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Molecular and Cellular Biology, March 1999, p. 1751-1758, Vol. 19, No. 3
0270-7306/99
Regulation of p53 Function and Stability by
Phosphorylation
Margaret
Ashcroft,
Michael
H. G.
Kubbutat, and
Karen H.
Vousden*
ABL Basic Research Program, NCI-FCRDC,
Frederick, Maryland
Received 18 August 1998/Returned for modification 5 October
1998/Accepted 24 November 1998
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ABSTRACT |
The p53 tumor suppressor protein can be phosphorylated at several
sites within the N- and C-terminal domains, and several protein kinases
have been shown to phosphorylate p53 in vitro. In this study, we
examined the activity of p53 proteins with combined mutations at all of
the reported N-terminal phosphorylation sites (p53N-term), all of the
C-terminal phosphorylation sites (p53C-term), or all of the
phosphorylation sites together (p53N/C-term). Each of these mutant
proteins retained transcriptional transactivation functions, indicating
that phosphorylation is not essential for this activity of p53,
although a subtle contribution of the C-terminal phosphorylation sites
to the activation of expression of the endogenous p21Waf1/Cip1-encoding gene was detected. Mutation of the
phosphorylation sites to alanine did not affect the sensitivity of p53
to binding to or degradation by Mdm2, although alteration of residues
15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with
recent reports that phosphorylation at these sites inhibits the
p53-Mdm2 interaction. However, expression of the phosphorylation site
mutant proteins in both wild-type p53-expressing and p53-null lines
showed that all of the mutant proteins retained the ability to be
stabilized following DNA damage. This indicates that phosphorylation is
not essential for DNA damage-induced stabilization of p53, although
phosphorylation could clearly contribute to p53 stabilization under
some conditions.
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INTRODUCTION |
The p53 tumor suppressor protein
plays an important role in the cellular response to stress such as DNA
damage and hypoxia, and loss of p53 is associated with genomic
instability and tumor development (28). Activation of p53
can lead to cell cycle arrest, which can be reversed under some
circumstances, or apoptotic cell death (3). Both of these
responses prevent replication of cells with damaged DNA and are thereby
thought to protect cells from an accumulation of potentially oncogenic
genetic alterations. The ability of other signals, such as hypoxia or
deregulated proliferation, to activate a p53 response may allow the
elimination of cells which are already dividing under abnormal conditions.
One of the principal mechanisms by which p53 functions is as a
transcription factor, and many cellular genes have been identified which are transcriptionally regulated by p53. Broadly, these genes can
be divided into those likely to contribute to activation of a cell
cycle arrest, such as the gene encoding the cyclin-dependent kinase
inhibitor p21Waf1/Cip1, and those which are more likely to
play a role in mediating the apoptotic response, such as the genes
encoding Bax and KILLER/DR5 (3). Another important p53
target gene is Mdm2. Although the Mdm2 protein does not play
a direct role in mediating the p53 response, it is crucially involved
in negatively regulating p53 protein levels (26). Mdm2 binds
directly to p53 and targets it for degradation through the
ubiquitin-dependent proteolytic pathway (19, 24). In this
way, Mdm2 is responsible, at least in part, for maintaining low levels
of p53 under conditions of normal cell growth (5), although
Mdm2-independent degradation targeted by kinase-inactive JNK has also
recently been described (13).
Under most circumstances, signals which activate the p53 response lead
to rapid elevation of the p53 protein, principally through
stabilization of the protein, and activation of the DNA binding
function of p53. Regulation of p53 stability could be achieved by
regulating the ability of Mdm2 to target p53 for degradation, and the
DNA binding activity of p53 can be regulated independently by
alterations in protein conformation. In particular, the extreme C
terminus of p53 has been implicated in playing a negative regulatory role, maintaining p53 in a latent form which can be activated by
several mechanisms which modify this C-terminal region (15, 16,
20, 44).
Posttranslational modification of p53 by phosphorylation has been
proposed to be an important mechanism by which p53 stabilization and
function are regulated (34). p53 has been described as a substrate for many kinases in vitro, and phosphorylation of numerous serine and threonine residues within the N- and C-terminal regions of
the protein has been shown (40). Despite substantial
information on kinases which can phosphorylate p53 in vitro,
comparatively little is known about which sites in p53 are
phosphorylated and by which kinases in vivo. In recent studies using
phosphospecific antibodies, serines 15 and 37 were identified as sites
of phosphorylation in cells following DNA damage (45, 46),
with evidence that the ATM kinase may participate in the
phosphorylation of serine 15 in response to some, but not all,
activating signals (1, 6, 46). Phosphorylation of serine 33 in response to UV or ionizing radiation has also been shown recently
(43) with evidence that N-terminal phosphorylation of p53
can direct C-terminal acetylation of the protein. Phosphorylation at
the CK2 site (serine 392) has also been confirmed in vivo with evidence
that this site is phosphorylated following UV irradiation but not gamma
irradiation (4, 23).
Although there is substantial evidence that p53 can be phosphorylated
at several sites, the consequences of such phosphorylation for p53
function are still very poorly understood. Analyses of p53 proteins
from a number of different species carrying mutations at either one or
several of the potential phosphorylation sites have been carried out in
a number of cellular systems. Many of these studies have shown
contradictory and confusing results, probably due to variability in the
cell types and p53 proteins used. For example, mutation of the CK2 site
had no apparent effect on the growth-suppressive function of p53
(7, 10, 12), while mutation of this site reduced the ability
of p53 to suppress transformation (7, 37). There is also
evidence that phosphorylation of this site is important to maintain the
transcriptional activity of mouse p53 in contact-inhibited cells
(17). Similarly, although loss of phosphorylation of serine
15 in human p53 has been shown to extend the half-life of p53
(9), phosphorylation of this residue has also been suggested
to play a role in preventing interaction with Mdm2, leading to the
prediction that loss of this site might prevent stabilization of the
protein (38, 45). Phosphorylation of p53 by MEKK1/JNK has
also been reported to stabilize p53 by inhibiting interaction with Mdm2
(14). In general, functional studies such as these have
shown only subtle defects in phosphorylation site mutant p53, although
defects in the transcriptional activity of a p53 protein containing
several N-terminal phosphorylation site mutations have been reported
(32).
Since p53 has been shown to be phosphorylated at several sites within
the N- and C-terminal regions, it is possible that mutation of a number
of sites in combination is required for any global effects on the
regulation of p53 or on p53 activity itself. To address this problem
and to determine the requirement of phosphorylation for p53 function,
we created three human p53 mutant proteins in which all of the
potential N-terminal phosphorylation sites, all of the C-terminal
phosphorylation sites, or all of the N- and C-terminal sites were
altered in combination to nonphosphorylatable residues within the
full-length protein. We then analyzed these mutant proteins for the
ability to be phosphorylated in vivo, the ability to function in
transcriptional assays, and stability in response to DNA-damaging signals.
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MATERIALS AND METHODS |
Construction of mutant p53 cDNAs.
The human p53 N-terminal
(p53N-term) and C-terminal (p53C-term) phosphorylation mutant proteins
were generated independently by PCR mutagenesis. To generate the
p53N-term mutant protein, an initial PCR using forward primer
5'-GCCATGGAGGAGCCGCAGGCAGATCCTGCCGTCGAGCCCCCTCTGGCTCAGGAAGCATTTGCAGACC-3' (the underlined sequences are the mutated codons) and reverse primer 5'-GATGGCCATGGCGCGGACGCG-3' was performed. This was
subcloned into human Sp65p53Pro at the NcoI site and
oriented by using PvuII. This construct was used as the
template in a second PCR using forward primer
5'-ATAGAATCCGGGAGCAGGTAGCTGCTGGG-3' and reverse primer
5'-ATCATCCATTGCTTGGGCCGGCAAGGGGGCCAGAACG-3'.
The PCR product was subcloned into Sp65p53
210 at the
EcoRI/PflMI sites. The NcoI fragment
from the resulting construct was subcloned into either Sp65p53Pro or
Sp65p53C-term (described below) to generate Sp65p53N-term and
Sp65p53N/C-term, respectively. Sequencing analysis (ABI prism) was
carried out to confirm that all mutations had been made correctly.
To generate the p53C-term mutant protein, a PCR was carried out by
using forward primer 5'-ATAGAATCCGGGAGCAGGTAGCTGCTGGG-3', reverse primer
5'-CCATGGATCCGTCTGCGTCAGGCCCTTCTG-3', and the
p53 cDNA template pCB6+371A/376A/378A. The resulting PCR product was subcloned into Sp65 at the EcoRI/BamHI sites.
This construct was used as a backbone to subclone the
EcoRI/StuI fragment from pGem4Zp53-315A to
generate the final p53C-term mutant protein. Sequencing analysis (ABI
prism) was carried out to confirm that all mutations had been made correctly.
The 15A/37A, 15A/37D, 15D/37A, and 15D/37D mutant combinations were
generated in a similar manner by using forward primer
5'-GCCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTG
GCTCAG-3'
or
5'-GCCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTG
GATCAG-3'
and the appropriate cDNA template (pCB6+p53-37A or pCB6+p53-37D).
Sequencing analysis (ABI prism) was carried out to confirm that
all
mutations had been made correctly. Similarly, PCR mutagenesis
was
performed to generate the 18A, 18D, 20A, and 20D point mutations,
which
were subsequently subcloned into pCB6+ at the
EcoRI/
BamHI
sites.
To generate the

