PUMA Couples the Nuclear and Cytoplasmic Proapoptotic Function of p53

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Science  09 Sep 2005:
Vol. 309, Issue 5741, pp. 1732-1735
DOI: 10.1126/science.1114297


The Trp53 tumor suppressor gene product (p53) functions in the nucleus to regulate proapoptotic genes, whereas cytoplasmic p53 directly activates proapoptotic Bcl-2 proteins to permeabilize mitochondria and initiate apoptosis. Here, we demonstrate that a tripartite nexus between Bcl-xL, cytoplasmic p53, and PUMA coordinates these distinct p53 functions. After genotoxic stress, Bcl-xL sequestered cytoplasmic p53. Nuclear p53 caused expression of PUMA, which then displaced p53 from Bcl-xL, allowing p53 to induce mitochondrial permeabilization. Mutant Bcl-xL that bound p53, but not PUMA, rendered cells resistant to p53-induced apoptosis irrespective of PUMA expression. Thus, PUMA couples the nuclear and cytoplasmic proapoptotic functions of p53.

The antineoplastic function of p53 occurs primarily through the induction of apoptosis (1). p53 undergoes posttranslational modification in response to oncogene-activated signaling pathways or to genotoxic stress; this allows stabilization of p53, which accumulates in the nucleus and regulates target gene expression. Numerous genes are regulated by p53, such as those encoding death receptors [for example, FAS (CD95)] and proapoptotic Bcl-2 proteins (for example, BAX, BID, Noxa, and PUMA) (27). In parallel, p53 also accumulates in the cytoplasm, where it directly activates the proapoptotic protein BAX to promote mitochondrial outer-membrane permeabilization (MOMP) (810). Once MOMP occurs, proapoptogenic factors (for example, cytochrome c) are released from mitochondria, caspases are activated, and apoptosis rapidly ensues (11). Thus, p53 possesses a proapoptotic function that is independent of its transcriptional activity (1215).

If p53 directly engages MOMP in cooperation with BAX, no further requirement for p53-dependent transcriptional regulation of additional proapoptotic Bcl-2 proteins would be expected. Nevertheless, PUMA (p53–up-regulated modifier of apoptosis), a proapoptotic BH3-only protein, is a direct transcriptional target of p53. Furthermore, mice deficient in Puma are resistant to p53-dependent, DNA damage–induced apoptosis even though p53 is stabilized and accumulates in the cytoplasm (6, 1618). A better understanding of the distinct nuclear and cytoplasmic proapoptotic functions of p53 may reveal strategies for the prevention and treatment of cancer.

In addition to the ability of cytoplasmic p53 to directly activate BAX, cytoplasmic p53 also interacts with Bcl-xL (8, 9). This interaction has been suggested to be either inhibitory to MOMP induced by p53 and BAX (9); or an initiating apoptotic signal if p53 inactivates the antiapoptotic function of Bcl-xL (8). The association between cytoplasmic p53 and Bcl-xL could prevent p53 from activating BAX; therefore, we screened for potential regulators of the p53·Bcl-xL complex. We prepared cytoplasmic extracts from mouse embryonic fibroblasts (MEFs) after treatment with ultraviolet (UV) light. Several proteins were immunoprecipitated in a complex with Bcl-xL and were detectable after SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and visualization by silver stain (Fig. 1A). Several bands were identified as p53 or fragments of Bcl-xL (fig. S1A), but a major band with a relative molecular mass of 23 kD coprecipitated with Bcl-xL and was excised for tryptic digestion and analyzed by liquid chromatography and tandem mass spectrometry. Three tryptic peptides from the excised band were derived from murine PUMA (fig. S1A) (6). This band was absent when we precipitated Bcl-xL from UV-treated E1A- and H-rasG12V–expressing MEFs that did not express Puma (fig. S1B). Protein immunoblotting also confirmed association of PUMA and p53 with Bcl-xL (Fig. 1B). PUMA appeared to be the major regulator of the p53·Bcl-xL complex after UV treatment because no free cytoplasmic p53 was observed in Puma/ cytosol (fig. S1, C and D).

Fig. 1.

DNA damage–induced p53·Bcl-xL and PUMA·Bcl-xL complexes. (A) Proteins from cytosolic extracts prepared from wild-type or Puma/ MEFs treated with 5 mJ/cm2 UV were immunoprecipitated with an agarose-conjugated antibody to Bcl-xL, eluted, subjected to SDS-PAGE, and visualized by silver staining. Bands were excised and subjected to tryptic digestion and mass spectrometry. The asterisk (*) indicates a fragment of Bcl-xL or p53. (B) Cytosolic extracts were treated as in (A), but protein complexes were analyzed by Western blot. mIgG (mouse immunoglobulin G) is a control antibody.

