Interacts Directly with and Negatively Regulates Pro-apoptotic Bax*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 3, Issue of January 17, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A Interacts Directly with and Negatively Regulates Pro-apoptotic Bax* Received for publication, August 2, 2002, and in revised form, November 1, 2002 Published, JBC Papers in Press, November 7, 2002, DOI /jbc.M Masaya Nomura, Shigeomi Shimizu, Tomoyasu Sugiyama, Masashi Narita, Toshinori Ito, Hikaru Matsuda, and Yoshihide Tsujimoto ** From the Departments of Post-genomics & Diseases and Surgery, Osaka University Graduate School of Medicine and the Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation, 2-2 Yamadaoka, Suita, Osaka , Japan The Bcl-2 family of proteins comprises well characterized regulators of apoptosis, consisting of anti-apoptotic members and pro-apoptotic members. Pro-apoptotic members possessing BH1, BH2, and BH3 domains (such as Bax and Bak) act as a gateway for a variety of apoptotic signals. Bax is normally localized to the cytoplasm in an inactive form. In response to apoptotic stimuli, Bax translocates to the mitochondria and undergoes oligomerization to induce the release of apoptogenic factors such as cytochrome c, but it is still largely unknown how the mitochondrial translocation and proapoptotic activity of Bax is regulated. Here we report that cytoplasmic protein binds to Bax and, upon apoptotic stimulation, releases Bax by a caspase-independent mechanism, as well as through direct cleavage of by caspases. Unlike Bad, the interaction with is not dependent on the phosphorylation of Bax. In isolated mitochondria, we found that inhibited the integration of Bax and Bax-induced cytochrome c release. Bax-induced apoptosis was inhibited by overexpression of either or its mutant (which lacked the ability to bind to various phosphorylated targets but still bound to Bax), whereas overexpression of was unable to inhibit apoptosis induced by a Bax mutant that did not bind to These findings indicate that plays a crucial role in negatively regulating the activity of Bax. Regulation of programmed cell death, or apoptosis, is essential for normal development and for the maintenance of homeostasis in most metazoans. Various apoptotic signals eventually converge into a common death mechanism, in which members of the cysteine protease family (known as caspases) are activated and cleave various cellular proteins. In mammals, the mitochondria play an essential role in apoptosis by releasing apoptogenic factors including cytochrome c, Smac/ Diablo, and Omi/HtrA2 from the intermembrane space into the cytoplasm (1 3). Once in the cytoplasm, cytochrome c binds to Apaf-1, a mammalian homologue of Ced-4, that recruits and * This work was supported in part by a Scientific Research on Priority Areas grant, a Center of Excellence Research grant, a grant from the Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, and Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Both authors contributed equally to this work. ** To whom correspondence should be addressed. tsujimot@ gene.med.osaka-u.ac.jp activates initiator caspase-9, which subsequently activates effector caspase-3/caspase -7 (4, 5), whereas Smac/Diablo and Omi/HtrA2 facilitate caspase activation by interacting with and inhibiting IAPs, the endogenous caspase inhibitor family (3, 6, 7). The Bcl-2 family of proteins includes the best characterized regulators of apoptosis, comprising anti-apoptotic members, including Bcl-2 and Bcl-x L, and pro-apoptotic members that include multi-domain Bax and Bak and various single-domain BH3-only proteins (1, 8). Proteins of this family directly regulate the release of mitochondrial apoptogenic factors. Many of the pro-apoptotic family members, such as Bax, Bid, Bad, Bim, and Bmf, are localized in the cytoplasm, and apoptotic stimulation results in their translocation to the mitochondria and induction of the release of apoptogenic factors, probably by inactivating anti-apoptotic members of the family and activating multi-domain members like Bax and Bak (9 12). Translocation of the BH3-only proteins appears to involve various post-translational modifications. For instance, cytoplasmic Bid is cleaved by caspase-8 and then undergoes translocation to the mitochondria (13, 14). Dephosphorylation by calcineurin frees Bad from cytosolic and allows it undergo translocation to the mitochondria (15). Although the mechanism involved is still unknown, Bim and Bmf are freed from microtubular dynein motor complexes and myosin V actin motor complexes, respectively, during certain forms of apoptosis (16, 17). Bax has also been shown to undergo translocation and integration into the mitochondrial membrane during apoptosis (18 22), and the translocation process has been suggested to involve a conformational change of the Bax molecule, especially exposure of the C terminus (20, 21, 23). It has also been reported that translocation of Bax to the mitochondria is enhanced by caspases (20) or by intracellular alkalization (24) and is negatively regulated by Bcl-2 through a still unidentified mechanism (19, 21, 22). The proteins (seven isomers in mammals:,,,,, /, and ) are highly conserved cytoplasmic molecules that form homodimers and heterodimers and interact with various cellular proteins. These proteins seem to control various cellular processes by sequestering regulatory molecules (25). The proteins have also been implicated in signaling for apoptosis through interaction with apoptotic molecules such as Bad (26), ASK1 (27), and FKHRL1 (28). Furthermore, and are known to act as mitochondrial import stimulation factors (29) and appear to play a crucial role in intracellular protein trafficking, although the precise mechanism by which these isomers of participate in protein translocation is not yet understood. In the present study, we showed that protein was bound to Bax in the cytoplasm of living cells and that Bax This paper is available on line at

