Phosphorylation and hsp90 Binding Mediate Heat Shock Stabilization of p53*

Chuangui Wang and Jiandong ChenDagger

From the Molecular Oncology Program, H. Lee Moffitt Comprehensive Cancer Center and Research Institute, Tampa, Florida 33612

Received for publication, July 6, 2002, and in revised form, November 7, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The p53 tumor suppressor is stabilized and activated by diverse stress signals. In this study, we investigated the mechanism of p53 activation by heat shock. We found that heat shock inhibited p53 ubiquitination and caused accumulation of p53 at the post-transcriptional level. Heat shock induced phosphorylation of p53 at serine 15 in an ATM kinase-dependent fashion, which may contribute partially to heat-induced p53 accumulation. However, p53 accumulation also occurred after heat shock in ATM-deficient cells. Heat shock induced conformational change of wild type p53 and binding to hsp90. Inhibition of hsp90-p53 interaction by geldanamycin prevented p53 accumulation partially in ATM-wild type cells and completely in ATM-deficient cells. Therefore, phosphorylation and interaction with hsp90 both contribute to stabilization of p53 after heat shock.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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.

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.

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.

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.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENT

We would like to thank Dr. Kevin Brown for providing the ATM-deficient cells.

    FOOTNOTES

* This work was supported by American Cancer Society Grant RSG CNE-102445 and National Institutes of Health Grant CA88406 (to J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: H. Lee Moffitt Cancer Center, MRC3057A, 12902 Magnolia Dr., Tampa, FL 33612. Tel.: 813-903-6822; Fax: 813-903-6817; E-mail: jchen@moffitt.usf.edu.

Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M206697200

    ABBREVIATIONS

The abbreviations used are: MDM, murine double minute; ARF, alternative reading frame; ATM, Ataxia-telangiectasin-mutated protein kinase; E3, ubiquitin-protein isopeptide ligase; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; CPT, camptothecin; NC, normal control.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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