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INTRODUCTION |
The p53 tumor suppressor is an important regulator of cellular
response to stress, abnormal cell proliferation, and DNA damage. In
normal cells, p53 is maintained at a very low level because of rapid
degradation through the ubiquitin-dependent proteasome pathway (1). In response to different stress stimuli including ionizing
radiation, UV, and hypoxia, p53 is activated and stabilized as a
transcription factor. Activated p53 induces the expression of
p21WAF1, gadd45, bax,
p53AIP, PUMA, and others, which in turn induce cell cycle
arrest and apoptosis (2).
The level of p53 in cells is mainly regulated at the
post-transcriptional level by the
MDM21 oncoprotein.
MDM2 binds to p53 and promotes its ubiquitination by acting as a
ubiquitin E3 ligase (3-5). Expression of MDM2 is activated by p53 at
the transcription level (6, 7), forming a negative feedback loop to
maintain p53 at low levels. To date, most stress signals that activate
p53 are known to interfere with the ability of MDM2 to promote p53
degradation by phosphorylation of p53, induction of the MDM2 inhibitor
ARF, or inhibition of MDM2 expression (1, 2). Phosphorylation of MDM2
by ATM may also play a role in p53 activation after DNA damage (8). The MDM2-binding domain in the N terminus of p53 contains several phosphorylation sites (9, 10). Recent studies (11, 12) suggest
that after DNA damage, the phosphorylation of Ser-15 by the ATM kinase
and Ser-20 by human Chk2 plays important roles in p53 stabilization by
interfering with MDM2 binding.
Previous reports (13-17) show that heat shock (1 h at 43 °C) also
induces p53 accumulation in wild type p53 cell lines and contributes to
cell cycle arrest or apoptosis. Heat shock is clinically useful in
facilitating the treatment of certain tumors such as gliomas in
combination with chemotherapy or ionizing radiation (18-20).
Therefore, investigating the mechanism of p53 accumulation after heat
shock is important in understanding p53 response to a physiologically
relevant stress. A previous study (22) suggests that p53 accumulation
in response to heat is not the result of a general stress-induced
increase in protein synthesis, because heat causes a reduction in
overall cellular protein synthesis. Heat shock has been reported to
induce DNA damage (21), suggesting that it may stabilize and activate
p53 in part through the DNA damage response pathways. A recent study
shows (15) an increase in p53 mRNA level after heat treatment of
A-172 glioma cells, suggesting a mechanism of induction at the
transcriptional level.
Stabilization of p53 also occurs in tumor cells with mutated p53, which
result in an accumulation to high levels. Missense mutations of p53 in
the DNA-binding core domain cause conformational change and stable
association with molecular chaperones such as hsp70 and hsp90 (23, 24).
hsp90 binding has been shown to contribute to the accumulation of
mutant p53 and many other client proteins (25-27). Our recent studies
show (28, 29) that binding of hsp90 inhibits the ability of MDM2 to
promote p53 ubiquitination and degradation, resulting in the
stabilization of both mutant p53 and MDM2. hsp90 appears to inactivate
MDM2 by blocking the central domain of MDM2 normally involved in
regulation by ARF, thereby mimicking the effect of ARF to prevent
mutant p53 degradation (29).
Although the role of hsp90 in mutant p53 stabilization is well
established, there is no evidence to date that it contributes to the
stabilization of wild type p53 during stress. Molecular chaperones only
associate with certain proteins in a transient manner during folding or
during heat shock response. Wild type p53 does not form stable complex
with hsp90 in normal growth conditions. Because DNA damage does not
induce p53 conformational change, hsp90 is unlikely to play a role in
DNA damage response. However, p53 is a structurally unstable protein
and undergoes conformational change (denaturation) at temperatures
above 40 °C in vitro (30). Work by Graeber et
al. (31) also indicates that heat shock alters the conformation of
wild type p53, allowing the formation of the p53·hsc70
complex. Therefore, it is possible that hsp90 binding also interacts
with wild type p53 after heat shock and plays a role in stabilizing p53
through mechanisms similar to the stabilization of mutant p53.