We next determined the kinetics of the p53·Bcl-xL complex after UV-induced DNA damage in the absence or presence of PUMA expression (fig. S2C). Bcl-xL was immunoprecipitated at the indicated time points after UV treatment, and p53 association was determined. In the presence of PUMA expression (HCT116 p21/PUMA+/+, referred to as PUMA+/+), the amount of p53 associated with Bcl-xL decreased after UV treatment, and this temporally correlated with the induction of apoptosis (fig. S2C). In contrast, the amount of p53 associated with Bcl-xL remained unchanged after treatment in HCT116 p21/PUMA/ (PUMA/) cells, and they were resistant to apoptosis (fig. S2, A and C). Furthermore, UV-induced apoptosis in the HCT116 model system required BAX expression (fig. S2D).

To examine the possible effects of PUMA on the stability of the p53·Bcl-xL association, we added a complex of recombinant Bcl-xL and purified cytoplasmic p53 to cytosol samples from UV-irradiated HCT116 cells (Fig. 2A). Cytosol from UV-treated HCT116 p21/PUMA+/+ cells disrupted the complex, such that less p53 was associated with Bcl-xL, and Bcl-xL associated with PUMA from the cytosol. In contrast, cytosol from UV-treated HCT116 p21/PUMA/ cells allowed for p53 to remain associated with Bcl-xL. The p53·Bcl-xL complex was disrupted by incubation with a PUMA BH3 domain peptide or full-length PUMA protein but not by other proapoptotic Bcl-2 protein BH3 domain peptides (Noxa, BIM, BID, BMF, BAD, bNIP3, BIK, HRK, BAX, or BAK) or proteins (BID or BAX) (Fig. 2B). The relative dissociation constants for p53 (KD = 164 ± 54 nM) and PUMA (KD = 10 ± 4 nM) from Bcl-xL were determined by surface plasmon resonance (BIAcore) (Fig. 2C). The p53 released from Bcl-xL by PUMA still activated BAX and induced mitochondrial cytochrome c release (Fig. 2D). Thus, PUMA may function to liberate p53 from Bcl-xL.

Fig. 2.

PUMA-induced release of p53 from Bcl-xL and consequent apoptosis. (A and B) Recombinant 6xHisBcl-xL·p53 complexes (10 nM) were added to cytosol from 100 mJ/cm2 UV-treated PUMA+/+ or PUMA/ cells (A) or 10 μM BH3 peptides of the indicated proapoptotic Bcl-2 proteins or 50 nM proapoptotic Bcl-2 proteins (B). 6xHisBcl-xL was then isolated by affinity chromatography, and associated p53 and PUMA were detected by Western blot. (C) Dissociation constants (KD) for p53 and PUMA from immobilized Bcl-xLΔC were determined by surface plasmon resonance. The concentration range of p53 and PUMA analyzed was 0.02 to 1.28 μM and 0.01 to 1.25 μM, respectively. Data are presented as a real-time graph of response units (RU) against time. (D) Cytochrome c release from isolated mitochondria. Recombinant 45 nM 6xHisBcl-xL·p53 complexes (or 6xHisBcl-xL alone) were added to either 45 nM PUMA protein or 10 μM PUMA BH3 peptide, and supernatant containing p53 was then added to mitochondria in the presence of 120 nM recombinant, monomeric full-length BAX. Cytochrome c release was assayed by Western blot by its change in localization from the mitochondrial pellet (p) to the supernatant (s). (E) Apoptosis of HCT116 cells. Indicated HCT116 cell lines were treated with two 50 mJ/cm2 doses of UV, 72 hours apart, then incubated for an additional 24 hours before harvesting (“2 × UV”); or they were treated once with 50 mJ/cm2 UV and incubated for 24 hours before harvesting (“1 × UV”). Cells were analyzed for apoptosis, and cytoplasmic lysates were isolated. Proteins from cytoplasmic lysates were immunoprecipitated (IP) with an antibody to Bcl-xL and evaluated by Western blot. Equal loading is shown by a nonspecific (NS*) band. (F) Indicated HCT116 cell lines were transfected with the PUMA BH3 domain peptide or control peptide (top) or pCMV.Flag-PUMA or control vector (bottom), cultured overnight, treated with 75 mJ/cm2 UV, and analyzed 24 hours later for apoptosis. Error bars represent ±SD.

