Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9683–9688, August 1999 Cell Biology

Bcl-2 family members do not inhibit by binding the caspase activator Apaf-1

(cell death͞CED-4͞CED-9͞Bcl-xL͞caspase-9)

KOHJI MORIISHI*, DAVID C. S. HUANG,SUZANNE CORY, AND JERRY M. ADAMS†

The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Hospital, Victoria 3050,

Contributed by Suzanne Cory, June 11, 1999

ABSTRACT The Bcl-2 family of proteins regulates apo- mammalian relatives of CED-9 include the oncoprotein Bcl-2, ptosis, the cell death program triggered by activation of the first inhibitor of apoptosis to be identified (12), and its certain proteases (caspases). An attractive model for how close relatives, such as Bcl-xL, whereas the counterparts of Bcl-2 and its closest relatives prevent caspase activation is that EGL-1 comprise a number of more distant Bcl-2 relatives, such they bind to and inactivate an adaptor protein required for as Bax or Bim, that can bind to the prosurvival molecules and procaspase processing. That model has been supported by ablate their function (reviewed in ref. 13). reports that mammalian prosurvival Bcl-2 relatives bind the A central unresolved issue in cell death research is how the adaptor Apaf-1, which activates procaspase-9. However, the in prosurvival Bcl-2 family members prevent caspase activation. vivo association studies reported here with both overexpressed The death programs impaired in cells from mice lacking either Apaf-1 (14, 15) or caspase-9 (16, 17) suggest that both are and endogenous Apaf-1 challenge this notion. Apaf-1 could be essential components of the pathway controlled by the Bcl-2 immunoprecipitated together with procaspase-9, and the family (see Discussion). If so, the critical issue becomes how Apaf-1 caspase-recruitment domain was necessary and suffi- these family members keep Apaf-1 in a latent form. Two major cient for their interaction. Apaf-1 did not bind, however, to any types of models remain under debate. The indirect model of the six known mammalian prosurvival family members argues that Bcl-2, which resides on the endoplasmic reticulum, (Bcl-2, Bcl-xL, Bcl-w, A1, Mcl-1, or Boo), or their viral ho- nuclear envelope, and mitochondrial outer membrane, acts to mologs adenovirus E1B 19K and Epstein–Barr virus BHRF-1. maintain organelle function or integrity (18, 19). For instance, Endogenous Apaf-1 also failed to coimmunoprecipitate with Bcl-2 may prevent the escape of the Apaf-1 cofactor cyto- endogenous Bcl-2 or Bcl-xL, or with two proapoptotic relatives chrome c from the intermembrane space of the mitochondrion (Bax and Bim). Moreover, apoptotic stimuli did not induce (10, 20). Other evidence instead favors direct interaction Apaf-1 to bind to these family members. Thus, the prosurvival between the prosurvival molecules and the adaptor protein. Bcl-2 homologs do not appear to act by sequestering Apaf-1 Studies of C. elegans genes in heterologous overexpression and probably instead constrain its activity indirectly. systems have suggested that CED-9, CED-4, and CED-3 exist in a multiprotein complex, dubbed the apoptosome (21), in which CED-9 inhibits apoptosis by binding to CED-4, thereby Programmed cell death is critical for the physiologic removal preventing it from activating CED-3 (22–25). Upon induction of unwanted cells during development, for tissue homeostasis, of apoptosis, the binding of EGL-1 to CED-9 is envisaged to and in host defense mechanisms (1). Because deregulation of displace CED-4, allowing it to activate CED-3 (5, 7, 26). this program also underlies many pathological states, including Recently, evidence for analogous mammalian apoptosomes , autoimmunity, and degenerative diseases (2, 3), clar- has been reported by two laboratories (27–30). Coimmuno- ifying the biochemical mechanisms of apoptosis is a major goal precipitation studies with epitope-tagged molecules from tran- of current biological research. siently transfected cells led them to conclude that Bcl-xL (and The pathway to apoptosis has been conserved during evo- later also Boo) bound to Apaf-1. However, the similar in vivo lution. Genetic studies in the nematode Caenorhabditis elegans association studies reported here for Bcl-xL or its relatives, have identified three regulators required for the activation and Apaf-1 and procaspase-9, challenge that conclusion. We find execution of apoptosis (CED-3, CED-4, EGL-1) and one that Apaf-1 can interact with procaspase-9 but we have been critical inhibitor (CED-9) (4, 5). CED-3, a cysteine protease of unable to detect association between Apaf-1 and any of the six the caspase family (6), serves as the executioner by cleaving known mammalian prosurvival family members or two viral vital cellular substrates. Premature CED-3 activation is pre- homologs. Moreover, a new mAb to Apaf-1 has allowed us to cluded by its synthesis as a zymogen, which requires the show that endogenous Apaf-1 does not interact detectably with either overexpressed or endogenous Bcl-2 or Bcl-x . Apaf-1 adaptor protein CED-4 for autocatalytic processing (7). L also failed to associate with two proapoptotic family members. CED-4 activity normally is held in check by CED-9, but CED-9 Finally, we show that apoptotic stimuli did not induce associ- in turn can be neutralized by its distant relative EGL-1, which ation of Bcl-2 relatives with Apaf-1. These results suggest that directly associates with CED-9 (5). Bcl-2 and its prosurvival homologs do not function by seques- The mammalian apoptotic program, albeit more complex, is tering Apaf-1. closely analogous. The eight or so mammalian caspases that mediate apoptosis include both those that initiate the program, such as procaspase-9, and those the initiator caspases then MATERIALS AND METHODS process, such as procaspase-3, which cleave downstream tar- Expression Constructs. The expression vector pEF PGK- gets (6). Activation of procaspase-9 is promoted by Apaf-1, the puro and derivatives expressing wild-type (wt) or FLAG- only well-characterized mammalian homolog of CED-4, in the presence of the cofactors cytochrome c and dATP (8–11). The Abbreviations: wt, wild type; HA, hemagglutinin; CARD, caspase recruitment domain. The publication costs of this article were defrayed in part by page charge *Present address: Department of Veterinary Science, National Insti- tute of Infectious Diseases, 1–23-1, Toyama, Shinjuku-ku, Tokyo 162, payment. This article must therefore be hereby marked ‘‘advertisement’’ in Japan. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. E-mail: adams@. PNAS is available online at www.pnas.org. edu.au.