2 Negative Regulation of Bax by underwent dissociation from this protein by caspase-independent and -dependent mechanisms during apoptosis to induce apoptotic changes of the mitochondria, indicating that plays a crucial role in the negative regulation of Bax activity in living cells. EXPERIMENTAL PROCEDURES Antibodies and Chemicals An anti-human Bax (N20) polyclonal antibody that cross-reacted with mouse Bax was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and an anti-human Bax (Ab-3) monoclonal antibody (mab) 1 was obtained from Oncogene (Darmstadt, Germany). Anti-sheep (T16, cross-reacting with human ), anti-rat (C17, cross-reacting with human and mouse ), anti-human (C16), and an anti-human mab (H8) that recognized the N-terminal region of various family members were purchased from Santa Cruz Biotechnology. An anti-mouse / mab, an anti-xpress mab, and an anti-flag M2 mab were purchased from Calbiochem (La Jolla, CA), Invitrogen, and Sigma, respectively. An anti-cytochrome c mab (65981A) that cross-reacted with human cytochrome c was obtained from PharMingen (San Diego, CA). Anti-Fas antibody (CH-11) was purchased from MBL (Nagoya, Japan). The cleavable protein cross-linker dimethyl-3,3 -dithiobispropionimidate-2hcl (DTBP) was purchased from Pierce, [ 32 P]orthophosphate was obtained from Amersham Biosciences, and zvad-fmk was purchased from Peptide Inc. (Minoh, Japan). Other chemicals were purchased from Wako Co. (Tokyo, Japan). Construction of Plasmid Vectors DNAs encoding human ,,, and various mutants (K49E/V176D, D239A, 1 6, and with amino acid residues 1 161, and 1 239, respectively) were generated by the PCR using proofreading Pfu DNA polymerase (Stratagene, CA) and were subcloned into a recombinant protein-producing vector (pgex-1 T) and two mammalian expression vectors, puc-caggs (30) and pcdna3.1 (Invitrogen), with an N-terminal His tag and an anti- Xpress epitope. DNAs encoding HA-tagged mouse Bax and its mutants ( N, 1, BH3, 5/6, and C, lacking amino acid residues 1 20, 22 37, 63 72, , and , respectively) were generated by PCR and were subcloned into pbluescript SK ( / ) (Strategene, CA). DNA encoding FLAG-tagged human Bad was subcloned into puc-caggs. Protein Purification Recombinant His-tagged human Bax was expressed and purified as described previously (31). Recombinant human ,, and were expressed as GST fusion proteins in Escherichia coli (strain DH5 ) and were purified on a glutathione-sepharose column was released from GST by cleavage with thrombin and purified to homogeneity by MonoQ chromatography. Mock proteins were produced by the same method using empty plasmids. In some experiments, GST was used without cleavage of GST. Recombinant caspase-3, -7, and -8 were expressed as His-tagged proteins in E. coli (strain DH5 ) and purified on a Ni -nitrilotriacetic acid column. One unit was defined as the amount of enzyme that released 1 nmol of 7-amino-4-methylcoumarin in a buffer (50 mm Tris/HCl, ph 7.4, 1 mm EDTA, and 1 mm EGTA) containing 100 M of substrate (Ac-DEVD- MCA, Ac-IETD-MCA, or Ac-VEID-MCA) over 10 min at 30 C. All of the proteins were dissolved in a buffer composed of 20 mm Tris/HCl (ph 7.4), 2 mm MgCl 2,and1mM dithiothreitol. Mouse Bax and its mutants were produced using an in vitro translation method. Briefly, DNAs were transcribed/translated with a TNT T7 transcription/translation kit (Promega, Japan) in the presence of [ 35 S]methionine according to the manufacturer s instructions. Analysis of Protein-Protein Interaction For immunoprecipitation experiments, HeLa and NIH3T3 cells were incubated with 2 mm DTBP (a protein cross-linker) for 30 min. Then the cells were lysed and sonicated in lysis buffer (50 mm Tris/HCl, ph 7.4, mm KCl, 5 mm MgCl 2,1mM EGTA, and 0.2% Nonidet P-40) containing proteinase inhibitors. To investigate the interaction with exogenous Bax and Bad, 293T cells were transiently transfected with the expression plasmids using LipofectAMINE in the presence of zvad-fmk (100 M) to prevent Bax- or Bad-induced apoptosis. Then the cells were lysed, and the lysates were subjected to immunoprecipitation with the indicated antibodies, and the precipitates were analyzed by Western blotting. To detect binding between purified proteins, recombinant proteins were incubated for 8 h with either GST proteins or GST alone in The abbreviations used are: mab, monoclonal antibody; DTBP, dimethyl-3,3 -dithiobispropionimidate-2hcl; HA, hemagglutinin; GST, glutathione S-transferase; GFP, green fluorescent protein. l of the lysis buffer, and then these proteins were incubated with glutathione-sepharose for 3 h. After brief centrifugation, the beads were washed and resuspended in the SDS-PAGE sample buffer, as described elsewhere (32). The proteins were analyzed by Western blotting and autoradiography. Surface Plasmon Resonance The affinity between Bax and proteins was measured by surface plasmon resonance using a Biacore2000 (Biacore). Equivalent molar amounts of GST-mock and GST proteins were immobilized on the sensor chip (CM5; Pharmacia Corp.) by the amine-coupling method. Bax was added as the analite, and the affinity was calculated from the difference between the resonance units with GST proteins and those with GST-mock. Metabolic Labeling 293T cells were transfected with puc-caggshuman Bax DNA or puc-caggs-human FLAG-Bad DNA using LipofectAMINE (Life