II mutant combinations, the
NcoI and
PflMI fragments from pGem4Zp53

II were subcloned into
Sp65p53C-term
or Sp65p53N-term, respectively, to generate
Sp65p53

IIC-term and
Sp65p53

IIN-term. The Sp65p53

II-15A/37A and
Sp65p53

I-

II combination
mutant cDNAs were generated in a similar
manner. All

II mutant
combinations were confirmed by sequencing
analysis (ABI
prism).
Plasmids and antibodies.
The pCB6+ expression plasmid,
containing the cytomegalovirus (CMV) early promoter (27),
was used to generate all expression constructs. The human p53
I,
p53
II, p53
370 (C-terminal truncation), p53-37A, p53-37D, and
p53-315A mutant cDNAs were previously generated by N. Marston et al.
(30). The mouse Mdm2 expression plasmid has already been
described (pCOC Mdm2 X2) (18), as have the luciferase
reporter plasmids containing the p21Waf1/Cip1 promoter
sequence pWWP-luc (8), the Mdm2 promoter sequence pGL2NA(mdm2)-luc (21), and the Bax promoter
sequence pGL3Bax-luc (11). The pEGFP-N1 plasmid was
purchased from Clontech. p53-specific monoclonal antibodies PAb1801 and
D0-1 have been described previously (2, 48). A
p21Waf1/Cip1-specific antibody was purchased from Oncogene
Science. Monoclonal antibodies to green fluorescent protein (GFP) and
actin were purchased from Clontech and Chemicon International,
respectively, and used in Western blot analysis to assess protein
levels where indicated.
Cell culture and transfections.
The Saos-2 human
osteosarcoma line (null for wild-type p53), the MCF-7 human breast
tumor line (expressing wild-type functional p53), and p53-null mouse
embryo fibroblasts (MEFs) were maintained in Dulbecco modified Eagle
medium supplemented with 10% fetal calf serum (FCS) at 37°C in an
atmosphere of 10% CO2 in air.
For transient transfection of Saos-2 cells and MEFs, 8 × 10
5 cells were plated in 10-cm
2 dishes and
transfected the following day by calcium chloride
precipitation with 50 ng to 10 µg of either a vector control (pCB6+)
or a human wild-type
p53 or mutant p53 expression plasmid where
indicated. For luciferase
reporter assays, cells were cotransfected
with 5 µg of each reporter
construct. To assess Mdm2 effects,
cells were cotransfected with 9 µg
of mouse Mdm2. To assess transfection
efficiency and normalize protein
levels, transfections also included
either 1 µg of pEGFP-N1 or 5 µg
of pCB6+