9683 Downloaded by guest on September 28, 2021 9684 Cell Biology: Moriishi et al. Proc. Natl. Acad. Sci. USA 96 (1999)

tagged human Bcl-xL or human Bcl-2, mouse FLAG Bcl-w, unpublished results). Secondary horseradish peroxidase- mouse FLAG A1, FLAG E1B19K and FLAG BHRF-1 have conjugated goat to mouse, rat, and rabbit Ig were been described (31–33). Mcl-1 cDNA was amplified by PCR from Southern Biotechnology Associates. Immunoreactive from a mouse thymoma library and cloned into pEF FLAGA proteins were detected by enhanced chemiluminescence (ECL, PGKpuro (31) to generate pEF Mcl-1FLAG puro. The Amersham Pharmacia), with exposure times of Ͻ 5 min. pcDNA3 mouse Boo FLAG construct was a gift from C. Vincenz, University of Michigan, Ann Arbor (30). Human RESULTS caspase-9 cDNA, a gift from S. Kumar (Hanson Centre, Adelaide, Australia), and an inactivating C287A mutant gen- Apaf-1 (8) has several distinctive domains (Fig. 1A). The erated by PCR using splice overlap extension (34) were N-terminal CARD (caspase recruitment domain) motif is subcloned into pEF PGKpuro. C-terminal hemagglutinin followed by a region having extensive homology with C. elegans (HA)-tagged wt or truncation mutants of human Apaf-1 were CED-4, including the Walker A and B boxes needed for ATP generated by PCR using Apaf-1 plasmids isolated from a binding and a unique region without homology to any known human HeLa cDNA library (Stratagene) or provided by X.D. protein, whereas the C-terminal half is comprised of 12 or 13 Wang, Southwestern Medical Center, Dallas (8). They were repeats of the WD40 motif, which often is involved in protein cloned into derivatives of pcDNA3 (Invitrogen) that we de- association (8, 11). To explore the role of these domains in rived to introduce a C-terminal HA tag. Point mutants of Apaf-1 function, we constructed cDNAs that encode eight Apaf-1 (K149R and DD232͞233AA) were generated by splice Apaf-1 deletion mutants (Fig. 1A), as well as two full-length overlap extension (34), and pcDNA3 FLAGApaf-1 (27) was a proteins having a point mutation in the A box (K149R) or the gift from V. Dixit, Genentech, San Francisco. B box (DD232͞233AA) that should block ATP binding (38). All PCRs were performed by using proof-reading Pfu Turbo The CARD Region of Apaf-1 Allowed Its Association with DNA polymerase (Stratagene), and the sequences of all Procaspase-9. To identify the regions of Apaf-1 critical for constructs were verified by automated sequencing (ABI Per- interaction with procaspase-9, we transiently expressed a cat- kin–Elmer). Details of the oligonucleotides are available on alytically inactive mutant of procaspase-9 (C287A) together request. with the wt or mutant forms of Apaf-1 in the human embryonal Cell Culture, Transfection, Immunoprecipitation, and kidney cell line 293T, and detected the proteins by the Western Blot Analysis. The 293T, HeLa, and Jurkat cell lines C-terminal Flag epitope on procaspase-9 or the C-terminal were maintained as described (31–33, 35). Cell death was HA tag on Apaf-1 and its derivatives. Immunoblots of lysates induced by culturing cells in 1 nm to 1 ␮M staurosporine confirmed that each protein was readily detectable (Fig. 1B, (Sigma), 5–500 ng͞ml doxorubicin (Sigma), or 100 J͞m2 UV Lower). To reveal any association between them, procaspase-9 irradiation. was immunoprecipitated from the lysates with anti-Flag anti- Liposome-mediated DNA transfection of 293T human em- body. The immune complexes, captured on Protein G Sepha- bryonal kidney cells was performed essentially as described rose beads, were fractionated by electrophoresis and blotted (32). Briefly, 106 cells were plated in a 10-cm dish and 24 h later with anti-HA to reveal any coprecipitated Apaf-1. were transfected by incubation overnight with 2.5 ␮g of total As reported by others (27, 28), full-length Apaf-1 bound to plasmid DNA and 15 ␮l of Lipofectamine (GIBCO͞BRL) in procaspase-9 (Fig. 1B, Upper, lysate 1). Many, but not all, of the 5 ml of high-glucose DMEM