In this study, we investigated the role of p53 conformational change
and interaction with hsp90 in the stabilization of p53 after heat
shock. Furthermore, we examined the phosphorylation status of p53 after
heat shock treatment. Our results showed that heat shock induced p53
accumulation by promoting ATM-dependent serine 15 phosphorylation and complex formation with hsp90. Both mechanisms
contribute to p53 stabilization after heat shock. Our results
identified the first example of hsp90 participation in regulating wild
type p53 stress response.
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MATERIALS AND METHODS |
Cell Lines and Reagents--
Tumor cell lines A-172
(glioblastoma, wt p53), U2OS (osteosarcoma, wt
p53), DLD-1 (colon carcinoma, mt p53241S-F) were
obtained from the ATCC. MCF-7 (breast tumor, wt p53) cells
were provided by Dr. Arnold J. Levine. Epstein-Barr
Virus-immortalized human lymphocyte from normal control (NC-607)
and ATM patient (AT-29) were kindly provided by Dr. Kevin Brown
(Louisiana State University Health Science Center). A-172, MCF-7, U2OS,
H1299, and DLD-1 were cultured in Dulbecco's modified Eagle's medium
containing 10% (v/v) fetal bovine serum. NC-607 and AT-29 cells were
grown in RPMI 1640 medium with 20% fetal bovine serum. Geldanamycin
(Sigma) and okadaic acid (LC Laboratories) were dissolved in
dimethyl sulfoxide at 1 mM and used at 4 µM
and 0.1 µg/ml, respectively. MG132 was dissolved in ethanol and used
at a working concentration of 30 µM for 5 h.
Heat Shock--
Cells growing exponentially in 10-cm dishes at
37 °C were transferred to a CO2 incubator at 43 °C
for 2 h. After heat shock, the cells were cultured in a
CO2 incubator at 37 °C for various periods.
Immunoprecipitation and Western Blot--
Cells were lysed in
radioimmune precipitation buffer (1% sodium deoxycholate, 0.1% SDS,
1% Triton X-100, 50 mM Tris pH 7.4, 150 mM
NaCl, 1 mM phenylmethylsulfonyl fluoride), and 20-100
µg of protein were fractionated by SDS-PAGE and transferred to
Immobilon P filters (Millipore). The filter was blocked for 1 h
with PBS containing 5% nonfat dry milk, 0.1% Tween 20 and then
incubated for 1 h with 3G9 (MDM2) (32) or DO-1 (p53, BD
Biosciences) in PBS containing 5% nonfat dry milk. Bound primary
antibody was detected by incubating for 1 h with HRP
goat-anti-mouse IgG or HRP-protein A. The filter was developed using
the ECL-Plus reagent (Amersham Biosciences). hsp90 was detected with a
mouse anti-hsp90 antibody (StressGen). P53 phosphorylation was analyzed
for Ser-9, Ser-15, Ser-20, Ser-37, Ser-46, and Ser-392 using
anti-phospho-p53 polyclonal antibodies (Cell Signaling Technology). For
immunoprecipitation-Western blot analysis, cells were lysed in lysis
buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride) containing 10 mM molybdate
(to stabilize p53·hsp90 complex), and 1000 µg of protein were
precleared with protein A-Sepharose beads (Sigma) and
immunoprecipitated with p53 antibody Pab1801, Pab1620, and Pab240 for
4 h at 4 °C. The beads were washed with lysis buffer, and the
immunoprecipitate was fractionated by SDS-PAGE followed by Western blot
for p53 or hsp90. For detection of ubiquitinated p53 by Western blot,
the cells were lysed with Laemmli SDS sample buffer and immediately
boiled for 3 min. Before subjecting to SDS-PAGE, the lysates were
clarified by centrifugation at 14,000 × g for 15 min
at 4 °C.
