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Direct Activation of Bax by p53 Mediates Mitochondrial Membrane Permeabilization and Apoptosis

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Science  13 Feb 2004:
Vol. 303, Issue 5660, pp. 1010-1014
DOI: 10.1126/science.1092734

Abstract

The tumor suppressor p53 exerts its anti-neoplastic activity primarily through the induction of apoptosis. We found that cytosolic localization of endogenous wild-type or trans-activation–deficient p53 was necessary and sufficient for apoptosis. p53 directly activated the proapoptotic Bcl-2protein Bax in the absence of other proteins to permeabilize mitochondria and engage the apoptotic program. p53 also released both proapoptotic multidomain proteins and BH3-only proteins [Proapoptotic Bcl-2family proteins that share only the third Bcl-2homology domain (BH3)] that were sequestered by Bcl-xL. The transcription-independent activation of Bax by p53 occurred with similar kinetics and concentrations to those produced by activated Bid. We propose that when p53 accumulates in the cytosol, it can function analogously to the BH3-only subset of proapoptotic Bcl-2proteins to activate Bax and trigger apoptosis.

The induction of apoptosis is central to the tumor-suppressive activity of p53 (1). Upon activation by DNA damage–induced or oncogene-induced signaling pathways, p53 promotes the expression of a number of genes that are involved in apoptosis, including those encoding death receptors (2, 3) and proapoptotic members of the Bcl-2 family (4, 5). In most cases, p53-induced apoptosis proceeds through mitochondrial release of cytochrome c, which leads to caspase activation (6).

Although most of the effects of p53 are ascribed to its function as a transcription factor, reports have suggested that the protein can also induce apoptosis independently of new protein synthesis (710). However, these studies have relied on ectopic expression of p53 or overexpression of mutants that lack transcriptional activity. Transcription-independent induction of apoptosis by p53 requires Bax and involves cytochrome c release and caspase activation, all of which occur in the absence of a nucleus, suggesting that p53 has the capacity to engage the apoptotic program directly from the cytoplasm (11).

We therefore tested if endogenous p53 can engage the apoptotic program directly from the cytoplasm in the absence of p53-induced transcription. We used E1A/H-rasG12V–transformed wild-type p53 (p53wt), Bax–/–, and p53–/– mouse embryonic fibroblasts (MEF) and an inhibitor of nuclear import, wheat germ agglutinin (WGA). p53 accumulated in both the nucleus and cytoplasm of p53wt and Bax–/– MEF that were exposed to ultraviolet (UV) light; however, pretreatment of cells with WGA blocked the nuclear import of p53 such that p53 was found exclusively in the cytoplasm (Fig. 1A). WGA effectively inhibited UV-induced p53-dependent induction of several p53-responsive genes, assayed by real-time polymerase chain reaction (PCR) (mdm2, bax, p21WAF1/CIP1, and PUMA; fig. S1A) and Western blot (Bax and p21WAF1/CIP1; fig. S1B). Whereas p53-dependent gene expression was blocked by the inhibition of p53 nuclear accumulation, the induction of apoptosis by UV (UV dose responses in fig. S2) proceeded in p53wt MEF even though p53 was exclusively cytoplasmic (Fig. 1B). In contrast, Bax–/– MEF were resistant to UV-induced apoptosis in the presence of WGA, suggesting that cytosolic p53 cooperates with Bax to induce apoptosis in the absence of p53-dependent gene regulation (Fig. 1B). The expression of a sublethal concentration of Bax rescued apoptosis under these conditions (Fig. 1C). The decrease in apoptosis following UV with WGA may represent the requirement for p53-dependent regulation of PUMA (1214) or other apoptosis-promoting proteins in the cells without WGA. UV-induced apoptosis without WGA was observed in Bax–/– MEF (Fig. 1B) suggesting that transcription-dependent death is not Bax dependent in these cells.

Fig. 1.

Cooperation of endogenous cytoplasmic p53 with Bax to induce apoptosis in the absence of p53 nuclear activity. (A) UV-induced nuclear accumulation of p53 is blocked by WGA. E1A/H-rasG12V–expressing p53wt, Bax–/–, and p53–/– MEF were transfected with WGA by means of the Chariot protein delivery system (20 μg WGA per 100-mm2 plate), treated with 5-mJ UV for 8 hours, and processed into total (T), nuclear (N), and cytoplasmic (C) fractions before immunoprecipitation with an antibody to p53. Starting materials for the immunoprecipitations were subjected to Western blot for proliferating cell nuclear antigen (PCNA, a nuclear marker) and actin (a cytoplasmic marker). (B) UV-induced cytosolic p53 failed to induce apoptosis in the absence of Bax. Above MEF were treated as indicated, harvested, and stained with Annexin V–FITC before fluorescence-activated cell sorting analysis (FACS). (C) UV-induced cytoplasmic p53-initiated apoptosis was rescued after re-expression of Bax. Bax–/– MEF were transfected with a sublethal dose of Bax, cultured for 24 hours, and treated as indicated before Annexin V–FITC staining and FACS analysis. (D and E) Inhibition of nuclear import allows for trans-activation–deficient p53 (p53QS) to accumulate in the cytosol and induce death. Primary untransformed MEF were treated as described in (A) and (B).