gal where indicated.
Cells were washed on the day following
transfection, harvested
24 h later, and prepared for Western
blotting, flow cytometry,
or luciferase and

-galactosidase analyses
as previously described
(
41,
42). For stable transfection of
MCF-7 cells, 5 × 10
5 cells were plated in
10-cm
2 dishes and transfected as for transient transfection
with 10
µg of either a vector control (pCB6+) or a human p53

II or
p53

II
mutant expression plasmid where indicated. Cells were split at
1:20 into 15-cm
2 dishes (in duplicate) 24 h after
washing and selected with G418
(600 µg/ml) on the following day.
Selected clones were isolated
and expanded for further
analysis.
In vivo 32P-phosphate labeling of cells.
To
assess the relative phosphorylation of the mutant proteins in vivo,
Saos-2 cells were transiently transfected with 5 µg of either a pCB6+
control plasmid or a wild-type p53, p53-15A, p53N-term, p53C-term, or
p53N/C-term expression plasmid as described above. Approximately
4 h prior to harvesting, the cells were washed twice in 5 ml of
phosphate-free medium (per 10-cm2 dish) and then incubated
at 37°C in 3 ml of phosphate-free medium supplemented with 10%
dialyzed FCS containing 1 mCi of 32P-labeled sodium
orthophosphate for 4 h. Cells were lysed in
radioimmunoprecipitation assay lysis buffer (20 mM Tris [pH 8.0], 137 mM NaCl, 0.5 mM EDTA, 10% glycerol, 1% Nonidet P-40, 0.1% sodium
dodecyl sulfate [SDS], 1% deoxycholate) (ice cold), and p53 protein
was immunoprecipitated with PAb1801-protein A Sepharose beads. Samples
were separated by SDS-10% polyacrylamide gel electrophoresis (PAGE)
and transferred to nitrocellulose, and phosphorylated protein was
assessed by PhosphorImager analysis (Storm 860 PhosphorImager;
Molecular Dynamics Inc.). To assess immunoprecipitated p53 levels,
blots were probed with PAb1801. Additionally, for each experiment,
phosphorylated proteins were assessed directly from SDS-10% PAGE by
PhosphorImager analysis.
In vitro association assays.
To determine the ability of
each of the mutant proteins to bind to Mdm2 in vitro, an in vitro
association assay was performed by using in vitro-translated proteins.
The wild-type p53, p53N-term, p53C-term, and p53N/C-term proteins were
translated in the presence of 35S-Pro-Mix (Amersham) and
human flag-Mdm2 was translated in the absence of
35S-Pro-Mix (Amersham) by using a reticulocyte lysate-based
in vitro translation kit as directed by the manufacturer (Promega). A
5-µl volume of hot, in vitro-translated wild-type p53, p53N-term,
p53C-term, or p53N/C-term protein was mixed with 5 µl of cold, in
vitro-translated flag-Mdm2 protein and incubated for 30 min at 30°C.
A 500-µl volume of LSAB buffer (100 mM NaCl, 100 mM Tris [pH 8.0],
1% Nonidet P-40) supplemented with 3% bovine serum albumin was then
added to each sample. Samples were spun at full speed for approximately 5 min at room temperature and transferred to a fresh microcentrifuge tube. A 5-µl volume of anti-flag antibody was added, and samples were
incubated at 4°C for 2 h to overnight with gentle rotation. Flag-Mdm2 complexes were precipitated by the addition of 50 µl of
protein G Sepharose beads, and samples were incubated for 1 h at
4°C with gentle rotation. Immunoprecipitated complexes were washed
five times with LSAB buffer, resuspended in 50 µl of 2 × SDS
sample buffer, boiled for 10 min, and then assessed by SDS-10% PAGE.
To assess 35S-labeled protein, gels were dried and exposed
to Kodak film.
Induction of p53 proteins in MCF-7 cells and p53-null MEFs.
To assess DNA damage-induced stabilization of endogenous wild-type p53
and exogenous mutant p53 in MCF-7 clonal lines stably expressing the
II mutant combinations, cells were plated at 8 × 105 into 10-cm2 dishes. At least 24 h
after plating, cells were treated with actinomycin D (5 nM) or
irradiated with UV light at 50 J/m2. Cells were harvested
at 10 to 16 h for actinomycin D treatment and at 6 h for UV
treatment. Cells were harvested and lysed directly by the addition of
500 µl of 2 × SDS sample buffer. Samples were boiled, analyzed
by SDS-10% PAGE, and assessed by Western blotting using PAb1801 and
D01. Stabilization of mutant p53 in p53-null MEFs was carried out by
transiently transfecting cells with 500 ng of DNA and carrying out the
DNA damage protocols described above at 24 h posttransfection.
The half-life of the p53 protein in MCF-7 cells stably expressing the