lacking antibiotics. This step was Apaf-1 mutants also coimmunoprecipitated with pro- followed by addition of 5 ml of DMEM supplemented with caspase-9, as summarized in Fig. 1A. The interaction was not 20% FCS. The cells were harvested 18 h after transfection, prevented by mutation of the Walker A box or B box (lysates washed twice in cold PBS, then suspended in lysis buffer (20 2 and 3), nor by deletion of the unique or WD40 regions mM Tris⅐HCl pH 7.4͞135 mM NaCl͞1% Triton X-100͞10% (lysates 4 and 5). On the other hand, the CARD was absolutely glycerol, supplemented with 0.5 ␮g͞ml Pefabloc͞1 ␮g/ml each required. Indeed, that domain (residues 1–97) alone sufficed of leupeptin, aprotinin, soybean trypsin inhibitor, and pepsta- for interaction with procaspase-9 (lysate 6), and the remainder ͞ ͞ tin 50 mM NaF 5mMNa3VO4). Protein concentration was of Apaf-1 was unable to bind (lysate 7). These data suggest determined by the Micro BCA (bicinchoninic acid) Protein that, at least when coexpressed at high levels, the CARD Assay Kit (Pierce) according to the manufacturer’s protocol. region of Apaf-1 allows it to complex with procaspase-9 and For immunoprecipitation, the lysates (1 mg protein per the remainder of the molecule appears dispensable for the reaction) were precleared by incubation at 4°C for 1.5 h with association. 20 ␮l of 50% (vol͞vol) Protein G Sepharose 4 Fast Flow beads An unexpected technical problem noted during these studies (Amersham Pharmacia), followed by centrifugation at 13 K for and apparent in Fig. 1B is that some control immunoprecipi- 5 min at 4°C. The supernatant was mixed with excess (Ͼ1 ␮g) tates prepared without the anti-Flag antibody (lanes marked antibody at 4°C for 1.5 h, and then with 20 ␮l of 50% (vol͞vol) Ϫ) contained significant levels of Apaf-1. This background, Protein G Sepharose beads for 1.5 h at 4°C. Then the beads particularly evident with the N-terminal polypeptides contain- were washed 3–6 times with 0.5 ml of lysis buffer, and the ing only the CARD and CED-4 homology regions (lysates 4 bound proteins were eluted by boiling in 25 ␮l of loading buffer and 5), suggested that Apaf-1 could bind nonspecifically to the (0.5M Tris⅐HCl, pH 6.8͞4% SDS͞20% glycerol͞0.005% bro- Sepharose beads. Indeed, a direct test (Fig. 1C) showed that a mophenol blue) containing 50 mM DTT. The eluate was significant proportion of the truncated Apaf-1 (1–412) bound fractionated by SDS͞PAGE (Novex) and transferred to nitro- to these beads in the absence of antibody and remained bound cellulose membranes (Amersham Pharmacia). even after several washes. Neither Bcl-xL (Fig. 1C) nor pro- Immunoblotting was carried out in PBS containing 5% skim caspase-9 (not shown) bound significantly to the beads. In view milk powder and 0.05% Tween 20. Antibodies included mouse of the ‘‘stickiness’’ of Apaf-1, our protocols routinely included monoclonal anti-FLAG M2 (Sigma), mouse monoclonal an- preclearance of the lysates with Protein G Sepharose beads, ti-HA 16B12 (HA.11; Babco), rat monoclonal anti-HA 3F10 several wash steps and controls without the immunoprecipi- (Boehringer Mannheim), rabbit polyclonal anti-Bcl-x (Phar- tating antibody. Mingen), mouse monoclonal anti-Bcl-x 7B2.5 (gift of C. No Interaction Between Overexpressed Bcl-xL and Apaf-1. Thompson, University of Chicago), mouse monoclonal anti- In the apoptosome model, Apaf-1 in healthy cells is seques- human Bcl-2–100 (36), hamster monoclonal anti-human Bcl-2 tered in an inactive form by prosurvival Bcl-2 family members. 6C8 (37), mouse monoclonal anti-Bax B-9 (Santa Cruz Bio- In view of previous reports (27, 28), we first sought to detect technology), and rat monoclonal anti-human Apaf-1 2E12 (G. association of Apaf-1 with Bcl-xL, by using procaspase-9 as a Hausmann, L. A. O’Reilly, D.C.S.H., A. Strasser, and J.M.A., positive control. Full-length Apaf-1HA was transiently ex- Downloaded by guest on September 28, 2021 Cell Biology: Moriishi et al. Proc. Natl. Acad. Sci. USA 96 (1999) 9685