Northern Blot--
Total cellular RNA was isolated using the
RNeasy kit (Qiagen). Twenty micrograms of total RNA were fractionated
on a formaldehyde denaturing gel and transferred onto a Biotrans
membrane (ICN). The filter was hybridized with a random-primed probe
synthesized using a p53 cDNA fragment. Hybridization was carried
out in a buffer containing 1% SDS, 1 M NaCl, and 10%
dextransulfate for 18 h at 65 °C. The filter was washed with
2× SSC buffer (0.3 M NaCl, 0.03 M sodium
citrate) and exposed against film. For detection of
glyceraldehyde-3-phosphate dehydrogenase mRNA
(glyceraldehyde-phosphate dehydrogenase), the filter was stripped and
rehybridized with a full-length 1.2-kilobase human
glyceraldehyde-3-phosphate dehydrogenase cDNA probe.
Immunofluorescence Staining--
Cells cultured on chamber
slides were fixed with acetone/methanol (1:1) for 3 min at room
temperature, blocked with PBS containing 10% normal goat serum for 20 min, and incubated with anti-p53 monoclonal antibody Pab1801 and rabbit
anti-MDM2 polyclonal serum in PBS with 10% normal goat serum for
2 h. The slides were washed with PBS containing 0.1% Triton
X-100, incubated with fluorescein isothiocyanate-labeled
goat-anti-mouse IgG and rhodamine-labeled goat-anti-rabbit IgG in PBS
with 10% normal goat serum for 1 h, washed with PBS with 0.1%
Triton X-100, and mounted.
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RESULTS |
Accumulation of p53 in Response to Heat Shock--
To determine
the effect of heat shock on p53 and MDM2 expression levels, A-172 cells
were incubated at 43 °C for 2 h and then returned to 37 °C
for 4-16 h. The protein levels of p53 and MDM2 were measured by
Western blot. The results showed that p53 and MDM2 levels increased
significantly 4 h after heat shock (Fig. 1, A and B).
Quantitation of the level of p53 by loading titration indicated that
heat shock caused a 6 to 8-fold increase in p53 level 4 h after
termination of heat treatment (data not shown). We found that p53
levels were also induced by heat shock in two other cell lines, U2OS
and MCF-7 (data not shown). We also examined the localization of p53
and MDM2 in A-172 cells after heat shock by immunofluorescence
staining. The results confirmed the increase of p53 and MDM2 levels
4 h after heat shock and showed that p53 mainly accumulated in the
nucleus under our experimental conditions (Fig. 1C).

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Fig. 1.
Induction of p53 and its target MDM2 by heat
shock. A, A-172 cells were incubated at 43 °C for
2 h and then cultured at 37 °C for the indicated times. Protein
extracts were subjected to Western blot analysis using specific
antibodies. B, heat shock induced p53 target MDM2. A-172
cells were incubated at 43 °C for 2 h and analyzed for p53 and
MDM2 expression after returning to 37 °C for 4 h. C,
double immunofluorescence staining of p53 and MDM2 in A-172 cells
4 h after heat shock. Upper panels, p53; lower
panels, MDM2. Ctrl, control cells at 37 °C;
HS, cells treated with heat shock; MG132, cells
treated with 30 µM MG132 for 4 h; CPT,
cells treated with 1 µM camptothecin for 6 h.
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To investigate the mechanism of p53 accumulation, the mRNA levels
of p53 were determined by Northern blot. The results showed that p53
mRNA level in A-172 and MCF-7 cells did not change after heat shock
(Fig. 2A). Therefore,
increased p53 protein expression was not attributed to increase in
transcription. To test whether p53 was stabilized after heat shock,
A-172 cells were treated with heat shock and the proteasome inhibitor
MG132. The magnitude of p53 accumulation after MG132 treatment provided
an indication of how rapidly p53 was degraded by
ubiquitin-dependent proteasomes. The result showed that in
control cells, a 4-h treatment with MG132 caused a significant increase
in p53 level. However, after heat shock, MG132 treatment did not lead
to further increase of p53 level compared with heat shock alone (Fig.
