Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes
2001; Springer Nature; Volume: 20; Issue: 5 Linguagem: Inglês
10.1093/emboj/20.5.998
ISSN1460-2075
Autores Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle1 March 2001free access Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes Shawn B. Bratton Shawn B. Bratton Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK Search for more papers by this author Gail Walker Gail Walker Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK Search for more papers by this author Srinivasa M. Srinivasula Srinivasa M. Srinivasula Center for Apoptosis Research and Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA, 19107 USA Search for more papers by this author Xiao-Ming Sun Xiao-Ming Sun Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK Search for more papers by this author Michael Butterworth Michael Butterworth Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK Search for more papers by this author Emad S. Alnemri Emad S. Alnemri Center for Apoptosis Research and Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA, 19107 USA Search for more papers by this author Gerald M. Cohen Corresponding Author Gerald M. Cohen Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK Search for more papers by this author Shawn B. Bratton Shawn B. Bratton Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK Search for more papers by this author Gail Walker Gail Walker Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK Search for more papers by this author Srinivasa M. Srinivasula Srinivasa M. Srinivasula Center for Apoptosis Research and Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA, 19107 USA Search for more papers by this author Xiao-Ming Sun Xiao-Ming Sun Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK Search for more papers by this author Michael Butterworth Michael Butterworth Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK Search for more papers by this author Emad S. Alnemri Emad S. Alnemri Center for Apoptosis Research and Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA, 19107 USA Search for more papers by this author Gerald M. Cohen Corresponding Author Gerald M. Cohen Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK Search for more papers by this author Author Information Shawn B. Bratton1, Gail Walker1, Srinivasa M. Srinivasula2, Xiao-Ming Sun1, Michael Butterworth1, Emad S. Alnemri2 and Gerald M. Cohen 1 1Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester, LE1 9HN UK 2Center for Apoptosis Research and Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA, 19107 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:998-1009https://doi.org/10.1093/emboj/20.5.998 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, we have addressed the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome. We propose a multi-step process, whereby catalytically active processed or unprocessed caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3. We demonstrate that XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex. Introduction Apoptosis is a distinct form of cell death characterized by nuclear and cytoplasmic condensation, DNA fragmentation and externalization of membrane-associated phosphatidylserine. A class of cysteine proteases, known as caspases, produce these biochemical and morphological changes by selectively cleaving a number of structural and regulatory proteins at specific aspartate residues. In a general paradigm, either receptor- or stress-induced death signals stimulate oligomerization of specific adaptor molecules, such as FADD or Apaf-1, which subsequently recruit and promote trans-activation of ‘initiator’ caspases, such as caspases-8 and -9, respectively. Initiator caspases possess long prodomains that enable them to interact with death effector domains (DEDs) or caspase-activation recruitment domains (CARDs) present in these adaptor proteins. Once activated, initiator caspases are free to activate ‘effector’ caspases, such as caspases-3 and -7, which contain short prodomains and are primarily responsible for dismantling the cell during the execution phase of apoptosis (for reviews see Cohen, 1997; Earnshaw et al., 1999; Bratton et al., 2000). Cellular stress can stimulate the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, where it interacts with the adaptor protein Apaf-1 (Green and Reed, 1998). Apaf-1 contains at least three functional domains: (i) an N-terminal CARD, which binds the prodomain of caspase-9; (ii) a CED-4 domain required for Apaf-1 self-oligomerization; and (iii) a series of C-terminal