A tained substantial Apaf-1. In contrast, no evidence was found caspase-9 for specific interaction between Apaf-1 and either native CARD CED-4 unique WD40 repeats binding AB Bcl-xL or its tagged derivatives (the trace of Apaf-1 in certain N- -C + precipitates was also evident in the control lacking antibody). 1 97 412 601 1194 Inability to detect association could not be ascribed to poor 464 + expression of Bcl-xL, because its level was in fact higher than 412 + that of procaspase-9, as judged by analysis of the lysates with 90 412 - anti-Flag antibody (Fig. 2A, Lower). Moreover, both pro- 413 601 - caspase-9 and Bcl-xL were efficiently immunoprecipitated by the antibodies (data not shown). In case the C-terminal HA tag 457 1194 - on Apaf-1 had prevented the putative Apaf-1:Bcl-xL interac- 140 + tion, we also tested Apaf-1 with an N-terminal HA tag, or an 97 + N- or C- terminal Flag tag, but again we found no evidence for 98 1194 - interaction (data not shown). B Because the WD40 region of Apaf-1 may negatively regulate 1234 5 67 its action (8, 39, 40), we next tested whether removal of that domain would allow interaction with Bcl-x . An HA-tagged ip: + α-Flag + - + - + - + - + - + - + - L N-terminal portion of Apaf-1 (residues 1–412), including the Apaf-1 - 1-1194 - CARD and CED-4 homology domain, was transiently coex- 98-1194 - pressed in 293T cells with the Bcl-xL derivatives. Under α-HA - 1-464 conditions where Apaf-1(1–412) clearly interacted with pro- - 1-412 caspase-9 (lysate 2 in Fig. 2B), it did not interact specifically

1-97 - with either native Bcl-xL (lysate 4) or the tagged versions (lysates 3 and 5). Similarly, the C-terminal moiety of Apaf-1