Because Bcl-xL expression remains constant after UV treatment in HCT116 cells (Fig. 2A and figs. S2C and S3A), we hypothesized that if large amounts of stabilized p53 accumulate, a fraction of the cytosolic p53 might remain unassociated with Bcl-xL, eliminating the requirement for PUMA to release cytosolic p53 from Bcl-xL. After exposure of HCT116 p21/PUMA+/+ and HCT116 p21/PUMA/ cells to a single dose of UV, cytosolic p53 coimmunoprecipitated with Bcl-xL. In cells expressing PUMA, cytosolic p53 was also found unassociated with Bcl-xL, and this correlated with apoptosis (Fig. 2E). The Bcl-xL·p53 complex was stable for at least 72 hours after UV treatment in HCT116 p21/PUMA/ (fig. S3B). After a second UV treatment, cytosolic p53 not associated with Bcl-xL was observed in cells lacking PUMA, and these cells underwent apoptosis (Fig. 2E). As a control, HCT116 p53/ cells were resistant to two doses of UV. One explanation is that after a second UV treatment, the p53-sequestering activity of Bcl-xL was saturated, thus allowing p53 to engage MOMP. The requirement for PUMA expression in p53-dependent, UV-induced apoptosis was also overcome by introduction of the PUMA BH3 domain peptide or transient expression of PUMA (Fig. 2F) in HCT116 p21/PUMA/ cells. Introduction of PUMA BH3 domain peptide or transient expression of PUMA did not induce apoptosis in any of the cell lines, but sensitized the HCT116 p21/PUMA/ cells to UV-induced death. In contrast, neither PUMA BH3 domain peptide nor PUMA expression sensitized HCT116p53/ to UV-induced apoptosis. Therefore, PUMA is not sufficient for apoptosis or sensitization to UV-induced apoptosis in the absence of p53 (Fig. 2F). Consistent with this, neither PUMA protein nor the BH3 domain peptide directly permeabilized mouse liver mitochondria or induced apoptosis (fig. S4, A to C).

We also tested other BH3 domain peptides for sensitization to UV-induced apoptosis. The BAD and BIM BH3 domain peptides failed to sensitize PUMA-deficient cells to UV-induced apoptosis, which highlights the specificity of the PUMA BH3 domain in this pathway (fig. S4E). BAD or PUMA BH3 domain peptides enhanced staurosporine-induced apoptosis independently of p53, suggesting that PUMA may also coordinate with other stimuli to promote death (fig. S4D). Thus, although some BH3-only proteins directly activate proapoptotic multidomain Bcl-2 proteins, such as BAX, to induce MOMP, several BH3-only proteins, like PUMA, regulate specific apoptotic pathways by interfering with antiapoptotic Bcl-2 proteins such as Bcl-xL (19, 20).

Structural analyses indicate that p53 interacts with amino acid residues on Bcl-xL distinct from those that mediate the binding of Bcl-xL to BAK, BAD, or BIM (2124). Consistent with this, a mutant of Bcl-xL, Bcl-xLG138A, which does not bind to proapoptotic multidomain Bcl-2 proteins or BH3-only proteins (25), and Bcl-xLwt associated equally with p53, whereas BAX and PUMA bound Bcl-xLwt but not Bcl-xLG138A (Fig. 3A). Likewise, PUMA protein and PUMA BH3 domain peptide failed to disrupt the Bcl-xLG138A·p53 complex in vitro (Fig. 3B), whereas both effectively released p53 from Bcl-xLwt. Transient expression of either Bcl-xLwt or Bcl-xLG138A specifically blocked apoptosis induced by exogenous expression of p53 in E1A- and H-rasG12V–expressing Bcl-xL/ MEFs. In contrast, Bcl-xLG138A failed to inhibit apoptosis induced by BAX expression (Fig. 3C). The ability of Bcl-xLwt to block p53-induced apoptosis, but not that of Bcl-xLG138A, was overcome by transient expression of PUMA, leading to apoptosis (Fig. 3D, fig. S3C). Thus, the association between p53 and Bcl-xL appears not to be the signal to induce death (that is, p53 does not inactivate Bcl-xL to induce apoptosis). Rather, this complex requires an additional proapoptotic stimulus (such as PUMA) to release p53 and engage cell death.

Fig. 3.