We analyzed a transcriptionally inactive form of p53 that exclusively displays nuclear localization and does not support p53-dependent apoptosis (15). We reasoned that this mutant, Trp53L25Q,W26S (referred to as p53QS), might not induce apoptosis because it failed to be exported to the cytosol. When primary untransformed p53QS knock-in MEF were treated with WGA, p53QS was present in both the cytosol and nucleus (Fig. 1D). The cytosolic accumulation of p53QS was associated with cell death even in the absence of UV exposure (Fig. 1E). We speculate that wild-type p53 does not induce death upon WGA treatment without UV because of its proper regulation by MDM2 and other proteins, whereas p53QS accumulates with WGA alone (and therefore triggers apoptosis). As controls, wild-type MEF (primary and transformed) responded to 5-mJ UV by p53-dependent gene regulation and apoptosis (Fig. 1, B and E, and figs. S1 and S2), whereas p53–/– MEF were resistant to 5-mJ UV and WGA-induced effects (Figs. 1, B and E, and figs. S1 and S2).

These results suggest that it is the failure of p53 to localize in the cytosol, rather than a defect in p53-induced transcription of proapoptotic genes, that accounts for the loss of p53-mediated apoptosis in p53QS cells. Indeed, a stably expressed temperature-sensitive form of p53QS, localized to the cytosol, was reported to induce apoptosis upon stabilization (10). To test the direct cytoplasmic effects of p53 in the absence of p53-induced gene expression, p53UVIP, p53ΔPP (no proline-rich domain, amino acids 62 to 91), and p53QS were purified (fig. S3, A and B) and microinjected into HeLa cells stably expressing a cytochrome c–green fluorescent protein (GFP) fusion protein localized to mitochondria (16). To prevent new protein expression in response to p53 or p53 nuclear import, cycloheximide (CHX) or WGA was added, respectively. Although injection of immunoprecipitates from non–UV-treated cell lysates lacking p53 had no effect on these cells (or on isolated mitochondria, fig. S3D), p53UVIP and p53QS each induced cytochrome c release in the microinjected cells (Fig. 2A, quantitation in fig. S3C). Microinjected p53ΔPP failed to induce cytochrome c release (Fig. 2A and fig. S3C). The amounts of microinjected proteins were in the range of 4 to 40 fg per cell, consistent with the amount of p53 that was isolated from the cytosols of UV-treated MCF-7 cells (17).

Fig. 2.

Cooperation of mammalian and recombinant p53 with Bax to induce mitochondrial permeabilization. (A) Wild-type p53 (p53UVIP), p53ΔPP, and trans-activation–deficient p53 (p53QS) were immunopurified (fig. S3, A and B) and microinjected into HeLa cells stably expressing cytochrome c–GFP (green) (16) and pretreated with CHX (50 μg/ml) or WGA (3 μg/9.4 cm2) and 100 μM z-Val-Ala-Asp(OMe)-fluoromethylketone. To observe microinjected cells, fluorescently labeled dextran (red) was comicroinjected; control immunoprecipitates (anti-p53–purified material from untreated MCF-7 cells) were also tested. Greater than 400 cells were quantified for cytochrome c release (fig. S3C). CHX- and WGA-treated cells were analyzed 4 hours postinjection. (B and C) Effects of p53 on isolated mitochondria. Immunopurified p53UVIP (B) or p53Bac (C) was added to freshly isolated C57Bl/6 liver mitochondria (30) with and without recombinant full-length Bax (31) and incubated at 37°C for 60 min. Supernatants and mitochondrial pellets were then analyzed for cytochrome c by Western blot. The combination of Bax and BidN/C is a positive control for mitochondrial permeabilization. (D) Immunopurified p53ERtam or p53ΔPPERtam (fig. S3, A and B) was analyzed with or without 4-OHT for mitochondrial permeabilization as in (B). 10 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid pH 7.4, 1 mM ethylenediaminetetraacetic acid (HE) is the buffer for recombinant proteins. DMSO, dimethyl sulfoxide.