12345 6 7 containing the WD40 repeats (residues 457-1194) also failed to Apaf-1 Apaf-1 interact with the Bcl-xL (Fig. 2C). - 1-1194 98-1194 - Thus, in contrast to recent reports (27, 28), our immuno- precipitation experiments have not revealed any evidence of - 1-464 α-HA - 1-412 specific interaction between overexpressed Bcl-xL and Apaf-1, under conditions where association between Apaf-1 and pro- 1-97 - caspase-9 was readily demonstrable. It is highly unlikely that the putative Apaf-1:Bcl-xL complex was disrupted by the α-Flag - casp-9 Flag casp-9 Flag - immunoprecipitating antibody, because the diverse antibodies C tested included a polyclonal antiserum to Bcl-x (Fig. 2) and a TB monoclonal anti-Bcl-x antibody (data not shown), as well as the x 1 x 3 anti-Flag antibody, interacting with a tag at either end of Bcl-xL (Fig. 2). Moreover, we tested lysates prepared with other α-HA - Apaf-1 1-412 detergents (1% NP-40 or 1% digitonin) or in the absence of detergent (data not shown). Hence, the lack of interaction is α - Bcl-x -Bcl-x L unlikely to reflect epitope masking, antibody interference, or lysis conditions. Nor is it peculiar to the cell line used, because FIG. 1. Apaf-1 binds through its CARD to procaspase-9. (A) The cotransfected COS cells yielded identical results (data not wt and truncated human Apaf-1 proteins used in this study, all with a shown). Some of our experiments also included protocols that B C-terminal HA tag, and their binding to procaspase-9. ( ) The Apaf-1 closely replicated those reported to reveal such complexes (27, CARD (residues 1–97) is necessary and sufficient for binding to procaspase-9. 293T cells were transiently cotransfected with a vector 28), but no specific complexes were found. expressing mutant procaspase-9 Flag and ones expressing HA-tagged Endogenous Apaf-1 Did Not Interact with Any Prosurvival wt or mutant Apaf-1: wt (lane 1), Apaf-1 K149R (A box mutant) (lane Bcl-2 Family Member. Because all of the studies described 2), Apaf-1 KDD232͞233AA (B box mutant) (lane 3), Apaf-1 (1–464) above had been performed with epitope-tagged Apaf-1, it (lane 4), Apaf-1 (1–412) (lane 5), Apaf-1 (1–97) (lane 6), and Apaf-1 remained possible that the tag had inhibited interaction with (98–1194) (lane 7). Lysates (1 mg protein) were incubated in the Bcl-xL (although not with procaspase-9). The recent genera- presence (ϩ) or absence (Ϫ) of monoclonal anti-FLAG M2, and the ͞ tion of mAbs for Apaf-1 by L. O’Reilly (G. Hausmann, L. A. resulting immunoprecipitates were fractionated by SDS PAGE and O’Reilly, D.C.S.H., A. Strasser, and J.M.A., unpublished re- Upper blotted with monoclonal anti-HA 3F10 antibody ( ). Expression sults) enabled us to look for interactions with endogenous of procaspase-9 and Apaf-1 was confirmed by blots of lysates (Lower). The larger bands in lanes 4 and 5 represent aggregated Apaf-1 Apaf-1. The 2E12 mAb used here is specific for human Apaf-1 mutants. These data are representative of three experiments. Results and recognizes its CARD. Lysates of the human 293T cells with other Apaf-1 derivatives are summarized (A). (C) Apaf-1, transfected with vectors for Flag-tagged procaspase-9 or Bcl-xL particularly its N-terminal region, binds to Sepharose beads. Lysates were precipitated with anti-Flag antibody and the immune (0.5 mg protein) from 293T cells transfected with vectors expressing complexes were analyzed with the 2E12 antibody (Fig. 3A). ͞ Apaf-1 1–412 HA or Bcl-xL were mixed with 50% (vol vol) Sepharose Endogenous Apaf-1 coprecipitated with procaspase-9 (lysates CL4B beads (Sigma) at 4°C for 1.5 h, and the beads then were washed 1 and 2) but not detectably with Bcl-xL (lysates 3 and 4). once or three times with lysis buffer. Equivalent amounts of total (T) Irrespective of whether Bcl-xL bore an N- or C-terminal Flag and Sepharose-bound (B) lysate then were Western-blotted. The data tag, the levels of Apaf-1 in those anti-Flag immunoprecipitates are representative of two experiments. Full-length Apaf-1 yielded lower background binding (see text). were no greater than in the control without antibody (Fig. 3A, compare ϩ and Ϫ lanes). pressed in 293T cells together with either Flag-tagged pro- If there prove to be several mammalian CED-4 homologs, caspase-9 or Bcl-xL, either the native protein or derivatives Apaf-1 might preferentially interact with another Bcl-2 pro- with Flag at either end. Immunoprecipitates were prepared survival relative. To explore this possibility, we transiently with anti-Bcl-x or anti-Flag antibodies and blotted with an- expressed in 293T cells each of the five other known mam- ti-HA antibody (Fig. 2A, Upper). As expected, the pro- malian prosurvival family members (Bcl-2, Bcl-w, Mcl-1, A1, caspase-9 immunoprecipitate (lysate 2) reproducibly con- and Boo), as well as two viral functional homologs (adenovirus Downloaded by guest on September 28, 2021 9686 Cell Biology: Moriishi et al. Proc. Natl. Acad. Sci. USA 96 (1999)

A Apaf-1 HA B Apaf-1 1-412 HA C Apaf-1 457-1194 HA 12345 ip: α-Flag, 12 3 45 235 α ip: -Flag, Bcl-x + - + - + - + - + - ip: α-Flag + - + - + - Bcl-x + - + - + - + - + - α-HA - Apaf-1 α - Apaf-1 α-HA - Apaf-1 -HA 1-412 457-1194 12345 12345 235 - Apaf-1 α-HA α-HA - Apaf-1 1-412 α-HA - Apaf-1 457-1194