2B), indicating that it was already stabilized. This
suggested that the increased p53 level was because of reduced p53
degradation by the proteasomes.

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Fig. 2.
Stabilization of p53 by heat shock.
A, p53 and GADPH mRNA levels were determined by Northern
blot in control and heat shock-treated cells. B, p53 protein
levels were determined in A-172 cells 4 h after heat shock in the
presence or absence of 30 µM MG132. C, p53
ubiquitination level was determined by rapid lysis in SDS sample buffer
and Western blot. The amount of control loaded was increased 8-fold for
the 4-h time point to match the increased p53 in heat-treated
cells.
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Because p53 turnover is regulated by MDM2-mediated ubiquitination, we
examined the level of p53 ubiquitination by direct lysis in SDS sample
buffer, which preserved polyubiquitinated forms of p53. Western blot of
such lysate showed that the high molecular weight bands of
ubiquitinated p53 were significantly reduced immediately after heat
shock (Fig. 2C). There was significant recovery of p53
ubiquitination at 4 h after heat shock, which may be because of
high level MDM2 expression at this time point and the start of a
recovery process (Fig. 1B). Therefore, the ability of MDM2 to promote p53 ubiquitination was inhibited after heat shock, consistent with the increase in p53 stability.
Phosphorylation of p53 in A-172 Cells after Heat Shock and
Camptothecin (CPT) Treatment--
Because it is well established that
the stability of p53 is regulated by phosphorylation, we examined the
phosphorylation status of p53 at several different sites after heat
shock. The DNA-damaging agent CPT was used as a positive control.
Furthermore, to control for the increased levels of p53 protein after
heat shock, we also used MG132 treatment to induce p53 accumulation at
37 °C. Antibodies recognizing different phosphorylated serine residues on p53 were used in Western blot detection of phosphorylated p53. The results showed significant increase of phosphorylation level
at Ser-15 after heat shock (Fig. 3).
Phosphorylation of Ser-20 was also reproducibly induced by heat shock,
although it was less than that induced by CPT treatment. The
phosphorylation level of Ser-46 was induced by CPT but not by heat
shock. The anti-phospho-Ser-392 antibody also did not detect a change
after heat shock (Fig. 3), whereas the UV irradiation control induced moderate increase in signal as expected (Fig. 3, bottom
panels) (33). The results of Ser-9 and Ser-37 phosphorylation were
uninformative because they were not induced by heat shock or the CPT
control (data not shown). In summary, heat shock selectively induced
phosphorylation of p53 at Ser-15 and Ser-20, suggesting the involvement
of ATM and human Chk2 kinases.

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Fig. 3.
Phosphorylation of p53 at different serine
residues in response to heat shock and DNA damage. A-172 cells
were heated at 43 °C for 2 h and then cultured at 37 °C for
4 h. Control cells were treated with 30 µM MG132, 1 µM CPT, or 20 J/m2 UV for 6 h at
37 °C. Cells were lysed in buffer containing okadaic acid, and
identical amounts of protein extracts were subjected to Western blot
analysis using phospho-peptide-specific antibodies raised against
indicated sites on p53. Total levels of p53 were determined by Western
blot with a mixture of DO-1 and Pab1801.
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Phosphorylation of p53 at Ser-15 Is
ATM-dependent--
Ser-15 phosphorylation of p53 after DNA
damage is carried out by the ATM kinase (34). Therefore, we
investigated the role of ATM in Ser-15 phosphorylation after heat shock
using immortalized lymphocytes derived from normal (NC-607) and AT
patients (AT-29). The results showed that Ser-15 phosphorylation was
not induced by heat shock in ATM-deficient lymphocytes, whereas strong
induction was observed in normal lymphocytes (Fig.