WD-40 repeats thought to mediate protein− protein interactions (Zou et al., 1997). Apaf-1, when bound to cytochrome c, apparently hydrolyzes dATP/ATP and undergoes oligomerization via its CED-4 domains (Hu et al., 1998, 1999; Srinivasula et al., 1998). Simultaneously, the CARD domain recruits and facilitates processing of procaspase-9 (Li et al., 1997; Srinivasula et al., 1998; Qin et al., 1999). This complex of cytochrome c, Apaf-1 and caspase-9 is commonly referred to as the apoptosome. Reconstitution experiments using purified recombinant proteins indicate that the apoptosome is ∼1.4 MDa in size (Saleh et al., 1999; Zou et al., 1999), whereas in native cell lysates, Apaf-1 oligomerizes into an ∼700 kDa complex and, in addition to processed caspase-9, contains fully processed caspases-3 and -7 (p17 and p12 subunits) (Cain et al., 1999). Thus, the initial processing of effector caspases by caspase-9 and their subsequent autocatalytic processing appears to take place within the apoptosome. The specific mechanisms that govern these processes remain unclear. Caspases are inhibited by a number of viral and mammalian proteins, including inhibitor-of-apoptosis proteins (IAPs). These evolutionarily conserved proteins were first identified in baculoviruses and contain both an N-terminal tandem repeat of ∼70 amino acids, termed baculovirus IAP repeat (BIR) domain, and a C-terminal RING zinc-finger domain (Clem and Miller, 1994). Several IAP homologs containing one or both domains have been identified in mammalian cells, including X-linked IAP (XIAP or hILP), ML-IAP (livin), cellular inhibitor-of-apoptosis protein-1 (cIAP-1/hIAP-2), cIAP-2 (hIAP-1), neuronal apoptotic inhibitory protein (NAIP) and survivin (for review see Deveraux and Reed, 1999; Vucic et al., 2000). The anti-apoptotic mechanisms of some of these proteins are unknown; however, XIAP, ML-IAP, cIAP-1 and cIAP-2 appear to inhibit both stress- and death receptor-induced apoptosis through direct inhibition of distinct caspases (Deveraux et al., 1997, 1998; Vucic et al., 2000). In particular, XIAP inhibits Apaf-1-mediated activation of procaspase-9 and the activity of processed caspase-9, but not FADD-mediated activation of procaspase-8 or the activity of processed caspase-8. Perhaps more importantly, XIAP potently inhibits active caspases-3 and -7 in vitro. Thus, it is the ability of XIAP to inhibit active caspases-3 and -7 that suppresses death receptor-induced apoptosis, whereas its ability to inhibit both the apical and effector caspases protects against stress-induced apoptosis (Deveraux et al., 1997, 1998). Interestingly, XIAP inhibits active caspases-3 and -9 through distinct domains within the protein. The BIR2 domain, together with a few critical residues in the linker region between the BIR1 and BIR2 domains, is sufficient for inhibition of caspase-3, whereas the BIR3 domain inhibits caspase-9 (Takahashi et al., 1998; Sun et al., 1999, 2000). In this study, we used a cell-free model to define the requirements for the recruitment, processing and retention of caspases within the apoptosome. We propose a multi-step process, whereby catalytically active processed or unprocessed caspase-9 is required to recruit caspase-3 to the apoptosome via an interaction between the catalytically active cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3 required for its processing. Caspase-3 is subsequently processed within the apoptosome to its fully mature form and remains largely associated with the complex. In addition to binding active caspases-9 and -3, XIAP also associates with oligomerized Apaf-1. Thus, by associating with the apoptosome, XIAP appears not only to influence the activation of caspase-3 by caspase-9, but also to inhibit the release of active caspase-3 from the complex. Results and discussion The Apaf-1 apoptosome must contain caspase-9 in order to recruit caspase-3 to the complex Several genetic studies using ‘knockout’ mice and in vitro studies using cytochrome c/dATP have determined the apparent order of caspase activation following mitochondrial stress (Hakem et al., 1998; Kuida et al., 1998; Woo et al., 1998; Slee et al., 1999). However, none have examined the precise order in which caspases are recruited to the apoptosome, nor the molecular determinants required for this process. By systematically immunodepleting specific caspases from the lysate (Figure 1C) and replacing them with known caspase mutants (Figure 1A and B), we wished to determine if and how the presence of a given caspase within the apoptosome might influence the recruitment/presence