II mutant combinations was determined after treatment
with or
without actinomycin D (5 nM, 16 h) by radioactive pulse-labeling
of cells for 30 min and a chase in unlabeled medium for 0, 1,
2, or
4 h. Endogenous wild-type p53 and exogenous p53 were then
immunoprecipitated by using monoclonal antibody PAb1801 or PAb421
(the
two gave similar results). Quantification of labeled p53
protein was
carried out by using the Storm 860 PhosphorImager
(Molecular Dynamics
Inc.).
 |
RESULTS |
Phosphorylation site mutant p53.
Site-directed mutagenesis was
used to construct mutant p53 carrying alanine substitutions at the
known or predicted phosphorylation sites within the p53 N and C termini
(Fig. 1A). The N-terminal amino acids
serine 6, serine 9, serine 15, threonine 18, serine 20, serine 33, and
serine 37 were replaced in the mutant protein p53N-term. The mutant
protein p53C-term carried substitutions of serine 315, serine 371, serine 376, serine 378, and serine 392. A further mutant protein, for
which both the p53N- and p53C-term mutant proteins were combined, was
also created and called p53N/C-term. All of the mutant proteins were
efficiently expressed to levels comparable to that of wild-type p53
following transient transfection into p53-null Saos-2 cells (Fig. 1B).

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FIG. 1.
(A) Schematic representation of the human p53 protein
indicating the important functional domains (transactivation, proline
rich, sequence-specific DNA binding, oligomerization, and regulation of
DNA binding), conserved box regions I to V, and potential sites of
phosphorylation within the N- and C-terminal regions. To generate
N-terminal phosphorylation mutant p53 (N-term), Ser6, Ser9, Ser15,
Thr18, Ser20, Ser33, and Ser37 were mutated to Ala in combination. To
generate C-terminal phosphorylation mutant p53 (C-term), Ser315,
Ser371, Ser376, Ser378, and Ser392 were mutated to Ala in combination.
N- and C-terminal phosphorylation mutant p53 (N/C-term) was generated
by a combination of the above. (B) Western blot analysis (using
PAb1801) of Saos-2 cells transiently expressing wild-type p53 and the
p53N-term, p53C-term, and p53N/C-term mutant proteins after transient
transfection with 5 µg of the respective plasmid. Cells were
harvested at 24 h.
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In order to determine whether all of the major phosphorylation sites
had been altered in these mutant proteins, we carried
out
orthophosphate labeling following transient transfection in
Saos-2
cells. Efficient phosphorylation of the wild-type protein
was achieved
under these conditions (Fig.
2), with
each of the
mutants showing a reproducible reduction in phosphate
incorporation
in vivo. The p53N/C-term mutant protein, carrying
substitutions
of all the candidate phosphorylation sites under
investigation,
showed no detectable phosphorylation, indicating that
all of the
major kinase sites had been lost. Mutation of the N- or
C-terminal
sites resulted in a decrease, but no total loss, of
phosphorylation,
confirming that sites within both of these regions are
targets
for kinases in vivo. These results indicated, however, that the
N terminus of p53 is more extensively phosphorylated than the
C
terminus. Recent results have shown that serine 15 is a major
phosphorylation site of p53 in vivo, and in agreement with this,
we
have found that mutation of only this residue in the full-length
protein greatly reduces phosphorylation to almost the same extent
as
mutation of all of the other potential N-terminal phosphorylation
sites
together. However, there appears to be at least one other
phosphorylation site within the N-terminal region, in agreement
with a
recent study showing three phosphorylation sites within
the N-terminal
24 amino acids of p53 (
46).

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FIG. 2.
Relative phosphorylation of p53 mutant proteins in vivo.
Phosphorylation site mutant p53 proteins (5 µg) were transiently
transfected into Saos-2 cells. At approximately 4 h prior to
harvesting (at 24 h), the cells were washed and incubated in 5 ml
of phosphate-free medium (supplemented with 10% dialyzed FCS)
containing 1 mCi of 32P-labeled sodium orthophosphate (per
10-cm2 dish). Cells were lysed with
radioimmunoprecipitation assay lysis buffer, and p53 protein was
immunoprecipitated with PAb1801-protein A Sepharose beads. (A)
Phosphorylated protein was assessed by SDS-10% PAGE (top), and blots
were reprobed with PAb1801 to assess immunoprecipitated p53 levels
(bottom). (B) Phosphorylated protein was quantified by PhosphorImager
analysis and represented graphically by using absolute integrated
optical density units (I.O.D.). The graph represents the results of
four independent experiments.
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Activity of phosphorylation site mutant proteins.
Many
previous studies have examined the effect of mutating one of several
phosphorylation sites within p53 on the biological activity of the p53
protein, with varied results. Some analyses showed no effect of loss of
phosphorylation sites compared to the wild-type protein; others showed
subtle alterations in the ability to modulate cell cycle progression or
inhibition of cell growth. However, none of those previous studies
examined p53 mutant proteins that were simultaneously mutated at all
phosphorylation sites and were therefore unable to address the
consequences of complete loss of phosphorylation for p53 function. We
examined the activity of our mutant proteins in transactivation assays by using a series of p53-responsive promoters (Fig.
3). Each of the mutant proteins was as
efficient as wild-type p53 in activating transcription from the human
p21Waf1/Cip1, Mdm2, and bax promoters in luciferase
reporter constructs in these transient assays. By using Western blot
analysis and flow cytometry to estimate p53 expression levels and the
percentage of cells transfected, we estimated that p53 proteins are
overexpressed approximately fivefold per cell following transient
transfection with 500 ng of a p53 expression plasmid compared with the
levels seen following DNA damage of a wild-type p53-expressing cell
(data not shown). Each of the mutant proteins was also able to elevate levels of endogenous p21Waf1/Cip1 protein following
transient transfection into Saos-2 cells (Fig. 4), although slightly reduced activation
of p21Waf1/Cip1 by p53 proteins with C-terminal
phosphorylation site mutations was seen in several repeats of this
experiment. However, under the conditions used in this assay, we were
unable to detect any differences between the phosphorylation site
mutant proteins and wild-type p53 in the activation of cell cycle
arrest or apoptosis (data not shown).

