, along with the
standard deviation of triplicates.
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The ability of c-Abl to overcome the inhibitory effect of Mdm2 on
p53-mediated apoptosis (15, 19) was evaluated in Saos-2 cells, using a
transient transfection assay. The induction of apoptosis by p53 was
reduced more than 2-fold by co-expression of mdm2 (Fig.
6), which agrees with previous studies
using HeLa (15). However, co-expression of c-abl abolished
the effect of mdm2. Under these conditions c-Abl alone does
not affect p53-mediated apoptosis to any significant extent (Fig. 6).
Thus, c-Abl can alleviate the negative effect of Mdm2 on p53-mediated
apoptosis.

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Fig. 6.
c-Abl relieves the inhibitory effect of Mdm2
on p53-mediated apoptosis in Saos-2 cells. Saos-2 cells were
transfected with the indicated plasmid combinations of p53 (1.5 µg),
mdm2 (2.5 µg), and c-abl (4 µg). High amounts
of plasmids were used to enable p53-mediated apoptosis. The effect of
p53 on apoptotic cell death was determined as described previously
(15). Apoptosis induced by p53 alone was taken as 100%. The standard
errors from duplicates are indicated.
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DISCUSSION |
In this study we have investigated the mechanisms underlying the
cooperation between the growth inhibitory proteins p53 and c-Abl. The
synergistic effect of c-Abl on p53 transcriptional activation (10, 11)
has been confirmed and extended to include other physiological p53
responsive promoters. Importantly, this synergy was demonstrated
between endogenous p53 and c-Abl at physiological protein levels, hence
supporting its biological relevance. The enhancement of p53-mediated
transactivation by c-Abl is largely due to the ability of c-Abl to
increase the steady state level of p53 (Fig. 2). This is achieved by
neutralizing Mdm2-mediated degradation of p53 (Fig. 3). This novel role
for c-Abl provides a likely mechanistic explanation for the cooperation
between these two proteins (Fig. 1 and Refs. 10 and 11). This
explanation is supported by the findings that c-Abl neutralizes the
inhibitory effect of Mdm2 on p53-mediated transactivation and apoptosis
(Figs. 5 and 6). However, we cannot exclude the possibility that c-Abl may also enhance the biochemical activity of p53. The neutralization of
Mdm2 is important for extending the time window during which p53
remains functional, especially under conditions where there is no
apparent time lag between the induction of genes mediating growth
suppression and the induction of mdm2 (20). As shown here,
c-Abl neutralizes Mdm2, thereby providing the time delay required for
p53 to exert its biological effects. In this manner, c-Abl can modulate
the extent and duration of the p53 response.
The mechanisms by which c-Abl neutralizes the inhibitory effects of
Mdm2 are unclear. Since c-Abl is mostly nuclear, it may prevent
Mdm2-mediated cytoplasmic shuttling of p53, which is essential for p53
degradation (21). Alternatively, c-Abl may neutralize p53
destabilization by interfering with the degradation process. The
oncogenic Bcr-Abl can promote the ubiquitin-mediated degradation of Abi
(22). As c-Abl and Bcr-Abl have antagonistic effects on cell growth,
they may also have opposite effects on protein degradation.
Neutralizing the inhibitory effect of Mdm2 may prove to be an efficient
and common mechanism by which p53 is regulated. Indeed, recent reports
showed that ARF-INK4a (p19Arf) cooperates with p53 by
overcoming the inhibitory effects of Mdm2 (reviewed in Ref. 23). The
p19Arf pathway is induced by several oncogenes, such as E1A
(Ref. 23 and references therein). In addition, E1A can also stabilize
and activate p53 by selectively blocking the induction of
mdm2 gene expression by p53 (24) as well as by abrogating
the interaction between Mdm2 and p300, which can facilitate p53
degradation (25). It is yet to be determined whether c-Abl interferes
directly with Mdm2 function or whether it employ p19Arf,
p300, or yet another protein to neutralize Mdm2. Nevertheless, this
novel function of c-Abl is believed to be important for its growth
suppression function.