Technologies, Inc.), according to the supplier s protocol. The transfected cells were labeled for 24 h in phosphate-free RPMI 1640 medium with [ 32 P]orthophosphate (4 Ci/10 6 cells). Cell Fractionation Cell fractionation was performed using digitonin, as described previously (22). Briefly, after washing twice with phosphate-buffered saline, the cultured cells were collected and treated with 10 M digitonin for 5 min at 37 C. The cytosolic and organellar fractions were separated by centrifugation and lysed with RIPA buffer. As a result, more than 92% of cytosolic protein was recovered in the supernatant, and more than 95% of mitochondrial protein was localized to the pellet. The heavy membrane fraction enriched for mitochondria was prepared as follows. The cells were washed twice in phosphatebuffered saline, resuspended in isotonic buffer (20 mm potassium Hepes, ph 7.4, 1.5 mm MgCl 2,10mM KCl, and 250 mm sucrose), and then homogenized with a Dounce homogenizer. After separation of nuclei and unbroken cells by centrifugation at 600 g for 10 min, the post-crude nuclear supernatant was centrifuged at 10,000 g for 10 min to collect the heavy membrane fraction. Assessment of the Integration of Bax into the Mitochondrial Membrane Heavy membrane fractions enriched for mitochondria from cells or isolated rat mitochondria were incubated in 0.1 M Na 2 CO 3 (ph 11.5), and then were centrifuged at 200,000 g for 45 min to separate the supernatant and pellet as described elsewhere (21). Analysis of Bax Translocation and Cytochrome c Release in Vitro Mitochondria were prepared from the livers of male Donryu rats in Mt-A buffer (0.3 M mannitol, 10 mm potassium Hepes, ph 7.4, 0.1% fatty acid-free bovine serum albumin), as described previously (33). Recombinant (the indicated amounts) and rbax (1 g) were preincubated for 30 min at 25 C and then were added to the mitochondria (100 g) and incubated for a further 3 min at 25 C in100 l of Mt-B buffer (Mt-A buffer plus 100 M potassium phosphate and 4.3 mm succinate). Next, the mixture was then centrifuged to collect the mitochondria, and aliquots of mitochondria or supernatant were analyzed by Western blotting using anti-bax antibodies. To detect the release of cytochrome c, mitochondria were centrifuged, and the supernatant was analyzed by Western blotting with an anti-cytochrome c antibody. In Vitro Assay of the Dissociation of Bax from GST (2 g) and rbax (5 g) were preincubated for8hat4 C in ph 7.5 buffer, and then the mixture was incubated with glutathione-sepharose for 3 h. After brief centrifugation, the beads that bound GST and GST Bax complex were incubated with either cytosol (10 g) in ph 7.5 buffer for 12 h at 25 C, with 200 l of ph 6.5, ph 7.5, or ph 8.0 buffer (50 mm Tris/HCl, ph 6.5, 7.5 or 8.0, 0.2% Nonidet P-40) for 12 h at 4 C, or with caspase-8 (600 units) for 4hat37 C. After brief centrifugation, the beads were washed and resuspended in the sample buffer and then analyzed by Western blotting. GST (2 g) and rbax (5 g) were preincubated for 30 min at 4 C in10 l of ph 7.5 or ph 8.0 buffer (50 mm Tris/HCl, ph 7.5 or 8.0, 0.2% Nonidet P-40). The protein mixtures were then incubated for 8 h at 4 C in200 l of ph 7.5 or 8.0 buffer (50 mm Tris/HCl, ph 7.5 or 8.0, 0.2% Nonidet P-40) and mixed with glutathione-sepharose for 3 h. After brief centrifugation, the beads were washed, and rbax bound to the beads was analyzed by Western blotting (5 g) was incubated with caspase-3 (600 units) or mock protein for4hat37 C, rbax (2 g) was added, and then incubation was done for 12 h at 4 C. After addition of Ni resin, bound to rbax was collected and eluted with 0.3 M imidazole (ph 6.8). In Vitro Assay of Cleavage by Caspases Cytosolic fractions from healthy HeLa cells or recombinant were incubated for 5 h at 37 C with or without caspases and in the presence or absence of 200 M zvad-fmk. Then the cleavage of was detected by Western blotting. Analysis of Cell Death 293T cells were transiently transfected with human Bax DNA (0.2 g) with or without DNA expressing human

3 2060 Negative Regulation of Bax by FIG. 1. Interaction of Bax with a and b, interaction of Bax with in healthy cells. HeLa (a, left panel) and NIH3T3 (b) cells were preincubated with 2 mm DTBP (a cleavable protein cross-linker), and the lysates were immunoprecipitated (IP) with anti-bax polyclonal antibody (N20) ( Bax) or normal rabbit IgG. The immune complexes were analyzed by Western blotting using anti antibodies specific for the indicated isoform. The same experiment was also performed without DTBP in HeLa cells (a, right panel). lysate indicates the portion ( 1 10) of the total lysate that was subjected to immunoprecipitation. c and d, direct interaction between Bax and several isoforms (,, and ) with comparable affinities. c, recombinant Bax (rbax, 2 g) was incubated with 2 g of the indicated GST proteins or the equivalent amount of GST-mock protein for 8 h. Then GSH-Sepharose was added for 3 h and collected by centrifugation, after which bound rbax was analyzed by Western blotting. total indicates the total amount of rbax used. d, the indicated amount (15 g or30 g) of rbax or bovine serum albumin was run over a chip containing immobilized GST-mock protein, GST ,,or, and protein interactions were measured by surface plasmon resonance as described under Experimental Procedures. e, the amount of each isoform in HeLa cells. Lysates from healthy HeLa cells (10 g) and the indicated GST isoforms (25 ng each) were analyzed by Western blotting using antibodies specific for (left panel), (middle panel), and (right panel). The amount of each of the isoforms in 10 g of lysate was estimated by comparison with the GST