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FIG. 3.
Transcriptional activation by p53 phosphorylation mutant
proteins assessed by using a luciferase reporter assay. p53
phosphorylation mutant proteins (50 ng) were cotransfected into Saos-2
cells with 5 µg of either a human p21Waf1/Cip1, Mdm2, or
bax luciferase reporter construct. Five micrograms of pCB6+ gal was
included to assess transfection efficiency. Cells were harvested at
24 h, lysed, and assessed for luciferase and -galactosidase
activities. Each graph shows fold activation relative to that of the
pCB6+ control harvested at 24 h.
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FIG. 4.
Wild-type p53 and p53 phosphorylation mutant proteins (5 µg) were transiently transfected into Saos-2 cells and the cells were
harvested at 24 h for Western blot analysis to assess
p21Waf1/Cip1 protein levels by using an anti-p21
antibody.
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Regulation of p53 stability.
Although phosphorylation of p53
does not appear to be essential for p53 function, several studies have
suggested that phosphorylation of p53 plays an important role in
regulating p53 activities such as stability and DNA binding. p53
stability has recently been shown to be regulated through interaction
with Mdm2 (19, 24), and in vitro analysis showed that each
of the phosphorylation site mutant proteins was able to bind to Mdm2
(Fig. 5A). We then analyzed the
sensitivity of each of the mutant proteins to Mdm2-mediated degradation
in a transient assay (Fig. 5B) in which each mutant protein was
expressed in a p53-null cell line with or without the addition of
exogenous Mdm2. As previously reported, wild-type p53 was efficiently
degraded following expression of exogenous Mdm2, and mutations within
the N terminus of p53 which prevent interaction with Mdm2 (
I)
(24) or deletions within the C terminus of p53 which do not
interfere with Mdm2 binding (
370) (25) rendered the p53
protein resistant to degradation by Mdm2. All of the phosphorylation
site mutant proteins retained sensitivity to Mdm2-mediated degradation
in a manner essentially indistinguishable from that of wild-type p53.
It has recently been shown in an in vitro association assay that
phosphorylation of serines 15 and 37 by dsDNA-PK reduces the ability of
p53 to bind to Mdm2 (38, 45). This study also indicated that
alanine substitution at these sites enhances the binding of Mdm2 in
vitro, and it is therefore not surprising that the phosphorylation site
mutant proteins under investigation here (which also have alanine
substitutions) remain susceptible to Mdm2-mediated degradation. We
therefore assessed whether replacement of serines 15 and 37 with
aspartic acid (which mimics the charge of a phosphorylation group)
would render the protein resistant to Mdm2-mediated degradation when
transiently cotransfected into Saos-2 cells. Although individual
alteration of serine 15 or 37 to alanine or aspartic acid did not
significantly affect the susceptibility of the p53 protein to
Mdm2-mediated degradation, mutation of both serines 15 and 37 to
aspartic acid rendered the protein very slightly resistant to
degradation (Fig. 5C), consistent with the previous reports that
phosphorylation at these sites reduces Mdm2 binding. This effect was
very subtle, even when titration of smaller amounts of Mdm2 was used
(data not shown). Individual mutations of other phosphorylation sites in or close to the Mdm2 binding site (threonine 18 and serine 20) also
failed to affect the sensitivity of p53 to Mdm2-mediated degradation in
this assay (Fig. 5D).

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FIG. 5.
(A) In vitro association assay using in
vitro-translated, unlabeled, human flag-Mdm2 (cold) and
35S-labeled, in vitro-translated wild-type p53 and
p53N-term, p53C-term, and p53N/C-term mutant proteins. Complexes were
immunoprecipitated (IP) with anti-flag antibody in the presence of
protein G Sepharose beads, and radioactively labeled p53 proteins were
assessed by SDS-10% PAGE. (B) Western blot analysis (using PAb1801)
of Saos-2 cells after transient cotransfection of the phosphorylation
mutant p53 (3 µg), mouse Mdm2 (9 µg), and pEGFP-N1 (1 µg). Cells
were harvested at 24 h. The I mutant protein (deletion of
conserved box I, the region containing the Mdm2 binding site), which is
resistant to Mdm2-mediated degradation, and the 370 mutant protein
(C-terminal truncation mutant protein), which is partially resistant to
Mdm2-mediated degradation, were used as controls. Relative transfection
efficiency was monitored by expression of cotransfected GFP. (C)
Western blot analysis (using PAb1801) of Saos-2 cells after transient
cotransfection of the p53-15A/37A, -15A/37D, -15D/37A, and -15D/37D
combination mutant proteins (3 µg), mouse Mdm2 (9 µg), and pEGFP-N1
(1 µg). Cells were harvested at 24 h. Relative transfection
efficiency was monitored by expression of cotransfected GFP. (D)
Western blot analysis (using PAb1801) of Saos-2 cells after transient
cotransfection of the p53-20A, -20DD, -18, and -18D combination mutant
proteins (3 µg), mouse Mdm2 (9 µg), and pEGFP-N1 (1 µg). Cells
were harvested at 24 h. Relative transfection efficiency was
monitored by expression of cotransfected GFP.
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One attractive hypothesis is that phosphorylation of p53 plays an
important role in allowing the protein to become resistant
to
degradation, with the prediction that phosphorylation site
mutant
proteins would show a defect in the ability to become stabilized
following exposure to signals which normally activate a p53 response,
such as DNA damage. In our transient assays, there was no clear
indication that the phosphorylation site mutant proteins were
less
stable or were expressed at lower levels than the wild-type
protein. We
were concerned, however, that high levels of transient
expression were
masking a more subtle regulation of p53 stability
and therefore turned
to the analysis of cells stably expressing
the phosphorylation site
mutant proteins. Since wild-type p53
and all of the mutant proteins
under consideration in this study
induce cell cycle arrest and
apoptosis, we took advantage of the
observation that deletion of
conserved regions in the DNA binding
domain of p53 renders the protein
unable to arrest cell growth
but still responsive to normal regulation
of stability through
Mdm2 (
35). The N-term, C-term, and
15A/37A phosphorylation site
mutant proteins were therefore made in the
context of a deletion
of conserved region II (