4). Therefore, Ser-15 phosphorylation
after heat shock was dependent on ATM kinase.

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Fig. 4.
Accumulation of p53 and phosphorylation at
serine 15 in NC-607 (normal) and AT-29 (ATM-deficient)
lymphocytes. Cells were heated at 43 °C for 2 h and then
cultured at 37 °C for 4 h. Indicated amounts of total cellular
proteins were analyzed for p53 level by Western blot with a mixture of
DO-1 and Pab1801, and serine 15 phosphorylation level was determined by
phospho-Ser-15-specific Western blot.
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Similar to A-172 cells, heat shock also caused over 3-fold induction of
p53 protein level in normal lymphocytes. Although Ser-15
phosphorylation level did not change in ATM-deficient lymphocytes after
heat shock, p53 level was still moderately increased (<3-fold) (Fig.
4). Therefore, phosphorylation of Ser-15 by ATM contributed to but was
not absolutely required for p53 accumulation after heat shock. This
result also suggested that additional mechanisms contribute to p53
stabilization after heat shock.
p53 Conformational Change after Heat Shock--
To test the
hypothesis that heat shock caused wild type p53 conformational change,
p53 was precipitated by mutant-specific Pab240 and wild type-specific
Pab1620 antibodies (35). Pab240 recognizes hydrophobic peptide
sequences of p53 normally packed inside the DNA-binding core domain
(36); therefore, the exposure of the Pab240 epitope is indicative of
misfolded p53. The result showed that in control A-172 cells, p53 could
be precipitated efficiently by Pab1620 but weakly by Pab240 (Fig.
5A). Conversely, heat shock
significantly increased reaction with Pab240 when the cells were
harvested 2 h after heat shock treatment (Fig. 5A), although a significant fraction of p53 also retained Pab1620
reactivity. At 4 h after heat shock, which was the highest point
of p53 accumulation, p53 reactivity to Pab1620 increased strongly and
Pab240 reactivity reduced significantly. Therefore, there was rapid
recovery of p53 conformation back to the wild type status after heat
shock. The result also showed that change of p53 conformation
correlated with the level of p53 ubiquitination (Fig. 2C).
Immediately after heat shock, there was significant reduction of
ubiquitinated p53 species, which recovered to near control level 4 h after heat shock. Therefore, change in p53 conformation may also play
a role in stabilizing p53.

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Fig. 5.
Conformational change of wild type p53 and
interaction with hsp90 after heat shock. A, A-172 cells
were treated with heat shock, and p53 was immunoprecipitated by wild
type-specific Pab1620 and mutant-specific Pab240 using identical
amounts of cell lysate followed by Western blot with DO-1.
B, A-172 cells were immunoprecipitated with Pab1801 after
heat shock using identical amounts of cell lysate followed by
anti-hsp90 Western blot. Control A-172 and DLD-1 (mutant p53) cells
were maintained at 37 °C.
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Wild Type p53 Binds to hsp90 after Heat Shock--
hsp90 binding
is important for preventing degradation of many cellular proteins
including mutant p53. Disruption of hsp90 mutant p53 binding leads to
destabilization of p53 through ubiquitin-dependent proteasomes (25, 27, 29, 37). Because heat shock induced significant
conformational change in wild type p53, we asked whether hsp90 also
regulates wild type p53 stability by binding to p53 and preventing its
degradation by MDM2.
To test the binding between hsp90 and p53, conformation-independent
Pab1801 antibody was used to precipitate p53 from cell lysate harvested
2 and 4 h after heat shock. Coprecipitated hsp90 was detected by
Western blot of the p53 precipitate using hsp90-specific antibody. The
results showed that hsp90 did not coprecipitate with wild type p53 in
the control A-172 cultures without heat shock treatment (Fig.