proteins using densitometric analysis and is shown below the blots (in nanograms) or its mutants (0.5 g), plus 0.1 g of the green fluorescent protein (GFP) expression construct (pegfp-n1; Clontech). Transfected cells were incubated for 24 h at 37 C and stained with 1 M Hoechst 33342, after which the extent of apoptosis was calculated as the percentage of GFP-positive cells showing nuclear fragmentation relative to all GFP-positive cells. RESULTS Bax Interacts with Although translocation and integration of cytoplasmic Bax into the mitochondrial membrane is a critical step for its pro-apoptotic activity, the mechanism of action is poorly understood. To improve our understanding of the regulation of Bax, we searched for a molecule that interacted with Bax, modulated its activity, and found that Bax was bound to protein in HeLa cells (Fig. 1a). The same interaction between Bax and was also observed in NIH3T3 cells (Fig. 1b). Because there are several isoforms of (25), we next tested the interaction of Bax with other isoforms. As shown in Fig. 1a, Bax was also bound to and in HeLa cells, whereas there was no interaction with or (data not shown). Furthermore, it has recently been reported that Bax binds to (34). Although the interaction between Bax and was initially detected in the presence of a protein cross-linker (Fig. 1a, left panel), a FIG. 2. Regions of Bax essential for interaction with The indicated mutants of mouse Bax were produced by in vitro translation in the presence of [ 35 S]methionine and were incubated with GST or GST-mock protein. After GST and GST-mock protein were precipitated with GSH-Sepharose as described under Experimental Procedures, Bax bound to GST or GST-mock was analyzed by SDS-PAGE followed by autoradiography. A diagram of the Bax deletion mutants is also shown. These deletion mutants retained the regions shown by horizontal lines. 1 7 indicate the possible helices retained by Bax. similar level of binding was observed in the absence of the cross-linker (Fig. 1a, right panel). Recombinant His-tagged Bax (rbax) showed binding to GST-fused ,, and but not to GST (Fig. 1c), indicating that Bax directly interacted with these isoforms. Furthermore, surface plasmon resonance analysis revealed that ,, and all had a comparable affinity for rbax (Fig. 1d). Estimation of the amount of each endogenous isoform in HeLa cells by comparison with recombinant isoforms on Western blots revealed that 10 g of HeLa cell lysate contained 35, 22, and 10 ng of ,, and, respectively (Fig. 1e). According to these findings, although Bax interacted with ,, and, was the major isoform in HeLa cells, so we studied its role further. Although Bad is known to bind to (26), we were unable to detect any interaction between and the other proapoptotic Bcl-2 family members Bid or Bak, either by immunoprecipitation or by the surface plasmon resonance method (data not shown). Both the N- and C-terminal Regions of Bax Are Required for Interaction with We then attempted to determine the regions of Bax involved in binding to by employing an in vitro interaction assay using 35 S-labeled, HA-tagged mouse Bax mutants and GST , because the level of expression of the Bax mutants varied considerably in transfection experiments. Wild-type Bax and three of its deletion mutants ( 1 (lacking -helix 1), BH3 (lacking the BH3 region), and 5/6 (lacking the channel-forming -helices 5 and 6)) showed binding to GST , whereas Bax N and C (lacking the N- terminal 20 amino acids and C-terminal 22 amino acids, respectively) did not bind to GST (Fig. 2), suggesting that both the N- and C-terminal regions of Bax were involved in this binding process. Although it was reported that some detergents, such as Nonidet P-40, could enhance the conformational changes of Bax and increase its homodimerization and heterodimerization with other Bcl-2 family members (35), the interaction between Bax and was decreased rather than enhanced by addition of Nonidet P-40 (data not shown). Interaction of Bax with Is Independent of Bax Phosphorylation The proteins bind to various phosphorylated proteins, such as Raf-1 and Bad, via phosphorylated serine residues (26, 36), but these proteins are also known to bind to several nonphosphorylated proteins (37). Therefore, we tested whether phosphorylation of Bax was involved in its

4 Negative Regulation of Bax by FIG. 3. Interaction of unphosphorylated Bax with a, lack of Bax phosphorylation. 293T cells were transiently transfected with puc-caggs-bax (3 g) or with puc-caggs-flag-bad DNA (3 g) and incubated in phosphate-free medium containing [ 32 P]orthophosphate (4 Ci/10 6 cells). After 24 h, the cells were lysed and were immunoprecipitated (IP) with anti-bax or anti-flag antibodies or with normal rabbit IgG or mouse IgG. The immune complexes were analyzed by SDS-PAGE followed by autoradiography (top panel), as well as by Western blotting (WB) using anti-flag (second panel), anti-bax (third panel), and anti (bottom panel) antibodies. b, interaction of K49E/V176D with Bax, but not Bad. 293T cells were transiently transfected with puc-caggs-flag-bad (left panels) and puc-caggs-bax (right panels) DNA together with pcdna or its K49E/V176D mutant in the presence of zvad-fmk (100 M). After 24 h, the cells were lysed and immunoprecipitated (IP) with anti-flag antibody or NMI (left panels), as well as with anti-bax antibody or normal rabbit IgG (right panels). Then the immune complexes were analyzed by Western blotting using anti-xpress (to detect ), anti-flag (left panels) and anti-bax (right panels) antibodies. lysate indicates the portion ( 1 10) of each lysate that was subjected to immunoprecipitation. wt, wild type. FIG. 