II) (Fig.
6A), which abolishes the
growth-inhibitory
functions of p53 (
7). All of the

II
combination mutant proteins
were expressed to similar levels when
transiently transfected
into Saos-2 cells (Fig.
6B), and we therefore
stably transfected
each of these mutant proteins into MCF-7 cells,
which express
endogenous wild-type p53. Initial analysis of polyclonal
cell
lines showed that each of the exogenous mutant proteins was
expressed
to low levels in MCF-7 cells and that, like that of
endogenous
wild-type p53, the level of each mutant protein increased
following
DNA damage by actinomycin D or UV irradiation (data not
shown).
Clones of cells expressing the mutant p53 proteins were then
isolated
and subjected to closer analysis. Expression of the mutant
protein
was slightly higher than that of the endogenous protein,
possibly
because the mutant protein is expressed from the efficient CMV
promoter. It was clear that either actinomycin D treatment or
UV
irradiation resulted in rapid elevation of both endogenous
wild-type
p53 and exogenous p53

II which retained all of the wild-type
phosphorylation sites (Fig.
7A). The
p53

I-

II mutant protein
used as a control, which is unable to bind
Mdm2, was stable in
untreated cells, and protein levels were not
further increased
by either actinomycin D or UV treatment (Fig.
7A).
Importantly,
this observation indicates that the increase in p53

II
after actinomycin
D treatment and UV irradiation is not due to enhanced
activation
of the CMV promoter at these time points. We did note that
stable
expression of the p53

I-

II mutant protein in untreated
cells
occasionally resulted in a very slight elevation of endogenous
p53 protein levels compared to those seen in other uninduced lines
(Fig.
7A).

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|
FIG. 6.
(A) Schematic representation of the conserved box II
deletion mutant proteins. (B) Western blot analysis (using PAb1801) of
Saos-2 cells transiently expressing p53 II and the p53 II 15A/37A,
p53 II N-term, and p53 II C-term mutant proteins after transient
transfection with 5 µg of the respective plasmid. Cells were
harvested at 24 h.
|
|

View larger version (39K):
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[in a new window]
|
FIG. 7.
Stable expression of p53 II combination mutant
proteins in MCF-7 cells which express wild-type (wt) p53. Stable clones
were isolated, and p53 protein levels were assessed by Western blot
analysis using PAb1801 or D01 after treatment with either 5 nM
actinomycin D (Act; harvested at 10 h) or 50-J/m2 UV
(harvested at 6 h). (A) Western blot analysis of MCF-7 stable cell
lines expressing either the pCB6+ control (leftmost blot), p53 II
(middle blot), or p53 I- II (rightmost blot). Equal protein loading
was confirmed by actin expression. endo., endogenous; exo, exogenous.
(B) Western blot analysis of two independent MCF-7 stable cell lines
(clone 1, left; clone 2, right) expressing either p53 II15A/37A
(top), p53 IIN-term (middle), or p53 IIC-term (bottom). Equal
protein loading was confirmed by actin expression. (C) Western blot
time course analysis of actinomycin D (ActD) treatment (0, 3, and
6 h) of MCF-7 cells stably expressing either pCB6+ (control),
p53 II (clone 1), p53 II15A/37A (clone 1), and p53 IIN-term
(clone 1). All blots were reprobed with an antiactin monoclonal
antibody to assess protein levels. Equal protein loading was confirmed
by actin expression.
|
|
Having established that the MCF-7 cells could be used to measure DNA
damage-induced protein stabilization of both endogenous
and exogenous
p53, we examined several independently derived clones
of cells
expressing the phosphorylation site mutant proteins (Fig.
7B). In each
case, levels of the mutant p53 proteins were clearly
elevated in
response to either actinomycin D treatment or UV irradiation,
to levels
similar to those seen with the

II mutant protein alone.
Similar
results were also obtained for treatment with gamma irradiation
(data
not shown). Treatment of the cells with a proteasome-calpain
inhibitor
(
N-acetyl-Leu-norleuceinal) stabilized both the wild-type
and mutant p53 proteins in the absence of DNA damage to levels
which
were not further elevated by actinomycin D treatment or
UV irradiation
(data not shown). In an attempt to distinguish
more modest differences
in the rate of stabilization of the phosphorylation
site mutant
proteins, the time course of induction was analyzed.
All of the
exogenous p53 mutant proteins were induced at similar
rates over the
same time course as endogenous wild-type p53 (Fig.
7C). To confirm that
the increase in the wild-type and mutant
proteins following DNA damage
was due to an increase in stability,
the half-lives of the p53