5B). However, significant amounts of hsp90 coprecipitated
with the mutant p53 protein in DLD-1 cells at 37 °C and also with
wild type p53 in the heat-treated A-172 cells (Fig. 5B). The
binding between p53 and hsp90 was stronger at 2 h after heat shock
than 4 h, because more p53 was present in the 4-h lysate yet the
amount of hsp90 coprecipitated did not increase (Fig. 5B).
This was consistent with the significant recovery of wild type p53
conformation and ubiquitination level observed at this time point.
Therefore, heat shock induced transient association between wild type
p53 and hsp90.
Inhibition of hsp90 Prevents p53 Accumulation after Heat
Shock--
Binding between hsp90 and mutant p53 is known to cause p53
stabilization (25, 27, 29, 37). Therefore, we examined the role of
hsp90 on wild type p53 stability after heat shock. A-172, NC-607, and
AT-29 cells were treated with heat shock in the presence or absence of
the hsp90 inhibitor geldanamycin. The level of p53 was measured by
Western blot 4 h after heat shock. The results showed that without
heat shock, geldanamycin treatment did not significantly affect p53
level. However, geldanamycin prevented the accumulation of p53 after
heat shock in ATM-deficient AT-29 cells (Fig.
6A), suggesting that hsp90
function was important for p53 accumulation. The inhibitory effect of
geldanamycin was not as complete in A-172 and NC-607 cells, probably
because ATM-mediated phosphorylation also contributed to p53
stabilization.

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Fig. 6.
hsp90 inhibitor geldanamycin blocks p53
accumulation after heat shock. A, cells were treated
with heat shock in the presence or absence of 4 µM
geldanamycin (GA), and p53 levels were determined by Western
blot after 4 h. B, the effect of geldanamycin on
p53·hsp90 binding after heat shock was determined by treating
cells in the presence of 4 µM geldanamycin and
immunoprecipitation of p53 with Pab1801 4 h after heat shock.
Coprecipitation of hsp90 was detected by Western blot. The amounts of
cell lysate used for precipitation were adjusted to contain a similar
amount of p53.
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Geldanamycin binds to the ATP-binding site of hsp90 and often reduces
its binding to client proteins (26). To confirm that geldanamycin
blocked hsp90-p53 binding after heat shock, A-172, NC-607, and AT-29
cells were treated with heat shock and geldanamycin. hsp90-p53 binding
was detected by p53 immunoprecipitation followed by hsp90 Western blot.
The result showed that geldanamycin treatment reduced hsp90-p53
coprecipitation in the three cell lines (Fig. 6B).
Therefore, inhibition of hsp90-p53 binding after heat shock prevented
p53 accumulation.
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DISCUSSION |
The results described above show that short term exposure to
elevated temperature causes accumulation and activation of wild type
p53. Increased p53 expression level is not because of induction of p53
transcription, because its mRNA level does not change after heat
stress. Similar to other forms of p53 stress response, reduced ubiquitination and degradation appears to be the major cause of its
accumulation after heat shock. The stabilized p53 is functional in
inducing expression of downstream target genes and has been shown to
contribute to cell cycle arrest or apoptosis response after heat shock
(13, 15). Therefore, p53 activation after heat shock is a functionally
relevant response to environmental stress.
Ubiquitination and degradation of p53 is mainly regulated by MDM2. Many
p53 responses to environmental or cellular stresses cause p53
stabilization by interfering with MDM2 function. Phosphorylation of p53
and MDM2, inhibition of MDM2 expression, and inactivation of MDM2 by
expression of ARF have all been implicated in wild type p53
accumulation (1, 38). Furthermore, stabilization of mutant p53 involves
binding to molecular chaperones hsp90 (25, 27, 37). hsp90-mutant p53
complex is resistant to MDM2-mediated ubiquitination and degradation,
because MDM2 bound to p53 in such a complex is inactivated by hsp90
(29). hsp90 appears to conceal the ARF binding site on MDM2 in
hsp90·p53·MDM2 complexes and prevent MDM2 from ubiquitinating p53
and itself. As a result, both MDM2 and mutant p53 become stabilized in
tumor cells expressing mutant p53 (28). We previously showed that
dissociation of hsp90 by geldanamycin treatment restores the
ubiquitination and degradation of mutant p53 (29). Therefore,
hsp90-mutant p53 complex also causes p53 stabilization by
interfering with MDM2 activity.