During apoptosis, the release of cytochrome c and other proteins from the mitochondrial intermembrane space is controlled by the Bcl-2 family of proteins (18). A subset of these proteins, including Bid and Bim, share the third Bcl-2 homology domain (BH3) with other family members, and thus are called “BH3-only” proteins. These proteins activate another set of proteins, including Bax and Bak, which contain three of the BH domains (“BH123” or “multidomain” proteins), to permeabilize the mitochondrial outer membrane leading to the release of cytochrome c and other proteins from the intermembrane space. Apoptosis induced by p53 is dependent on Bax and Bak (19), and the lack of Bax in MEF partially circumvents p53-mediated suppression of transformation by oncogenes (4, 20, 21).

Thus, we examined the effect of p53 on isolated mitochondria with and without Bax. Neither recombinant monomeric Bax nor native p53UVIP induced cytochrome c release on its own; however, incubation of mitochondria with both proteins caused cytochrome c release (Fig. 2B). Highly purified, non–stress induced, baculovirus-expressed p53 (p53Bac) also induced cytochrome c release in the presence of Bax (Fig. 2C). These results suggest that neither stress-associated p53 modifications nor other proteins synthesized or activated upon stress are required for this p53 effect. Although the mouse liver mitochondria have associated Bak (22), this appeared to be insufficient to support the p53-induced cytochrome c release. Therefore, this previously unobserved cytoplasmic activity of p53 seems to require Bax.

A fusion protein composed of p53 and the steroid-binding domain of the estrogen receptor, mutated to respond to tamoxifen (p53ERtam), functions in cells to trigger p53-dependent gene expression and apoptosis (23). We observed that the transient expression of this fusion protein in transformed wild-type MEF caused apoptosis upon addition of 4-hydroxytamoxifen (4-OHT), which was not inhibited by CHX (fig. S4A). A mutant form of p53 lacking the proline-rich domain (p53ΔPPERtam) failed to induce apoptosis with and without 4-OHT. p53ERtam, expressed in MEF lacking either Bax alone or Bax and Bak, did not trigger cell death unless Bax was ectopically expressed at subapoptotic levels (fig. S4B). Therefore, the transcription-independent activity of p53 appears to be more dependent on Bax in MEF, as suggested by our previous studies (11).

Purified p53ERtam or p53ΔPPERtam incubated with mitochondria had no effect with or without 4-OHT (Fig. 2D). However, mitochondria released cytochrome c when incubated with recombinant Bax plus active p53ERtam (i.e., with 4-OHT); the p53ΔPPERtam lacked this effect. Therefore, the function of p53ERtam on Bax appears to require the proline-rich domain of the p53 molecule.

We also tested the effect of p53 on the membrane permeabilizing activity of Bax in a lipid vesicle system that faithfully mimics mitochondrial outer membrane permeabilization (24). No effects of recombinant monomeric Bax, recombinant activated Bid (BidN/C), p53UVIP, or p53Bac were observed on the release of labeled dextrans from liposomes when added alone, whereas the addition of Bax and BidN/C together permeabilized the liposomes (24). The addition of native p53UVIP with Bax also caused permeabilization of the liposomes and release of fluorescein isothiocyanate (FITC)–dextran. In the liposome system, 5 nM p53UVIP or p53Bac was effective (Fig. 3A); similar concentrations permeabilized isolated mitochondria (Fig. 2, B and C). In contrast, p53ΔPP had substantially reduced permeabilization activity in this system (Fig. 3B). p53ERtam also functioned in the liposome system; as little as 130 pM p53ERtam showed effects in the presence of 4-OHT and recombinant Bax (fig. S5). In contrast, the p53ΔPPERtam mutant had no effect with or without 4-OHT.

Fig. 3.

Cooperation of mammalian and recombinant p53 with Bax to permeabilize synthetic liposomes. (A and B) Liposomes formed from defined lipid mixtures containing fluorescein (F)–dextran (24) were combined with indicated concentrations of p53UVIP, p53Bac, p53ΔPP, or p53QS with and without recombinant full-length Bax (31). The combination of Bax and BidN/C is a positive control for liposome permeabilization. Maximal release is determined by 1% CHAPS solubilization of liposomes. (C) Liposomes were combined with full-length Bax (0.25 ng) and either 45 nM p53 (all types indicated) or BidN/C in the presence of 1 nM BMH before SDS–polyacrylamide gel electrophoresis (PAGE) and Western blot analysis for Bax. Recombinant monomeric Bax (determined by gel filtration) is primarily dimeric after SDS-PAGE analysis (25).