II,
p53

I-

II, and p53

IIN-term mutant
proteins and the respective
endogenous wild-type proteins for
each line were assessed by
radioactive pulse-labeling of the cells
in the absence of actinomycin D
and after treatment with actinomycin
D (Table
1). The results show that both the
p53

II and p53

IIN-term
proteins are stabilized to comparable
degrees by an increase in
half-life following treatment, from
approximately 1 to 3 h, indicating
that mutation of all of the
phosphorylation sites within the N-terminal
region of p53 does not
impair stabilization of the protein after
DNA damage. Furthermore,
these results also confirm that the elevated
levels of p53

I-

II
protein observed in undamaged cells (Fig.
7A, rightmost blot) is due to
increased stability compared with
the control, as shown previously for
p53

I (
24), and that the
half-life of the p53

I-

II
protein does not increase after DNA
damage.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Half-lives of endogenous wild-type p53 and exogenously
expressed p53 mutant proteins in MCF-7 cells in the absence of or after
treatment with actinomycin Da
|
|
Since p53 forms tetramers, it is possible that stabilization of the
mutant proteins expressed in MCF-7 cells may be enhanced
by
hetero-oligomerization with the stabilized wild-type protein
after DNA
damage. The observation that overexpression of the exogenous
p53

I-

II protein had only a very slight effect on the stability
of
endogenous p53 in these cells (Fig.
7A) indicated that this
was
unlikely to be a significant factor in these studies. However,
to rule
out this possibility, we analyzed the stability of mutant
p53 in cells
lacking endogenous wild-type p53. Preliminary evidence
suggested that
Saos-2 cells do not retain a normal DNA damage
response, and we
therefore analyzed the stability of mutant p53
in p53-null MEFs (Fig.
8). As expected, the transcriptionally
inactive p53

II mutant protein was stably expressed in these cells
even in the absence of DNA damage (Fig.
8A), consistent with its
inability to activate expression of Mdm2. We therefore analyzed
the
expression of the p53 phosphorylation site mutant proteins
containing a
wild-type DNA binding domain (Fig.
1), which retained
transcriptional
activity, in transient assays. These studies confirmed
that both the
p53N-term and p53C-term mutant proteins could be
stabilized in response
to actinomycin D treatment and UV irradiation,
like wild-type p53,
while the p53

I mutant protein was stable
under all conditions (Fig.
8B and C). Interestingly, although
induction of mutant p53 levels in
response to actinomycin D treatment
was essentially identical to that
of the wild-type protein, stabilization
of the p53N-term mutant protein
in response to UV irradiation
was slightly less than that observed for
wild-type p53 (Fig.
8C).