Flag Bcl-x α - Flag Bcl-xL α - L -Bcl-x - -Bcl-x - Bcl-xL Bcl-xL - casp-9 Flag α-Flag - casp-9 Flag - casp-9 Flag - Flag Bcl-x α-Flag α-Flag L - Flag Bcl-xL - Flag Bcl-xL

FIG. 2. Overexpressed Bcl-xL and Apaf-1 do not interact. Tests with full-length Apaf-1 (A), its N-terminal moiety (B), or its C-terminal moiety (C). 293T cells were transiently transfected with vectors encoding (A) Apaf-1 HA, (B) Apaf-1 (1–412) HA, or (C) Apaf-1 (457–1194) HA, together with either a vector control (lane 1) or vectors encoding mutant caspase-9 FLAG (lane 2), FLAG Bcl-xL (lane 3), Bcl-xL (lane 4), or Bcl-xL FLAG (lane 5). Proteins were immunoprecipitated in the presence (ϩ) or absence (Ϫ) of anti-FLAG M2 or polyclonal anti-Bcl-x, fractionated by SDS͞PAGE and then blots were analyzed with anti-HA 3F10 (Upper). Expression of the constructs was documented by Western blots on lysates (Lower). Data shown are representative of three experiments.

E1B 19K and Epstein–Barr virus BHRF-1). Western blots Jurkat lymphoid cells. When Apaf-1 was immunoprecipitated confirmed adequate expression and precipitation of each of from the HeLa cell lysates with mAb 2E12, neither Bcl-2 nor these Flag-tagged proteins (arrowed bands in Fig. 3B, Lower). Bcl-xL was detected (data not shown). In the converse exper- However, none of their immunoprecipitates contained endog- iment, both prosurvival proteins were precipitated with each enous Apaf-1, although it clearly interacted with transfected of two antibodies and none of the immunoprecipitates con- procaspase-9 (Fig. 3B, Upper). tained Apaf-1 (Fig. 4A, Upper). Lysates from Jurkat cells gave Endogenous Apaf-1 Did Not Associate with Endogenous the same result. Importantly, endogenous Bax did coprecipi- Bcl-2 or Bcl-xL. To avoid any possible artefacts associated with tate with the prosurvival molecules under these conditions overexpression, we next tried to detect interaction of endog- (Fig. 4A, Lower). The formation of Bcl-2͞Bax heterodimers enous Apaf-1 with endogenous Bcl-2 or Bcl-xL in two human appears to involve a conformational change in Bax induced by lines containing substantial Apaf-1: HeLa epithelial cells and detergents in the cell lysates (41). In case detergents had interfered with interaction of Bcl-2 (or Bcl-xL) with Apaf-1, we A repeated all these experiments with lysates made in the 1 2 3 4 absence of detergents but still observed no association of ip: + α-Flag + - + - + - + - endogenous Apaf-1 with either prosurvival protein (Fig. 4B). α-Apaf-1 Apoptotic Stimuli Did Not Induce Apaf-1 to Associate with Bcl-2 Family Members. An alternative hypothesis for control 123 4 of Apaf-1 would be that Bcl-2 prosurvival relatives sequester - casp-9 Flag Apaf-1 after a death stimulus, thereby serving as a ‘‘sink’’ to α-Flag - Flag Bcl-xL prevent activated Apaf-1 from initiating the caspase cascade. To test this possibility, we subjected HeLa cells to a cytotoxic α-Apaf-1 dose of UV radiation and immunoprecipitated Bcl-2 and B Bcl-xL from lysates made without detergent. As reported with another cytotoxic stimulus (42), Bax underwent a change that allowed it to associate with the prosurvival proteins, but no

casp-9 Bcl-2 Bcl-w Mcl-1 A1 Boo E1B 19K BHRF-1 Apaf-1 was discernible in the immune complexes (Fig. 4C).

