It has long been appreciated that many mutant p53 species display unusually stable physical association with the heat shock protein hsc70 both in tissue culture cells and in human tumor specimens (7
). This association has been reported to regulate p53 conformation in vitro (12
) and correlates with increased transforming activity in intact cells (13
). In the present study, we made use of a temperature-sensitive p53 mutant and the selective hsp90-binding agent GA to establish a role for multiple molecular chaperones, not just hsc70, in modulating the stability, localization, and function of mutant and wild-type p53 within a conserved cellular background. The biologic relevance of these findings was then confirmed by demonstrating a similar pattern of chaperone interactions with the mutant p53 species expressed by some human breast cancer cell lines.
Although the biochemical activity of many molecular chaperones is well established, much less is known about how chaperones act together in large heteromeric complexes to regulate the posttranslational function of diverse kinases, receptors, and transcription factors (29
). The best-studied example of chaperone-mediated conformational regulation in eukaryotic cells is that of steroid hormone receptors, which require the interaction of multiple chaperones to acquire or maintain a state competent to bind hormone (30
). Through a series of ATP-dependent reactions, an immature hormone receptor complex that contains hsp90, hsp70, and at least the two cochaperones hip (p48) and hop (p60) is maintained in dynamic equilibrium with a more favored mature complex which is competent to bind hormone (4
). This mature complex lacks hsp70, hip, and hop but contains at least two new proteins, p23 and one of the three large immunophilins FKBP52, FKBP54, and Cyp40. Upon hormone binding, the receptor is no longer found in association with chaperone proteins and becomes active as a transcription factor (8
). Treatment with GA has been shown to block the transition of steroid receptors from immature to mature complexes, thus preventing hormone binding (36
) and resulting in enhanced ubiquitination and proteasome-mediated degradation of the hormone-binding protein (38
We now report that analogous to steroid receptors, p53 coprecipitated in lysate with multiple molecular chaperones in addition to hsp70 when cells were maintained under conditions favoring mutant p53 conformation and function (Fig. ). As recently reported by Sepehrnia et al. for A1-5 cells (34
) and Selkirk et al. for T47D breast cancer cells (33
), we also found hsp90 coprecipitating with mutant p53. In addition, however, we detected coprecipitation of p23 and the large immunophilin Cyp40, components characteristic of more mature steroid receptor complexes. Immunodepletion studies indicated that at least 70% of the p53 in A1-5 cells grown under mutant temperature conditions was associated with a p23-containing chaperone complex (Fig. ), while a significantly smaller fraction appeared to be associated with hsp70. Such a pattern is consistent with that reported for unliganded progesterone receptors (35
). Upon a temperature shift, coprecipitation of all of these chaperones was markedly reduced (Fig. ), yielding a p53 species capable of acting as a transcription factor (Fig. and ).
Treatment of A1-5 cells with GA resulted in an effect on p53 similar to that observed with steroid receptors, namely, loss of mature complex components and enhancement of intermediate components, as indicated by the increased hop signal seen in Fig. A, lane 4. Despite these similarities, however, differences also were apparent. Specifically, hsp90 is present in both intermediate and mature progesterone and glucocorticoid receptor complexes (36
), while with mutant p53, we found that hsp90 was lost in complexes from GA-treated A1-5 and breast cancer cells even though p53 appeared to be trapped in intermediate, hop-containing complexes (Fig. to ). In this respect, mutant p53 appeared to behave more like transforming tyrosine kinases such as v-Src. With these targets, association with hsp70 and hsp90 is detectable under control conditions and hsp90 association is disrupted by GA (39
). While chaperone interactions appear relatively well conserved, differences do exist in the associations seen with specific targets, the functional significance of which is unclear at this time (29
As with the glucocorticoid receptor, trapping of p53 in an apparently intermediate complex by GA stimulated its degradation (Fig. and ) but did not render it active as a transcription factor (Fig. to ). The concept that association with other proteins can modulate the degradation of p53 is supported by recent studies demonstrating such a role for mdm-2 (15
). GA-stimulated degradation of mutant p53 in our system, however, does not appear to be mediated through mdm-2, as no induction of the protein could be detected following GA treatment of A1-5 cells. Our finding that GA failed to restore transcription factor activity to mutant p53 species agrees with a previous study reporting that although GA treatment alters the conformation of mutated p53 as measured by IP with conformation-specific antibody, it only partially restores its ability to bind a consensus DNA sequence (3
). It seems most likely that GA’s failure to restore p53 function as a transcription factor results from the continued association of p53 with chaperones such as hsp70 and hop in GA-treated cells. These persistent associations may impair oligomerization or posttranslational modifications such as phosphorylation which are required for p53 activity as a transcription factor (21
). It is also possible that GA interferes directly with the function of some of the various kinases which have been proposed to be involved in activating p53 such as casein kinase II or raf-1 (reviewed in reference 25
At this point, we do not know whether the chaperone components that we have identified coprecipitate with p53 as a single complex or several distinct complexes (Fig. ), but in vitro reconstitution experiments in reticulocyte lysate may be able to address this issue directly in future studies. It is not possible to comment on the stoichiometry of the components observed in our coprecipitation experiments for two reasons. First, complexes are isolated by IP under nonequilibrium conditions, which may allow for their gradual dissociation during the procedure. Second, inherent variation in the affinity of the antibodies used to detect components by Western blotting makes it impossible to directly compare the absolute amounts of each protein detected.
It is interesting to speculate that the extended chaperone interactions we have observed with mutant p53 actually represent a pathologic exaggeration of normal, physiologic interactions of wild-type p53 with the chaperone machinery. Due to its intrinsic conformational lability, the turnover and function of wild-type p53 could be regulated to a significant extent by ongoing posttranslational interactions with components of the chaperone machinery. Such interactions may become detectable by coimmunoprecipitation only when they become pathologically extended as a consequence of mutation of the target. Under normal conditions, their transient nature may serve to modulate the presentation of p53 for degradation by the ubiquitin-proteasome system. Because they involve heat shock proteins, these same interactions could also serve as sensors of cell stress or damage. Altering their levels and availability by insults such as ionizing radiation or alkylating agents could lead to p53 stabilization and provide a mechanism for its rapid activation as a transcription factor in response to cellular damage. Consistent with this proposal, cellular stresses that do not involve DNA damage have been shown to induce p53 activation (20
). In addition, salicylate concentrations which inhibit the heat shock response have been shown to inhibit p53 activation in response to UV irradiation and the chemotherapeutic agent adriamycin (5
In summary, we have shown that several mutant p53 species, but not wild-type p53, are stably associated with a conserved group of molecular chaperones. Mutant p53 molecules, presumably due to specific alterations in conformation, appeared to be retained within this molecular chaperone machinery, leading to their mislocalization and protection from degradation. Alteration of specific chaperone interactions by GA treatment resulted in destabilization of mutant proteins, supporting the view that posttranslational interaction with certain chaperone heteroprotein complexes may stabilize a target while interaction with others may actually stimulate its degradation (10
). Taken together, our findings demonstrate that chaperone proteins play an important role in modulating the function of many mutant p53 species and suggest that they could be involved in regulating the activity of wild-type protein in response to cellular stress.