4. Dissociation of Bax from and integration into the mitochondria during apoptosis. a, dissociation of Bax from during apoptosis. HeLa cells were incubated for the indicated time with 200 M VP16 in the absence or presence of 200 M zvad-fmk. Equivalent aliquots of the cells were lysed and immunoprecipitated with anti-bax antibody, and the immune complexes were subjected to Western blotting using anti (top panel) or anti-bax antibodies (middle panel). Aliquots of the cell lysates were also subjected to Western blotting with anti antibody (lower panel). The extent of apoptosis is also shown in the figure. The remaining cells were fractionated into organellar and cytosolic fractions using 10 M digitonin, and each fraction (5 g of protein) was analyzed by Western blotting with anti-cytochrome c antibody. b, translocation of Bax from the cytosol to the mitochondria was not inhibited by zvad-fmk during VP16- induced apoptosis. HeLa cells were incubated for the indicated time with 200 M VP16 in the absence or presence of 200 M zvad-fmk. Heavy membrane (HM) and cytosolic fractions were prepared by differential centrifugation without alkaline treatment. Then the fractions were analyzed by Western blotting using anti-bax antibody. c, integration of Bax, but not , into the mitochondrial membrane. HeLa cells were incubated for 24 h with 200 M VP16 in the absence or presence of 200 M zvad-fmk. Heavy membrane (HM) fractions were prepared by differential centrifugation, incubated in ph 7.5 buffer ( ) or in 0.1 M Na 2 CO 3 (ph 11.5) ( ) on ice for 30 min, and then centrifuged at 200,000 g for 45 min to yield a supernatant (S) and a pellet containing heavy membranes (P). The fractions were analyzed by Western blotting using anti-bax and anti antibodies. Equivalent amounts of the HM fraction from VP16 and VP16 zvad fmk cultures were analyzed, but a much larger amount of the fraction from VP16 cultures was assessed. d, dissociation of Bax from induced by the cytosol of apoptotic cells. GST (2 g) and rbax (5 g) were preincubated for 8 h at 4 C, and then the mixture was incubated with glutathione-sepharose for 3 h. After brief centrifugation, the beads retaining Bax complex were incubated with the indicated cytosol (10 g) for 12 h at 25 C. After brief centrifugation, the beads were washed. Then the combined supernatants (sup) and the beads resuspended in sample buffer were analyzed by Western blotting using anti-bax and anti antibodies. total indicates the amount of rbax in the pellet after incubation with glutathione-sepharose. interaction with As shown in Fig. 3a, although Bax was bound to , we could not detect any phosphorylation of Bax in 293T cells when overexpressed Bax was labeled with [ 32 P]orthophosphate, a result consistent with previous reports (38). Under the same experimental conditions, we readily detected phosphorylation of Bad (Fig. 3a), which is known to be phosphorylated before binding to (26). Furthermore, the immunoprecipitated Bax did not react with antibodies specific for phosphoserine or phosphothreonine (data not shown). These results indicated that phosphorylation of Bax was not necessary for interaction with To further confirm that phosphorylation of Bax did not play an essential role in the interaction with , we examined the binding of Bax to a mutant of ( 49 /V176D) that had lost the ability to bind to various target phosphoproteins, including Raf-1 and ASK1 (27). As shown in Fig. 3b, whereas wild-type was co-immunoprecipitated with both Bax and FLAG-Bad, K49E/V176D was co-immunoprecipitated with Bax but not with FLAG-Bad, supporting our hypothesis that phosphorylation of Bax was not necessary for interaction with Bax Is Negatively Regulated by and Dissociates during Apoptosis in Both a Caspase-dependent and Caspase-independent Manner To obtain some insight into the biological significance of the interaction of Bax with , we next examined whether this interaction was altered during the apoptotic process. As shown in Fig. 4a, treatment with VP16 (etoposide) caused the amount of interacting with Bax to decrease markedly, and a large fraction of Bax was translocated to the mitochondria (Fig. 4b) with the release of cytochrome c (Fig. 4a). To test whether Bax in the mitochondrial fraction was stably integrated into the mitochondrial membrane, mitochondrial fractions were treated with an alkaline solution (ph 11.5) that only saved Bax, showing stable integration into the membrane. Before incubation with VP16, a very small amount of Bax was found in the heavy membrane fraction (Fig. 4b), half of which was stably integrated into the mitochondrial membrane (Fig. 4c). Note that a much larger amount of VP16 sample was analyzed than that of VP16 in Fig. 4c. On the other hand, the majority of Bax was found in the heavy membrane fraction after VP16 treatment (Fig. 4b), most of which was stably integrated into the membrane (Fig. 4c). In the presence of zvad-fmk, which completely inhibited caspase