ip: + α-Flag - + - + - + - + - + - + - + - + Lysate Treatment with staurosporine or doxorubicin yielded the same result (data not shown). α-Apaf-1 A B C HC + UV ip antibody ip antibody ip antibody α-Flag LC, pG lysate Apaf-1 2E12 none Bcl-2-100 Bcl-2 6C8 Bcl-x 7B2 Bcl-x poly none Bcl-2-100 Bcl-x 7B2 Apaf-1 2E12 none Bcl-2-100 Bcl-x 7B2 Apaf-1 2E12 FIG. 3. Endogenous Apaf-1 does not interact with Bcl-xL or the other prosurvival homologs. (A) Tests with Bcl-xL and procaspase-9. α-Apaf-1 Lysates from 293T cells transfected with wt procaspase-9 FLAG (lane 1), procaspase-9 C287A FLAG (lane 2), FLAG Bcl-xL (lane 3), or α-Bax Bcl-xL FLAG (lane 4) were immunoprecipitated by using monoclonal anti-FLAG M2, fractionated by SDS͞PAGE and then analyzed by FIG. 4. Endogenous Apaf-1 does not interact with endogenous Western blotting with monoclonal anti-Apaf-1 2E12. (B) Tests with Bcl-2 or Bcl-xL in HeLa cells. Lysates prepared in the presence of other prosurvival Bcl-2 homologs. Lysates from 293T cells transfected Tritan X-100 from healthy cells (A), or without detergent from healthy with constructs expressing Flag-tagged mutant procaspase-9, Bcl-2, cells (B), or those dying from UV treatment (C) cells were immuno- Bcl-w, Mcl-1, A1, Boo, E1B19K, or BHRF-1 were immunoprecipitated precipitated with anti-Apaf-1 2E12, or the indicated anti-Bcl-2 or with anti-FLAG M2, and the blot was probed with anti-Apaf-1 2E12 anti-Bcl-x antibodies. The blots were probed with anti-Apaf-1 2E12 (Upper). Probing of the same blot (Lower) with anti-Flag M2 con- (Upper) or anti-Bax B-9 (Lower). Viability of the healthy cells was 98% firmed expression and immunoprecipitation of each of the transfected and remained 90% for the dying cells at this time point (6 h after Bcl-2 homologs (open arrows). HC, heavy chain; LC, light chain; PG, exposure to UV irradiation). Data shown are representative of three protein G. Data shown are representative of four experiments. experiments. Downloaded by guest on September 28, 2021 Cell Biology: Moriishi et al. Proc. Natl. Acad. Sci. USA 96 (1999) 9687