Association of a BH3-domain binding mutant of Bcl-xL with p53 and inhibition of p53-induced death. (A) 6XHisBcl-xLΔCwt or 6XHisBcl-xLΔCG138A (10 ng) was combined with 10 ng of p53wt in phosphate-buffered saline (PBS) and incubated at 25°C for 2 hours. Ni2+ beads were used to capture the complexed proteins before SDS-PAGE and Western blot analysis for p53 and Bcl-xL (left). “Beads” represents Ni2+ beads incubated alone with p53wt in PBS. (Right) Full-length BAX and PUMA (10 ng) were also examined. (B) Recombinant 10 nM 6xHisBcl-xLwt or G138A·p53 complexes were added to 50 nM PUMA or 10 μM PUMA BH3 peptide and analyzed by Western blot for association with p53 or PUMA. (C and D) E1A/H-rasG12V–expressing Bcl-xL/ MEFs were transiently transfected with the indicated plasmids (pcDNA3.control, pcDNA3.p53wt, pcDNA3.BAX, pCMV.FLAG-PUMA, pcDNA3.Bcl-xLwt or pcDNA3.Bcl-xLG138A), and apoptosis was analyzed 24 hours later. Error bars represent ±SD.

The effect of Bcl-xLG138A on p53-dependent apoptosis was further examined in E1A- and H-rasG12V–expressing Bcl-xL/ MEFs reconstituted to stably express Bcl-xLwt or Bcl-xLG138A at normal levels (Fig. 4A, fig. S3D) (26). Bcl-xL/ MEFs expressing Bcl-xLG138A demonstrated no protection against p53-independent death stimulation induced by cycloheximide, and their response was similar to that of Bcl-xL/ control cells. Stable expression of Bcl-xLwt inhibited cycloheximide-induced apoptosis, and Bcl-xLG138A expression also conferred resistance to p53-dependent, UV-induced apoptosis, suggesting that Bcl-xLG138A can efficiently and specifically inhibit p53-dependent death (Fig. 4A). In extracts from the stable cell lines, immunoprecipitation of Bcl-xLwt or Bcl-xLG138A captured cytosolic p53 (Fig. 4B). Cytosolic p53 that not was immunoprecipitated with Bcl-xL was observed only in UV-treated Bcl-xLwt cells (Fig. 4B). In both cases, PUMA was expressed after UV treatment, but coprecipitated only with Bcl-xLwt, not Bcl-xLG138A. Because Bcl-xLG138A binds cytoplasmic p53, but not BAX or PUMA, and is similarly deficient in binding other proapoptotic BH3-only proteins, the simplest interpretation of these results is that Bcl-xLG138A inhibits p53-dependent apoptosis by sequestering cytoplasmic p53 and prevents it from activating BAX to promote MOMP. The nuclear functions of p53 are unaffected by Bcl-xLwt or Bcl-xLG138A, as evidenced by the equivalent expression of PUMA after UV treatment (Fig. 4B). Thus, cytoplasmic p53 appears to mediate apoptosis under physiological conditions.

Fig. 4.

Bcl-xLG138A reconstitutes Bcl-xL deficiency for p53-dependent apoptosis. (A) E1A/H-rasG12V–transformed wild-type MEFs expressing pBABE.control, or Bcl-xL/ MEFs stably expressing either pBABE.control, pBABE.Bcl-xLwt, or pBABE.Bcl-xL G138A were treated with the indicated doses of cycloheximide (top) or UV (bottom) and analyzed 24 hours later for apoptosis. Error bars represent ±SD. (B) Proteins from the cytosol of 5 mJ/cm2 UV-treated E1A/H-rasG12V–transformed Bcl-xL/ MEFs stably expressing either pBABE.Bcl-xLwt or pBABE.Bcl-xLG138A were immunoprecipitated for Bcl-xL. Bcl-xL–coprecipitated p53 (Bound) and PUMA, and non-Bcl-xL–associated p53 (Free) and PUMA in the cytosol after Bcl-xL immunoprecipitation were analyzed by SDS-PAGE and Western blot. (C) Proposed model of p53-dependent, DNA damage–induced apoptosis. After p53 stabilization, p53 accumulates in the nucleus to directly regulate the expression of proapoptotic genes, such as BAX and PUMA. Likewise, p53 accumulates in the cytoplasm and directly binds to Bcl-xL. Once p53-dependent expression of PUMA occurs, PUMA binds to Bcl-xL, which releases p53 to directly activate BAX and induce MOMP.

Our studies support a model of p53-dependent, DNA damage–induced apoptosis that includes both nuclear and cytoplasmic functions of p53 (Fig. 4C). After p53 stabilization, p53 accumulates in the nucleus to directly regulate the expression of proapoptotic genes, such as BAX and PUMA. p53 also accumulates in the cytoplasm, directly binds to Bcl-xL, and awaits a secondary death signal. Once p53-dependent expression of PUMA occurs, PUMA binds to Bcl-xL, releasing p53 to directly activate BAX and induce MOMP. Our results may explain why Bcl-xL–deficient animals display sensitivity to DNA damage, whereas Puma deficiency promotes resistance to numerous p53-dependent apoptotic stimuli (16, 26, 27).

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