5 2062 Negative Regulation of Bax by FIG. 5.Inhibition of the mitochondrial integration of Bax by GST , GST (50 g), and the equivalent amount of GST-mock were incubated with or without rbax (25 g) for 3 h and then were incubated with the mitochondria (1 mg) for 20 min at 25 C. Supernatants and pellets were obtained by centrifugation. The pellets were incubated in 0.1 M Na 2 CO 3 (ph 11.5) on ice for 30 min, followed by centrifugation, and then each pellet was analyzed by Western blotting using anti-bax antibody (a). Cytochrome c in the supernatant was also analyzed by Western blotting using an anti-cytochrome c antibody (b). total represents the total amount of rbax used (a) or an equivalent aliquot of mitochondria (b). activation, dissociation of Bax from was only partly inhibited (Fig. 4a), indicating that dissociation occurred in both a caspase-independent and caspase-dependent manner. As shown in Fig. 4c, integration of Bax into the mitochondrial membrane was also partly inhibited by zvad-fmk, whereas translocation of Bax to the mitochondria was not affected by this caspase inhibitor (Fig. 4b). Note that the amount of caspase-independent dissociation of Bax was well correlated with the caspase-independent mitochondrial integration of Bax (Fig. 4, a and c). The findings obtained using cell lysates were similar to those obtained with living cells. As shown in Fig. 4d, lysates from VP16-treated cells were more efficient at causing Bax to dissociate from than lysates from normal cells. Interestingly, this dissociation was partially inhibited in the presence of the caspase inhibitor zvad-fmk, indicating that dissociation of Bax from occurred via both caspase-dependent and -independent mechanisms in the cell lysates (Fig. 4d), as it did in living cells (Fig. 4a). All of these results suggested that had a role in the sequesteration of Bax. The dissociation of Bax from during apoptosis suggested that was a negative regulator of Bax. To test this possibility, we examined whether affected the mitochondrial translocation of Bax using isolated mitochondria. As shown in Fig. 5, the addition of recombinant (or ) protein inhibited the integration of Bax into the mitochondrial membrane (Fig. 5a) as well as Bax-induced release of cytochrome c (Fig. 5b). Caspases Directly Cleave to Release Bax To investigate the mechanism of the caspase-dependent dissociation of Bax from , we examined whether caspases were able to cleave As shown in Fig. 6a, and , but not , were cleaved during apoptosis, and their cleavage was completely inhibited by the caspase inhibitor zvad-fmk. When recombinant caspase-3 was added to normal cell lysates, was cleaved in a z-vad-fmk-sensitive manner (Fig. 6b). Furthermore, recombinant caspase-3 also cleaved recombinant (Fig. 6c), indicating that is a direct target of caspase-3. As shown in Fig. 6c, was also cleaved by caspase-7 and caspase-8. In contrast, was cleaved by caspase-3 in the presence of cell lysate but not in its absence (Fig. 6, b and c), indicating that is not a direct target of caspases, unlike To identify the caspase cleavage site in , various Asp to Ala mutants were produced. Among them, only the D239A mutant was not cleaved during apoptosis, indicating that Asp 239 (not present in and ) is the caspase cleavage site. As shown in Fig. 4a, when was cleaved during apoptosis, cleaved was not co-immunoprecipitated with Bax, suggesting that cleaved has a decreased affinity for Bax. In fact, full-length r interacted with rbax, whereas this interaction was greatly diminished when r was FIG. 6.Caspase-dependent dissociation of Bax from a, cleavage of and during apoptosis. HeLa cells were incubated for 24 h with or without 200 M VP16 in the absence or presence of 200 M zvad-fmk. Equivalent aliquots of the cells were lysed and analyzed by Western blotting using anti ,, and antibodies. The arrows indicate cleaved b and c, direct cleavage of , but not , by caspases. b, the cytosolic fraction of HeLa cells was incubated with ( ) or without ( ) recombinant caspase-3 (600 units) in the presence ( ) or absence ( ) of200 M zvad-fmk for 5hat37 C. The lysates were analyzed by Western blotting using anti and anti antibodies. The arrows indicate cleaved c, recombinant , , and Bid were incubated with 600 units of caspases-3, -7, and -8 in the presence ( ) or absence ( )of200 M zvad-fmk for 5 h at 37 C. Each protein was analyzed by Western blotting using specific antibodies. Cleavage of Bid was performed to confirm caspase activity. The arrow indicates cleaved d, determination of the site of cleavage of by caspases. Top panel, structure of The -helix is indicated at the top, and the C-terminal amino acid sequence is indicated below.an asterisk indicates the site of cleavage by caspases. Bottom panel, cleavage of , but not D239A, during apoptosis. HeLa cells were transiently transfected with puc-caggs-his (wild) and puc-caggs-his D239A (D239A) for 24 h and treated with ( ) or without ( ) -Fas antibody (CH-11, 0.25 g/ml) for 18 h. Then the lysates were analyzed by Western blotting using anti-xpress (to detect ) and anti-bid (as a positive control) antibodies. e, interaction of Bax with but not with after cleavage by caspases. Recombinant (5 g) was treated with caspase-3 (600 units) or the mock protein for 4 h at 37 C (input), and then rhis-bax (2 g) was added at 4 C for 12 h. By addition of Ni resin, rbax and interacting were collected. The proteins were eluted from the Ni resin with 0.3 M imidazole (ph 6.8) and were analyzed by Western blotting (bound) with anti antibody and anti-bax antibody. f, dissociation of Bax from by caspase treatment. GST (5 g) and rbax (2 g) were incubated at 4 C for 12 h. After collecting the Bax complex with GSH-Sepharose, caspase-8 (600 units) was added, and incubation was done at 37 C for 4 h. Bax released from and bound to GST was detected by Western blotting using anti-bax antibody. cleaved by caspase-3 (Fig. 6e). Furthermore, when the complex formed by GST and rbax was treated with caspase-8, rbax was released, and this release was completely inhibited by a caspase inhibitor (Fig. 6f). These results indicated that caspase-dependent dissociation of Bax from could be ascribed to cleavage of by caspases. Bax Dissociates from under Basic and Acidic Conditions It has been demonstrated that in the early phase of apoptosis induced by a variety of stimuli, including cytokine deprivation, cytoplasmic alkalization occurs and induces a conformational change of Bax that results in its integration into the mitochondria (24). Therefore, we examined whether alkal-