of other caspases. Using gel filtration chromatography we initially characterized the formation of the apoptosome in control and caspase-depleted lysates (Figure 2). In unactivated control lysates, Apaf-1 eluted primarily in its monomeric form (fractions 15–21, Figure 2A), whereas procaspases-9 and -3 eluted as homodimers (fractions 16–22, Figure 2A). Following dATP activation, Apaf-1 oligomerized into ∼700–1400 kDa apoptosome complexes, containing both unprocessed and processed caspase-9, as well as fully processed caspase-3 (fractions 4–7, Figure 2B). A second complex of active caspase-3, which we formerly termed the ‘micro-apoptosome’, was also observed (fractions 9–12, Figure 2B). These results were essentially the same as previously reported (Cain et al., 1999). Figure 1.Reconstitution of immunodepleted lysates with recombinant wild-type and mutant caspases-9 and -3. (A) Recombinant wild-type and fully processed caspase-9 (lane 1), non-cleavable unprocessed D315/330A caspase-9 (lane 2) and catalytically inactive C287A caspase-9 (lane 3) were isolated and purified from bacteria and their purity was determined by Coomassie Blue staining. Each caspase-9 protein was incubated with (lanes 7–9) or without (lanes 4–6) recombinant active caspase-3 (100 nM) for 1 h at 37°C. The samples were subsequently separated by SDS–PAGE and immunoblotted using a polyclonal anti-caspase-9 antibody. (B) The non-cleavable D175A caspase-3 (lanes 1, 3 and 5) and catalytically inactive C163A caspase-3 (lanes 2, 4 and 6) mutants were purified from bacteria, Coomassie-stained to assess purity and exposed to purified recombinant caspase-8 (100 nM) for 1 h at 37°C. In the D175A caspase-3 preparation, two minor internal translation products of ∼27 and ∼29 kDa were present (Fernandes-Alnemri et al., 1996), as well as a minor bacterial contaminant (*). (C) THP.1 lysates were immunodepleted of caspase-9 or -3 and (D) reconstituted with their corresponding wild-type or mutant caspases (200 nM). The reconstituted lysates were dATP-activated for 1 h at 37°C and assayed for DEVDase activity as described in Materials and methods. The anti-caspase-3 antibody recognized a non-specific band (*) in lysates, which served as a fortuitous loading control. Download figure Download PowerPoint Figure 2.The Apaf-1 apoptosome sequentially recruits, activates and retains caspases-9 and -3. (A) Unactivated control lysates, (B) dATP-activated immunoprecipitated (IP) control lysates, (C) dATP-activated caspase-9-depleted lysates or (D) dATP-activated caspase-3-depleted lysates were fractionated by gel filtration, as described in Materials and methods. Each fraction was mixed with 10× SDS loading buffer, separated by SDS–PAGE and immunoblotted for Apaf-1, caspase-9 and/or caspase-3. The arrows represent either the unprocessed or processed form(s) of the caspases; a non-specific band (*) was detected by the anti-caspase-3 antibody. Download figure Download PowerPoint In order to determine whether the absence of caspase-9 had any effect on the recruitment of caspase-3 to the apoptosome, we immunodepleted lysates of procaspase-9 (Figure 1C) and activated these lysates with dATP. Apaf-1 oligomerized into an apoptosome complex (data not shown), but caspase-3 was not readily recruited to the complex and was completely unprocessed (fractions 4–7 and 16–22, compare Figure 2B and C) and inactive (Figure 1D). These data strongly suggest that Apaf-1 could oligomerize into a complex in the absence of procaspase-9, but that caspase-9 must be present to both recruit and activate caspase-3 (fractions 4–7, compare Figure 2B and C). The inability of the Apaf-1 complex to recruit caspase-3, in the absence of caspase-9, was not a consequence of inappropriate oligomerization of Apaf-1, as this complex produced significant DEVDase activity when incubated with a mixture of partially purified pro-caspases-9 and -3 obtained from THP.1 cell lysates (data not shown). We next immunodepleted lysates of procaspase-3 (Figure 1C) and activated these lysates with dATP. Removal of caspase-3 had no effect on Apaf-1 oligomerization (data not shown) or the recruitment of caspase-9 to the apoptosome (fractions 4–7, Figure 2D). However, since no active caspase-3 was present (Figure 1D), there was a slight alteration in the processing of caspase-9, in that less of the p37 form of caspase-9 was observed in the apoptosome (fractions 4–7, Figure 2D). Thus, Apaf-1 can oligomerize into a functional complex independent of caspase-9, but normally functions sequentially to recruit and activate caspases-9 and -3. Catalytically active processed or unprocessed caspase-9 is required for recruitment of caspase-3 to the apoptosome Since the presence of caspase-9 in the apoptosome was critical for the recruitment of caspase-3, we asked several questions: (i) does caspase-9 form a binding site through which caspase-3 must interact? (ii) does caspase-9 require processing to effectively recruit caspase-3? and (iii) does caspase-9 have to be catalytically active in order to recruit caspase-3? To address these questions, we replaced lysates depleted of endogenous procaspase-9 with wild-type caspase-9, previously processed to its p35 form in bacteria (p35 casp-9), non-cleavable procaspase-9 (D315/330A casp-9) or catalytically inactive procaspase-9 (C287A casp-9) (Figure 1A) and activated these lysates with dATP. In agreement with Stennicke et al. (1999), we found that processing of caspase-9 was not required for its activity, as D315/330A casp-9 was nearly as effective as p35 casp-9 at reconstituting DEVDase activity (Figure 1D). Moreover, the catalytically inactive C287A casp-9 mutant was completely incapable of supporting effector caspase activation (Figure 1D). When the lysates, reconstituted with wild-type or mutant caspase-9 proteins, were examined by gel filtration, Apaf-1 was always present as an oligomerized apoptosome complex (data not shown). In the p35 casp-9 reconstitution experiments, caspase-9 readily bound to oligomerized Apaf-1 (fractions 4–7, Figure 3A), indicating that oligomerized Apaf-1 was capable of recruiting and utilizing previously processed caspase-9. The apoptosome containing recombinant p35 casp-9 also recruited and activated endogenous caspase-3, which remained largely associated with the complex (fractions 5–6, Figure 3A). Although the concentrations of p35 casp-9 used in these experiments probably exceeded the normal endogenous concentrations of caspase-9, this reconstituted system did not completely activate all of the available procaspase-3 compared with control lysates (compare Figures 3A and 2B). Therefore, potential modulators of caspase-9 activity may have been removed during immunodepletion of endogenous procaspase-9; alternatively, p35 casp-9 may not fully mimic endogenous active caspase-9. Nevertheless, the ability of p35 casp-9 to associate with Apaf-1 in the apoptosome and to recruit and process caspase-3 verified the usefulness of this in vitro model (Figure 3). Figure 3.Recruitment of caspase-3 to the Apaf-1 apoptosome requires catalytically active processed or unprocessed caspase-9. Caspase-9-depleted lysates were reconstituted with (A) fully processed wild-type p35 caspase-9, (B) non-cleavable D315/330A caspase-9 or (C) catalytically inactive C287A caspase-9. Following dATP activation for 1 h at 37°C, the reconstituted lysates were fractionated and immunoblotted for caspases-9 and -3, as described in Materials and methods. The arrows represent either the unprocessed or processed form(s) of the caspases; a non-specific band (*) was detected by the anti-caspase-3 antibody. Download figure Download PowerPoint Next, we examined lysates reconstituted with the non-cleavable D315/330A casp-9 mutant. Although unprocessed caspase-9 does support DEVDase activity in dATP lysates (Stennicke et al., 1999; Figure 1D), the involvement of the apoptosome in this process has not been formally demonstrated. Indeed, we found that D315/330A casp-9 associated normally with oligomerized Apaf-1 following dATP activation, and readily recruited and activated caspase-3 (fractions 4–7, compare Figures 3B and 2B). Moreover, the active caspase-3 remained primarily associated with the apoptosome. Therefore, caspase-9 need not be processed in order to recruit and activate caspase-3 within the apoptosome. Finally, we examined lysates reconstituted with the catalytically inactive C287A casp-9 mutant. Although this mutant could not support effector caspase activation (Figure 1D), we questioned whether C287A casp-9 was unable to recruit caspase-3 to the apoptosome or was simply unable to activate it. Following dATP activation, C287A casp-9 bound to oligomerized Apaf-1 but was incapable of effectively recruiting procaspase-3 to the apoptosome (fractions 4–6, Figure 3C). The fact that C287A casp-9 could not undergo normal autocatalytic processing to form a fully processed caspase-9 enzyme was not responsible for its lack of ability to recruit caspase-3. Indeed, both D315/330A casp-9 and C287A casp-9 are largely present within the apoptosome as unprocessed caspase-9 enzymes (fractions 4–6, Figure 3B and C), but only D315/330A casp-9 is capable