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|
FIG. 8.
Stable and transient expression of p53 proteins in
p53-null MEFs. (A) Western blot analysis of MEFs stably expressing
either a vector control (pCB6+) or p53 II with or without treatment
with 5 nM actinomycin (ActD). (B) Western blot analysis of MEFs
transiently expressing a vector control (pCB6+) or the indicated p53
proteins without DNA damage or following treatment with actinomycin D
(Act; 5 nM) or UV irradiation (50 J/m2). (C) Western blot
analysis of MEFs transiently expressing a vector control (pCB6+),
wild-type p53, or p53N-term without DNA damage or following treatment
with actinomycin D (ActD; 5 nM) or UV irradiation (50 J/m2).
|
|
 |
DISCUSSION |
In this study, we have asked whether phosphorylation of p53 is
essential for the transcriptional activation, cell cycle arrest, or
apoptotic function of the protein. To address this question, we
generated a series of human p53 phosphorylation site mutant proteins
which were unable to become phosphorylated within the N-terminal and/or
C-terminal regions of the protein. By using transient overexpression
assay systems, we showed that the transcriptional activation function
was retained by all phosphorylation site mutant proteins, including one
mutant protein which completely lacked any detectable phosphorylation,
although a reduced activity of p53 proteins mutated at the C-terminal
phosphorylation sites could be detected. These results are consistent
with suggestions that C-terminal phosphorylation enhances p53 DNA
binding activity (33), although another study has recently
shown an association of decreased C-terminal phosphorylation with
increased sequence-specific DNA binding by p53 (49). Our
results show that phosphorylation of p53 is not essential for
transcriptional activation by p53, and indeed, numerous reports over
the last 5 years have contained similar findings for mutations at
single or double sites within the N- or C-terminal region of the
protein (reviewed in reference 36). However, it
seems likely that phosphorylation of p53 modulates p53 activities in a
more subtle manner. Previous studies have suggested that loss of
phosphorylation sites results in reduction, but not loss, of activity
(7, 9) or that loss of phosphorylation sites differentially
affects functions such as transcriptional activation in a cell
type-specific manner (29). Furthermore, evidence from one
study suggests that mutation of certain combinations of sites may be
important for a clear phenotype (32). While we have shown
that phosphorylation is not essential for the transcriptional function
of p53, it is possible that subtle levels of regulation of p53 function
through phosphorylation would not be seen in these transient
overexpression assays. We believe that our mutant proteins will be
useful in future studies using stable expression systems to address
this question.
The second point addressed by our study concerns the importance of
phosphorylation for the regulation of p53 stability. Many recent
studies have pointed to Mdm2 as a key regulator of p53 stability in
normal cells, and inhibition of Mdm2-mediated degradation of p53
following DNA damage is likely to play an important role in activating
a p53 response. Inhibition of the interaction of p53 with Mdm2 prevents
degradation, and recent studies have shown that phosphorylation of
serines 15 and 37 reduced the ability of p53 to bind to Mdm2, with the
prediction that this might impair Mdm2-mediated degradation and thus
lead to stabilization of p53 (38, 45). The observation that
there are several phosphorylation sites either in or close to the Mdm2
binding domain of p53 makes this an extremely attractive model, since
many kinases which might phosphorylate p53 at these sites could be
activated by DNA damage or other forms of stress. Loss of the ATM
kinase has been associated with an inability to stabilize p53 in
response to certain signals, and recent studies have shown that ATM can
phosphorylate p53 at serine 15 (1, 6) in response to DNA
damage. Analysis of the sensitivity of our phosphorylation site mutant
proteins to Mdm2 revealed no significant difference from the wild-type
protein; each mutant protein bound Mdm2 in vitro and was sensitive to
Mdm2-mediated degradation in transient assays in vivo. This might be an
expected result when considering the model in which phosphorylation of p53 plays a role in allowing the protein to become resistant to Mdm2;
each of our phosphorylation site mutant proteins with alanine substitutions would be constitutively sensitive to degradation. We
therefore also examined mutant p53 with an aspartic acid substitution at serine residue 15, 18, 20, or 37 in order to mimic phosphorylation of these residues and potentially generate a stable protein. Although each of the single aspartic acid substitution mutant proteins retained
full sensitivity to Mdm2-mediated degradation in our assay, the 15D/37D
double mutant protein showed slight but reproducible resistance to
Mdm2. Although the protein is clearly not completely resistant to Mdm2,
like p53
I, this result suggests that multiple N-terminal
phosphorylations could contribute to the stabilization of p53 in
response to some activating signals.
A clear prediction for a role of phosphorylation in modulating
sensitivity to Mdm2-mediated degradation in response to DNA damage is
that the p53 proteins mutated at the critical phosphorylation sites
should be unable to be stabilized in response to the normal activating
signals. Our study shows that proteins mutated at the N- or C-terminal
phosphorylation sites are stabilized following either actinomycin D
treatment or UV irradiation to similar extents and with kinetics
similar to those of the protein containing wild-type phosphorylation
sites in stably expressing MCF-7 cells. Furthermore, this stabilization
can be attributed to an increase in half-life after DNA damage, where
the p53
IIN-term mutant protein is stabilized to the same degree as
the p53
II control. While these results do not preclude a role for
phosphorylation in the regulation of p53 stability, they show that
stabilization of p53 in response to these DNA-damaging treatments can
be carried out in the absence of any detectable N- or C-terminal
phosphorylation of p53. One important point to consider when examining
cells expressing both wild-type and mutant p53 proteins is that p53 is
known to oligomerize, and hetero-oligomers of p53 proteins that can
bind Mdm2 with proteins that have lost the ability to bind Mdm2 (such
as p53
I) show reduced Mdm2 binding (31). This presents
the possibility that stabilization of wild-type p53 in cells expressing
the phosphorylation site mutant proteins could result in indirect
stabilization of mutant p53 through oligomerization with the wild-type
protein. This does not appear to be a significant concern in this
system, however, since expression of the p53
I-
II protein, which
fails to bind Mdm2 and is stable in the absence of any activating
signal, does not markedly stabilize endogenous wild-type p53 protein
levels (Table 1). Furthermore, the phosphorylation site mutant proteins were also shown to be stabilized in response to DNA damage in p53-null
MEFs. It is of interest that although actinomycin D treatment consistently stabilized the p53 mutant proteins as efficiently as
wild-type p53, analysis of p53-null MEFs provided some indication that
loss of the N-terminal phosphorylation sites may reduce, although not
abolish, stabilization in response to UV irradiation.
Taken together, our results show that phosphorylation of p53 is not
absolutely required for regulation of p53 stability, although it may
play a contributory role under some conditions, such as UV irradiation.
The observation that p53 can be stabilized in response to DNA damage
without any detectable N- or C-terminal phosphorylation demonstrates
that other mechanisms which allow p53 to become resistant to
degradation are likely to exist. This is likely to reflect resistance
to Mdm2-mediated degradation, although resistance to other regulators
of p53 stability, such as JNK (13), may also play a role.
Studies of both p53 and Mdm2 have shown that the binding between the
two proteins is necessary but not sufficient for degradation (24,
25) with evidence that regions of both proteins distant and
distinct from the binding sites are important for degradation. This has
been confirmed in recent studies showing that the p14ARF
protein can inhibit Mdm2-mediated degradation of p53 without disrupting
the p53-Mdm2 complex (22, 39, 47). Although
p14ARF does not play a clear role in mediating the
stabilization of p53 in response to DNA damage, it is possible that
several proteins function like p14ARF to stabilize p53 in
response to various types of stress. Our data suggest that such a
mechanism may also contribute to the stabilization of p53 in response
to DNA damage, either independently of or in cooperation with
phosphorylation of the p53 protein.
 |
ACKNOWLEDGMENTS |
We are very grateful to all members of the Vousden lab for advice
and encouragement. We also thank Thanos Halazonetis and Carol Prives
for helpful discussions.
This work was sponsored by National Cancer Institute, DHHS, under
contract with ABL.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: ABL Basic
Research Program, NCI-FCRDC, Building 560, Room 22-96, West 7th St.,
Frederick, MD 21702-1201. Phone: (301) 846-1726. Fax: (301) 846-1666. E-mail: vousden{at}ncifcrf.gov.
 |
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