6 Negative Regulation of Bax by FIG. 7.Caspase-independent dissociation of Bax from a, decrease of Bax binding to by treatment at ph 8.0. Upper panel, the interaction between rbax and GST at ph 7.5 or 8.0 was analyzed using surface plasmon resonance as in Fig. 1d. Lower panel, GST (2 g) and rbax (5 g) were preincubated for 30 min at 4 C at the indicated initial ph and then were mixed for 8hat4 C in buffer at the indicated terminal ph. The mixture was then incubated with glutathione-sepharose for 3 h. After a brief centrifugation, the beads were washed, and the amount of rbax and on the beads was analyzed by Western blotting. total indicates the total amount of rbax used. b and c, the ph-dependent dissociation of Bax from GST (2 g) and rbax (5 g) were preincubated at 4 C for8h, and then the mixture was incubated with glutathione-sepharose for 3 h. After brief centrifugation, the beads were treated in buffer (ph 7.5, ph 8.0 (b), or ph 6.5 (c)) at 4 C for 12 h. The amount of rhis-bax and on the beads was then analyzed by Western blotting. ization had an influence on the interaction of Bax with As shown in Fig. 7a, the interaction of Bax with was weaker at ph 8.0 than at ph 7.5. Treatment of Bax, but not , with an alkaline solution (ph 8.0) decreased their affinity (Fig. 7a), supporting the earlier finding that alkalization induced a conformational change of Bax (24). Dissociation of Bax from at an alkaline ph (Fig. 7b) implies that cytoplasmic alkalization during apoptosis may be one of the initial caspase-independent mechanisms promoting dissociation of the complex between Bax and Dissociation of Bax from was also observed at an acidic ph of 6.5 (Fig. 7c). It has been reported that cells show cytoplasmic acidification in the early phase of apoptosis induced by staurosporine and anti-fas antibodies (39), so acidification may also be a trigger for the dissociation of Bax from Inhibits Bax- and Fas-induced Apoptosis Finally, we examined the physiological role of the interaction between and Bax in the regulation of apoptosis. If negatively regulates Bax, overexpression of could be expected to inhibit Bax-induced apoptosis. As shown in Fig. 8, (a, left panel, and b), apoptosis induced by transfection of Bax DNA was significantly reduced by co-transfection of DNA, and integration of Bax into the mitochondrial membrane was also inhibited. Importantly, a mutant form of (K49E/V176D) that bound to Bax but not to phosphorylated targets, including Bad (Fig. 3b), also inhibited Bax-induced apoptosis (Fig. 8a, left panel), suggesting that the inhibition was due to direct association with Bax and not to the influence of various other binding proteins, including Bad, Raf-1, and forkhead protein. Furthermore, a caspase-cleaved mutant of (1 239) with a weak affinity for Bax (Fig. 6f) caused less inhibition of Bax-induced apoptosis, whereas another mutant ( ) that did not bind to Bax (data not shown) could not inhibit such apoptosis (Fig. 8a, middle panel). Incomplete suppression of Bax-induced apoptosis by overexpression of was probably due to the abundance of endogenous Moreover, as shown in Fig. 8a (right panel), did not inhibit apoptosis induced by Bax N, to which it did not FIG. 8.Involvement of in the regulation of apoptosis. a, inhibition of Bax-induced apoptosis by co-transfection of T cells were transfected with puc-caggs-human or its mutant (0.5 g), with or without puc-caggs-ha-tagged Bax (0.2 g) or a Bax mutant ( N), together with the GFP expression construct (0.1 g) (to identify DNA-transfected cells). After 24 h, apoptosis was assessed from the nuclear morphology by Hoechst staining of GFP-positive cells. The data are expressed as the means S.D. (n 4). b, prevention of Bax translocation to the mitochondria by expression of A similar experiment to that described in a was conducted. DNA-transfected cells were fractionated, and the extent of Bax translocation to the mitochondria was assessed by Western blotting using anti-bax antibody. c, efficient translocation of Bax N to the mitochondria. 293T cells were transfected with puc-caggs-ha-tagged Bax (0.2 g) or a Bax mutant ( N) and then were fractionated. The extent of Bax translocation to the mitochondria was assessed by Western blotting using anti-ha antibody. d, inhibition of Fas-mediated apoptosis by a caspasenoncleavable mutant of HeLa cells were transfected with puc- CAGGS-human or its mutant (0.4 g) together with the GFP expression construct (0.1 g) (to identify DNA-transfected cells). After 24 h, the cells were treated with 0.25 g/ml of anti-fas antibody (CH-11), and apoptosis was assessed after another 12 h from the nuclear morphology by Hoechst staining of GFP-positive cells. The data are expressed as the means S.D. (n 4). bind (Fig. 2). Consistent with the inability of to sequester Bax N, we found that Bax N showed efficient translocation to the mitochondria (Fig. 8c) and induced more apoptosis than wild-type Bax (Fig. 8a). These results suggested that inhibits Bax-induced apoptosis in an interaction-dependent manner. Next, we examined whether overexpression of could inhibit apoptosis induced by an anti-fas antibody. Because we found that caspase-8 cleaved to release Bax, we also tested a caspase-resistant (noncleavable) mutant of ( D239A). As shown in Fig. 8d, overexpression of D239A significantly inhibited apoptosis induced by anti-fas antibody, suggesting that the cleavage of by caspases could facilitate Fas-induced apoptosis. DISCUSSION Bax is mainly found in cytoplasmic and/or peri-mitochondrial locations in living cells, and apoptotic stimulation causes its stable integration into the mitochondrial membrane, along with the induction of cytochrome c release (18 22, 31, 40, 41). However, it is still poorly understood how Bax remains inactive in healthy cells. Although it has been suggested that Bax exists as a monomer in the cytoplasm of healthy cells and forms dimers or oligomers on the mitochondrial membrane during apoptosis (10, 21), the present study clearly showed that a significant fraction of Bax interacts with in living cells and that this interaction negatively regulates Bax by seques-