of recruiting caspase-3 to the apoptosome. Thus, the catalytically active cysteine present in the active site of caspase-9 appears to be critical for recruitment of caspase-3 to the apoptosome. Caspase-9 recruits caspase-3 to the apoptosome through recognition of a critical aspartate (D175) residue in the effector caspase Next, we wished to determine which residues in caspase-3 might be critical for its recruitment to the complex. As caspase-3 is processed at the IETD175↓S site, located between its large and small subunits (Nicholson et al., 1995; Fernandes-Alnemri et al., 1996), we questioned whether this residue in caspase-3 might be required for its recruitment to the apoptosome. Therefore, we immunodepleted lysates of procaspase-3 (Figure 1C) and reconstituted them with D175A casp-3, which, as expected, did not support DEVDase activity following dATP activation (Figure 1D). Examination of the lysates by gel filtration revealed that Apaf-1 was oligomerized (data not shown) and caspase-9 was processed normally (fractions 4–7, Figure 4A). However, almost all the D175A casp-3 mutant remained outside the apoptosome as the unprocessed proenzyme (fractions 16–22, Figure 4A). Thus, recruitment of caspase-3 to the apoptosome ultimately requires an interaction between the active site cysteine in caspase-9 and the D175 residue in caspase-3. As with all proteases, caspases appear to bind their substrates very tightly in the transition state, but only weakly (Km in the micromolar range) in the ground state (Stennicke and Salvesen, 1999). Therefore, the absence of the C287 residue in caspase-9 or the D175 residue in caspase-3 probably inhibits formation of a normal tetrahedral intermediate necessary for substrate catalysis and, in effect, physically prohibits recruitment of procaspase-3 to the apoptosome. Figure 4.Caspase-9 recruits caspase-3 to the Apaf-1 apoptosome via recognition of a critical aspartate (D175) residue. Caspase-3-depleted lysates were reconstituted with (A) unprocessable D175A caspase-3 or (B) catalytically inactive C163A caspase-3. Following dATP activation for 1 h at 37°C, the reconstituted lysates were fractionated and immunoblotted for caspases-9 and -3, as described in Materials and methods. The arrows represent either the unprocessed or processed form(s) of the caspases; a non-specific band (*) was detected by the anti-caspase-3 antibody. Download figure Download PowerPoint Both active and inactive caspase-3 remain associated with the apoptosome as long as caspase-3 is processed At this point, all our experiments indicated that catalytically active processed or unprocessed caspase-9 was required for caspase-3 recruitment to the apoptosome, based on the presence of active, processed caspase-3 within the complex. Consequently, we questioned whether caspase-3 must be active or merely processed in order to maintain its association with the apoptosome. Therefore, we reconstituted caspase-3-depleted lysates with catalytically inactive C163A casp-3. Following dATP activation, Apaf-1 oligomerized normally (data not shown) and recruited and processed endogenous caspase-9 (fractions 4–7, Figure 4B). Most importantly, however, the C163A casp-3 mutant was efficiently recruited to the apoptosome (fractions 4–7, Figure 4B), as this mutant contained the critical D175 residue required for normal caspase-3 recruitment. Since the endogenous processed caspase-9 was present in the apoptosome, C163A casp-3 was processed to its p20 form and remained associated with the apoptosome complex (fractions 4–7, Figure 4B). However, since the p20 form of C163A casp-3 was inactive (Figure 1D), it did not remove its prodomain through autocatalytic processing to generate its p17 form (Han et al., 1997). Therefore, the processing of caspase-3, and not its activity, was required for its retention within the apoptosome. The C163A casp-3 mutant, unlike wild-type caspase-3 or D175A casp-3, was present in significant amounts within the apoptosome as the unprocessed proform of the enzyme. The C163A casp-3 mutant was not fully processed, possibly because it had a somewhat different conformation from wild-type caspase-3, or perhaps within the context of the apoptosome, active caspase-3 participated in its own initial processing between its large and small subunits. Highly active, recombinant caspase-8 (100 nM) was also incapable of fully processing C163A casp-3 in the same period of time (lane 6, Figure 1B). Nevertheless, since the products of most enzymic reactions do not remain associated with the active site of the enzymes, we deemed it likely