Molecular chaperones such as hsp70 and hsp90 recognize exposed
hydrophobic regions in misfolded proteins, nascent polypeptides, and
ligand binding sites of steroid hormone receptors (26, 39). Wild type
p53 is a structurally unstable protein and readily undergoes conformational change above 40 °C in vitro (30). Point
mutations in the DNA-binding domain disrupt proper folding and cause
structural change at physiological temperatures. Different mutations
lead to different temperature sensitivities; some mutations (such as the Val-138) remain functional and conformationally wild type at
32 °C and lose activity at 39 °C (40). Many hot spot mutations found in human tumors cause significant conformational changes and
complete loss of activity at physiological temperature (41). Therefore,
it is conceivable that heat shock also causes transient conformational change of wild type p53 similar to the misfolding caused
by structural mutations, resulting in binding by hsp90.
We suggest that heat shock causes a transient misfolding of wild type
p53, which attracts binding by hsp90. hsp90 binding would prevent p53
aggregation and facilitate the refolding process. However, hsp90
binding would also prevent MDM2-mediated degradation in a manner
similar to stabilization of mutant p53 by hsp90 at 37 °C. Unlike
mutant p53, which cannot fold properly even in the presence of
molecular chaperones, heat-denatured wild type p53 can be efficiently
refolded by chaperones at 37 °C. Therefore, the conformational
change after heat shock is transient, and p53 level begins to decrease
several hours after heat shock. This correlates with return of
normal p53 ubiquitination level.
However, hsp90 appears not to be the only regulator of p53
stabilization after heat shock, because the hsp90 inhibitor
geldanamycin did not cause a complete block in p53 accumulation in ATM
wild type cell lines. We found that heat shock also induced
phosphorylation of p53 at Ser-15 and Ser-20, which are sites important
for regulating the stability and transcription activity of p53 (1).
Phosphorylation of Ser-15 and Ser-20 is dependent on ATM kinase
activity and ATM-mediated activation of human Chk2 kinase, suggesting
that heat shock can activate the ATM kinase and induce p53 accumulation
in part through phosphorylation. A previous study (21) shows
that heat shock causes low levels of DNA damage. Our results also
showed that heat shock only induced phosphorylation of p53 on Ser-15
and Ser-20, whereas the DNA-damaging agent camptothecin induced
stronger phosphorylation of Ser-15 and Ser-20 as well as an additional
phosphorylation at Ser-46. This is consistent with the requirement of
severe DNA damage for inducing Ser-46 phosphorylation (42). Therefore, heat shock may activate ATM through inducing low level of DNA damage,
which in turn activates p53 by phosphorylation of p53 or MDM2 (8). DNA
damage-mediated dephosphorylation of MDM2-acidic domain may also play a
role in stabilization of p53 (43). During the course or our study,
Miyakoda et al. (17) also reported that phosphorylation of
p53 at Ser-15 occurs after heat shock in an ATM-dependent
fashion, which is consistent with our finding.
In summary, ATM-mediated phosphorylation and conformation-mediated
hsp90 binding cooperate to cause p53 accumulation in response to heat
stress. Our results identify the first example in which hsp90 plays a
role in regulating wild type p53 stability in response to stress.
Conformational change of p53 has been suggested to occur in the absence
of heat stress based on change of reactivity to conformation-specific
antibodies (44). Therefore, it will be interesting to further
investigate whether hsp90 plays a role in regulating p53 stability in
other physiological processes.