7 2064 Negative Regulation of Bax by tering it to the cytoplasm. Among seven isoforms, , (this study), and (34) also bind to Bax and probably play a redundant role. It has been suggested that Bax undergoes a conformational change during apoptosis, on the basis of its increased susceptibility to proteolytic cleavage (20) and binding with some antibodies (42). These changes can be explained by our proposal that Bax dissociates from during apoptosis. Our finding that both N- and C-terminal regions of Bax were required for its interaction with is also consistent with previous observations that translocation of Bax to the mitochondria is stimulated by N-terminal deletion (Fig. 8c and Ref. 20) as well as by mutation of charged residues in the N- and C-terminal regions (23, 24). We also showed that Bax dissociates from under both alkaline and acidic conditions, which is consistent with the previous observation that translocation of Bax to the mitochondria is enhanced at an alkaline ph (24). Taken together, it seems likely that a significant fraction of Bax is sequestered by cytoplasmic in healthy cells, whereas apoptotic stimuli cause the dissociation of Bax from and translocation to the mitochondria. The proteins are highly conserved cytoplasmic molecules that interact with various cellular proteins and that are thought to be involved in the regulation of various cellular processes, including apoptotic signal transduction (25). It has been reported that different isoforms of sequester different pro-apoptotic molecules through a phosphorylated serine residue on the target molecule and thus inhibit apoptosis (e.g. for ASK1 (27), for Bad (26), and for FKHRL1 (28)). The different proteins therefore appear to protect cells from apoptosis at various steps of the death signaling pathway by sequestering different pro-apoptotic proteins (37). Most of the binding proteins interact with via phosphorylated serine or threonine residue (25, 36). In contrast, we showed that the interaction of Bax with occurs in a phosphorylation-independent manner, based on the observations that phosphorylation of Bax was undetectable (Fig. 3a) and that Bax still bound to a mutant form of ( 49 / V176D) lacking the ability to bind to various phosphoprotein targets (Fig. 3b). It has been reported that also interacts with nonphosphorylated proteins such as ADP-ribosyltransferase Exoenzyme S (ExoS) from Pseudomonas aeruginosa (43). Our preliminary study suggested that a region from -helix 7 to the C terminus of , the three-dimensional structure of which could not be identified (possibly because of its high flexibility) (44, 45), was crucial for the interaction with Bax (data not shown). -Helix 7 and the more C-terminal -helix 8 comprise the box-1 region, where phosphorylated target proteins mainly bind by hydrophobic interaction (36, 46, 47), and this region probably undergoes a conformational change upon binding of a phosphoprotein to It is therefore conceivable that Bax binds to in healthy cells, and unidentified phosphoprotein(s) may interact with to release Bax after an apoptotic stimulus is delivered. We showed that Bax dissociates from by caspaseindependent and -dependent mechanisms. For the caspaseindependent process, one possible trigger is alteration of cytosolic ph (acidification or alkalization), which has been shown to occur in the early phase of apoptosis (24, 39), and indeed we found that this induced the dissociation of Bax from Bax also underwent dissociation after the direct cleavage of by caspases. The caspase-dependent dissociation of Bax from and subsequent integration of Bax into the mitochondrial membrane probably represents a positive feedback loop for death signal transduction. In death receptor-mediated apoptotic signaling, however, casapase-8 (which cleaves ) is activated upstream of the mitochondria, so the caspasedependent dissociation of Bax from acts as an initial trigger for apoptotic mitochondrial changes. When HeLa cells were treated with anti-fas antibody, cleavage of , dissociation of Bax from , and integration of Bax into the mitochondria were observed simultaneously (data not shown). Furthermore, overexpression of caspase-uncleavable mutant (D239A) conferred stronger resistance to Fas-mediated apoptosis than overexpression of wild-type (Fig. 8d), suggesting that cleavage of by caspase-8 is one of the crucial steps in Fas/TNF-mediated apoptosis. An essential role of Bax in Fas-mediated apoptosis has been shown by genetargeting studies, because hepatocytes of Bax/Bak-deficient mice are resistant to Fas-mediated apoptosis, whereas hepatocytes from Bax-deficient, Bak-deficient, and wild-type mice are all equally sensitive to Fas-mediated apoptosis (11). In summary, we investigated the mechanisms by which translocation of Bax into the mitochondrial membrane is regulated and found that plays a crucial role in sequestering Bax to the cytoplasm, where apoptotic stimulation causes it to release Bax in both a caspase-independent and -dependent manner. 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