We then examined the activation of Bax as indicated by Bax oligomerization (25) after treatment with p53 in the liposome system with the cross-linking reagent, 1,6-bismaleimidohexane (BMH). Bax oligomers were induced by all wild-type p53 proteins and p53QS, similar to BidN/C at equimolar concentrations (Fig. 3C); both p53ΔPP and p53ΔPPERtam failed to induce Bax oligomers.

Two proteins, Bid and Bim, activate Bax in this manner, and both are BH3-only members of the Bcl-2 family (19, 26). These are thought to act in a “hit-and-run” manner to induce a conformational change, triggering their oligomerization (27). We were unable to detect a physical binding of p53 and Bax (22). However, BH3-only proteins, including Bid and Bim, bind to Bcl-2 and Bcl-xL (26), and the latter proteins block apoptosis through the sequestration of proapoptotic Bcl-2 members. Some BH3-only proteins (e.g., Bad) do not activate Bax, but instead appear to promote apoptosis by releasing Bid and Bim from Bcl-2 or Bcl-xL (25, 28).

p53 has been reported to bind to Bcl-xL and Bcl-2 (9), which might allow for Bak activation by releasing it from these inhibitors. Although p53 could activate Bax in the absence of any other proteins (Fig. 3), we tested if p53 has this additional BH3-like activity to release proapoptotic proteins sequestered by Bcl-xL. For this experiment, we used p53ERtam to determine if this binding might depend on p53 conformation. p53ERtam but not the p53ΔPPERtam mutant was bound by glutathione S-transferase (GST)–Bcl-xL only in the presence of 4-OHT (Fig. 4A). Endogenous Bcl-xL was associated with immunoprecipitated active p53ERtam (Fig. 4B), and this association required the p53 proline-rich domain. Such binding released BidN/C or Bax that was previously bound to Bcl-xL (Fig. 4C). Native p53 displaced BidN/C or Bax sequestered by Bcl-xL at an equimolar concentration (Fig. 4D), whereas concentrations of BidN/C or Bax that were 50 times higher were required to effectively displace native p53 from Bcl-xL (Fig. 4E).

Fig. 4.

Liberation of proapoptotic Bcl-2 proteins sequestered by Bcl-xL after interaction with p53. (A) Active p53ERtam but not p53ΔPPERtam directly binds recombinant Bcl-xL. p53ERtam or p53ΔPPERtam cytosols were combined with GST–Bcl-xL with or without 100 nM 4-OHT. Complexes were detected by SDS-PAGE and Western blot analysis. (B) Active p53ERtam but not p53ΔPPERtam binds to endogenous Bcl-xL. H1299 cells stably expressing p53ERtam or p53ΔPPERtam were treated with or without 100 nM 4-OHT for 24 hours and the p53ERtam or p53ΔPPERtam was immunoprecipitated. Complexes were detected by SDS-PAGE and Western blot analysis. (C) Release of full-length Bax or BidN/C complexed with Bcl-xL in the presence of p53ERtam but not p53ΔPPERtam. Immunopurified p53ERtam or p53ΔPPERtam was combined with complexes of GST–Bcl-xL/Bax or GST–Bcl-xL/BidN/C with or without 100 nM 4-OHT. Liberated proteins were detected by SDS-PAGE and Western blot analysis. (D) Release of full-length Bax or BidN/C complexed with Bcl-xL in the presence of a low concentration of native p53UVIP. GST–Bcl-xL (10 nM) was incubated with 100 nM BidN/C or Bax and washed to remove all unbound protein; 10 nM p53 [or bovine serum albumin (BSA)] was added. Liberated proapoptotic proteins were detected. (E) The association between Bcl-xL and p53 is disrupted by Bax or BidN/C only at high molar excess. GST–Bcl-xL (10 nM) was incubated with 100 nM p53UVIP and washed to remove all unbound protein. Indicated concentrations of Bax or BidN/C were added. Liberated p53 was detected.

The capacity of p53 to directly activate Bax to permeabilize mitochondria permits an uninterrupted pathway leading from DNA damage, for example, to the mitochondrial release of cytochrome c, caspase activation, and apoptosis. The previously unobserved function of p53, which is analogous to that of a BH3-only protein, in addition to its function as a transcription factor, demonstrates an emerging complexity that exists within components of the apoptotic machinery. Other examples include cytochrome c, which functions both in electron transfer and caspase activation, and Bcl-2 family members that have roles in the regulation of metabolism that are distinct from their apoptotic function (29). This multifunctionality of proteins is a source of complexities that, together with those governing gene regulation and RNA splicing, serve as controls in life-long decisions concerning cellular proliferation, differentiation, and death.

Supporting Online Material

www.sciencemag.org/cgi/content/full/303/5660/1010/DC1

Materials and Methods

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Figs. S1 to S5

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