that the proform of C163A casp-3 in the apoptosome was associated with active processed caspase-9 and that the processed p20 form of C163A casp-3 was associated with some other protein in the complex. Processed caspases-9 and -3 associate with Apaf-1 and XIAP in the apoptosome Since XIAP interacts directly with active caspases-3 and -7 in vitro and is the most potent caspase inhibitor of all known IAPs (Deveraux et al., 1998), and given that XIAP binds processed C163A casp-3 with significantly greater affinity than unprocessed C163A casp-3 (Sun et al., 1999), we speculated that XIAP might physically associate with the apoptosome and hold processed caspase-3 within the complex. Consequently, we examined normal lysates for the presence of this IAP. In unactivated lysates, XIAP was present in ∼200–700 kDa complexes. Following dATP activation, most of these complexes increased in size to ∼350–700 kDa, with the majority co-eluting with the apoptosome and some with the ‘micro-apoptosome’ complexes (Figure 5A). In activated lysates, XIAP was also cleaved into several fragments, including the BIR3-RING fragment (Deveraux et al., 1999), and each co-eluted with the apoptosome (Figure 5A). Figure 5.Co-immunoprecipitation of Apaf-1, caspase-9, caspase-3 and XIAP. (A) Control and dATP-activated THP.1 lysates were fractionated by gel filtration. The fractions were separated by SDS–PAGE and immunoblotted for XIAP using an antibody raised against its C-terminus. Control and dATP-activated lysates (∼15 mg/ml) were immunoprecipitated with an antibody against (B) caspase-9 or (C) caspase-3 and the resulting immuno complexes were recovered by centrifugation. The supernatants (S) and washed immunocomplexes (P) were separated by SDS–PAGE and western blotted (WB) for Apaf-1, XIAP or Hsp60. (D) Lysates were pretreated with DEVD·CHO (200 nM) for 1 h at 4°C and subsequently dATP-activated at 37°C for 1 h. The lysates were fractionated by gel filtration and analyzed by SDS–PAGE/immunoblotting for caspases-9 and -3, as described in Materials and methods. The arrows represent either the full-length proteins or, in the case of XIAP, various cleavage products. Bands corresponding to the light chain (LC) or heavy chain (HC) of the immunoprecipitated antibodies are also shown. Separation of the LC and the BIR3-RING fragment was difficult, but this fragment was only apparent in caspase-9 immunocomplexes obtained from dATP-activated lysates. Non-specific bands (*) were detected by the XIAP or caspase-3 antibodies. Download figure Download PowerPoint Co-elution of Apaf-1, caspase-9, caspase-3 and XIAP in the same high molecular weight fractions suggested that they might be present within the same complex (Figures 2B and 5A). To test this hypothesis, we performed immunoprecipitation experiments using both control and dATP-activated lysates, predicting that relevant apoptosome interactions would occur primarily in activated lysates. Immunoprecipitation of caspase-9 revealed an association with both Apaf-1 and XIAP that occurred primarily in dATP-activated lysates (compare lanes 2 and 4, Figure 5B). Caspase-9 presumably associated with Apaf-1 through CARD–CARD interactions (Li et al., 1997; Qin et al., 1999) and with XIAP through active site–BIR3 interactions (Deveraux et al., 1999; Sun et al., 2000). All of the immunodetectable caspase-3/-8 cleavage products of XIAP contained the BIR3 domain and each was co-immunoprecipitated with caspase-9 (compare lanes 2 and 4, Figure 5B). This observation was significant, because all of these cleavage products, including the BIR3-RING fragment (Deveraux et al., 1999), were associated primarily with the apoptosome fractions (fraction 5, Figure 5A). Based on these experiments, we speculated that XIAP might associate with oligomerized Apaf-1, at least in part, through an interaction with processed caspase-9 (model 1, Figure 7). Similarly, immunoprecipitation of caspase-3 revealed an interaction with both Apaf-1 and XIAP (compare lanes 2 and 4, Figure 5C). Since caspase-3 does not contain an obvious domain, such as a CARD, which would enable it to bind Apaf-1 directly, it was not immediately clear how caspase-3 might associate with Apaf-1. The association of caspase-3 with XIAP was more readily interpretable. Caspase-3 associated primarily with full-length XIAP in dATP-activated lysates (and possibly with undetectable BIR1–BIR2 fragments), but, in contrast to caspase-9, did not associate with the BIR3 fragments (compare lanes 2 and 4, Figure 5B and C). This was consistent wi
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