Yet another model would be that Apaf-1 binds to the confocal microscopy and subcellular fractionation indicate proapoptotic members of the Bcl-2 family. For instance, the that neither overexpressed nor endogenous Apaf-1 co-localize change in Bax might allow it to trigger Apaf-1 activation. with Bcl-2 or Bcl-xL (G. Hausmann, L. A. O’Reilly, D.C.S.H., However, immunoprecipitates of Apaf-1 did not contain Bax A. Strasser, and J.M.A., unpublished results). Thus, we have (Fig. 4 A and B), even after induction of cell death (Fig. 4C). found no support for the hypothesis that prosurvival Bcl-2 Similarly, in both healthy Jurkat cells and those induced to die, homologs physically sequester Apaf-1. Our observation that Apaf-1 was not found in association with Bax or with Bim, a Apaf-1 can bind nonspecifically to Sepharose beads, particu- representative of the second major class of proapoptotic Bcl-2 larly after removal of its WD40 repeat region (Fig. 1C), may family members (data not shown). in part explain the apparent interaction between Apaf-1 and Bcl-2-like proteins reported previously. The evidence for DISCUSSION interaction of the homologous C. elegans proteins appears stronger, but as yet it all derives from overexpression in Recently the strong biochemical evidence that Apaf-1 is the heterologous cell types (yeast or mammalian cells). It will be adaptor protein controlling procaspase-9 activation (9, 11, 39) important to learn whether endogenous CED-9 and CED-4 has been firmly supported genetically by the similar pheno- interact in the worm. types of mice lacking Apaf-1 or caspase-9 (14–17). Our Is it plausible that Apaf-1 is not the bona fide functional immunoprecipitation data confirm that Apaf-1 can interact homolog of CED-4, and that a true, as-yet-undiscovered, with procaspase-9 and show that the Apaf-1 N-terminal mammalian counterpart is regulated by the Bcl-2 family? CARD is necessary and sufficient for the association (Fig. 1) Certainly Apaf-1 has domains not present in CED-4, so its when either protein is overexpressed. We currently are testing regulation may well differ from that of CED-4. Indeed, C. whether these proteins interact in healthy cells when both are elegans CED-4 does not appear to require cytochrome c to at physiological levels. Presumably, the binding of dATP to activate CED-3 (38). On the other hand, the phenotype of mice Apaf-1 is not required for its association with procaspase-9, because Apaf-1 with mutated A or B boxes bound as well as deficient in Apaf-1 argues strongly that Apaf-1 is an essential, nonredundant component of the pathway regulated by Bcl-2, the wt protein (Fig. 1B). Regions of Apaf-1 other than the Ϫ͞Ϫ CARD appeared not to contribute to the interaction. Their because cells from Apaf-1 mice are refractory to several roles remain less well defined, although the CED-4 region may of the death stimuli inhibitable by Bcl-2 and behave normally be required for oligomerization (7, 11), whereas the WD40 toward the signals from ‘‘death receptors’’ such as CD95 (14, repeat region may play a negative regulatory role (8, 39, 40). 15), a pathway largely independent of Bcl-2 control (35). Biochemical studies on the key apoptotic players from C. Nevertheless, another mammalian CED-4 homolog might elegans have suggested that both CED-9 and CED-3 interact participate. Two molecules with trace homology to CED-4 with CED-4 in healthy cells. The apoptosome model (21) have been described very recently (44, 45), but their roles suggests that CED-9 holds CED-4 in an inactive form until a remain to be established. death stimulus induces expression of EGL-1, which binds to Our results strongly suggest that the regulation of Apaf-1 by CED-9, thereby releasing CED-4 to activate proteolytic activ- Bcl-2 is indirect rather than direct. More specifically, Bcl-2 and ity of CED-3 (5, 26). The central issue addressed in this paper its prosurvival homologs may prevent the release of apopto- is whether, in an analogous manner, the mammalian prosur- genic factors from mitochondria (18, 19) and, in view of their vival Bcl-2 family members function by binding to and neu- distribution in the cell, presumably also from the nucleus and tralizing Apaf-1. In experiments equivalent in design to those endoplasmic reticulum. Cytochrome c is thus far the strongest described here, two laboratories have reported that overex- candidate; it usually is released from mitochondria of cells pressed Apaf-1 binds to Bcl-xL (27, 28) and the newly described undergoing apoptosis and serves as an Apaf-1 cofactor (10, 20, Boo͞Diva (29, 30), whereas one experiment has suggested 46–48), probably by forming a multimeric complex with binding to Bcl-2 (43). Apaf-1 and procaspase-9 (11). On the other hand, cytochrome Our coimmunoprecipitation results are in conflict with c release is unlikely to be the sole and critical step regulated those reports. We have been unable to detect any interaction by Bcl-2 and its prosurvival relatives because Bcl-2 (and Bcl-xL) of Apaf-1 with Bcl-xL or any other prosurvival Bcl-2 family retains antiapoptotic activity in cells microinjected with cyto- member (Figs. 2–4). As well as testing full-length Apaf-1, we chrome c (49, 50), cytochrome c loss from mitochondria can be tested its N-terminal and C-terminal moieties. We excluded reversed (51), and none was released during killing by a Bcl-xL trivial technical explanations such as the presence or location peptide antagonist (52). Cytochrome c release may, in fact, of epitope tags, the cell line, and the lysis and immunopre- serve mainly to amplify the apoptosis process (53), and Bcl-2 cipitation conditions. Importantly, in case the epitope tag on may control a crucial, as-yet-unknown, upstream initiating Apaf-1 had inhibited interaction with Bcl-xL, we also looked step. for interaction with endogenous Apaf-1 by using a new Apaf- 1-specific mAb, but found no evidence for interaction with We thank S. Novakovic, J. Beaumont, and S. Ahmad for expert transfected Bcl-2, Bcl-xL, Bcl-w, A1, Mcl-1, Boo, or their viral technical assistance and Drs. J. Trapani and S. Kumar for comments counterparts adenovirus E1B19K and Epstein–Barr virus on the manuscript. The anti-Apaf-1 antibodies were generated by Drs. BHRF-1 (Fig. 3). Moreover, we found no association of L. O’Reilly and A. Strasser. We thank Drs. A. Strasser, L. O’Reilly, H. endogenous Apaf-1 with endogenous Bcl-2 or Bcl-xL, nor with Puthalakath, L. O’Connor, and G. Hausmann for advice and discus- Bax or Bim, representatives of the two classes of proapoptotic sions, and V. Dixit, S. Kumar, C. Thompson, C. Vincenz, X.D. Wang, family members (Fig. 4). Importantly, no detectable associa- and T. Willson for generous gifts of cell lines, antibodies, cDNA libraries, and expression vectors. K.M. was a recipient of a fellowship tion with Bcl-2 or Bcl-xL was induced by several cytotoxic stimuli (Fig. 4 and data not shown). from the Japan Science and Technology Corporation; D.C.S.H. is a A plausible sequestration model would seem to require that Special Fellow of the Leukemia Society of America. This work was supported by the National Health and Medical Research Council a great proportion, if not all, of the adaptor resides in a tight (Canberra, Reg. Key 973002) and U.S. National Cancer Institute complex with the prosurvival family members. No evidence for Grant CA80188. the existence of such complexes with Apaf-1 was found here, and it is unclear whether the Apaf-1 complexes reported by 1. Vaux, D. L. & Korsmeyer, S. J. (1999) Cell 96, 245–254. others (27–30, 43) would account for more than a few percent 2. 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