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• W2040289871 abstract "Article15 December 2003free access IAP-antagonists exhibit non-redundant modes of action through differential DIAP1 binding Anna Zachariou Anna Zachariou The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, Fulham Road, London, SW3 6JB UK Search for more papers by this author Tencho Tenev Tencho Tenev The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, Fulham Road, London, SW3 6JB UK Search for more papers by this author Lakshmi Goyal Lakshmi Goyal Howard Hughes Medical Institute, Rockefeller University, New York, NY, 10021 USA Search for more papers by this author Julie Agapite Julie Agapite Howard Hughes Medical Institute, Rockefeller University, New York, NY, 10021 USA Search for more papers by this author Hermann Steller Hermann Steller Howard Hughes Medical Institute, Rockefeller University, New York, NY, 10021 USA Search for more papers by this author Pascal Meier Corresponding Author Pascal Meier The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, Fulham Road, London, SW3 6JB UK Search for more papers by this author Anna Zachariou Anna Zachariou The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, Fulham Road, London, SW3 6JB UK Search for more papers by this author Tencho Tenev Tencho Tenev The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, Fulham Road, London, SW3 6JB UK Search for more papers by this author Lakshmi Goyal Lakshmi Goyal Howard Hughes Medical Institute, Rockefeller University, New York, NY, 10021 USA Search for more papers by this author Julie Agapite Julie Agapite Howard Hughes Medical Institute, Rockefeller University, New York, NY, 10021 USA Search for more papers by this author Hermann Steller Hermann Steller Howard Hughes Medical Institute, Rockefeller University, New York, NY, 10021 USA Search for more papers by this author Pascal Meier Corresponding Author Pascal Meier The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, Fulham Road, London, SW3 6JB UK Search for more papers by this author Author Information Anna Zachariou1, Tencho Tenev1, Lakshmi Goyal2, Julie Agapite2, Hermann Steller2 and Pascal Meier 1 1The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, Fulham Road, London, SW3 6JB UK 2Howard Hughes Medical Institute, Rockefeller University, New York, NY, 10021 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6642-6652https://doi.org/10.1093/emboj/cdg617 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Drosophila inhibitor of apoptosis protein DIAP1 ensures cell viability by directly inhibiting caspases. In cells destined to die this IAP-mediated inhibition of caspases is overcome by IAP-antagonists. Genetic evidence indicates that IAP-antagonists are non-equivalent and function synergistically to promote apoptosis. Here we provide biochemical evidence for the non-equivalent mode of action of Reaper, Grim, Hid and Jafrac2. We find that these IAP-antagonists display differential and selective binding to specific DIAP1 BIR domains. Consistently, we show that each DIAP1 BIR region associates with distinct caspases. The differential DIAP1 BIR interaction seen both between initiator and effector caspases and within IAP-antagonist family members suggests that different IAP-antagonists inhibit distinct caspases from interacting with DIAP1. Surprisingly, we also find that the caspase-binding residues of XIAP predicted to be strictly conserved in caspase-binding IAPs, are absent in DIAP1. In contrast to XIAP, residues C-terminal to the DIAP1 BIR1 domain are indispensable for caspase association. Our studies on DIAP1 and caspases expose significant differences between DIAP1 and XIAP suggesting that DIAP1 and XIAP inhibit caspases in different ways. Introduction Programmed cell death, or apoptosis, is an essential process of animal development and is important for normal morphogenesis and tissue architecture (Meier et al., 2000a). Apoptosis signalling culminates in the activation of a set of highly specific cysteine proteases called caspases. Generally, caspases are ubiquitously expressed as catalytically inactive zymogens consisting of an N-terminal pro-domain, a large (p20) and a small (p10) catalytic subunit. Upon induction of apoptosis the caspase zymogen is proteolytically processed at inter-domain sites generating the catalytically active protease (Shi, 2002). While initiator caspases are activated through an auto-proteolytic process, cleavage and activation of downstream effector caspases require the proteolytic activity of initiator caspases. Once activated, caspases cleave a host of structural and regulatory proteins resulting in the coordinated disassembly of the cell (Shi, 2002). Activation of the caspase cascade is tightly controlled by members of the evolutionarily conserved Inhibitor of APoptosis (IAP) protein family. IAPs are characterized by the presence of one or more BIR (Baculovirus IAP Repeat) domains (Salvesen and Duckett, 2002). The BIR module is essential for the anti-apoptotic activity of IAPs as it is required for caspase binding. For example, a region containing the BIR2 domain of the mammalian IAP XIAP specifically interacts with the effector caspases-3 and -7, while XIAP's BIR3 domain binds to the initiator caspase-9. Direct interaction of XIAP to the catalytic pocket of caspase-3 and caspase-7 results in the steric occlusion of these caspases blocking their access to substrates. Residues within a small segment N-terminal to XIAP's BIR2 domain are essential for the binding of XIAP to activated caspase-3 and -7 (Shi, 2002). Importantly, these residues are highly conserved in human IAPs that potently inhibit caspases. In particular, residue Asp148 of XIAP represents the major anchoring point for IAP–caspase association. Consequently, Asp148 is predicted to be strictly conserved in IAPs capable of binding to caspases (Huang et al., 2001). In addition to the BIR domain some IAPs also contain a C-terminal RING finger domain that provides these IAPs with E3 ubiquitin–protein ligase activity (Salvesen and Duckett, 2002). Although XIAP has a RING finger domain, this module is dispensable for XIAP's caspase- neutralizing activity as XIAP mutants lacking the RING finger domain are fully functional in suppressing caspase- mediated cell death. Mere physical association of XIAP with activated caspases seems to be entirely sufficient to suppress caspases. In complete contrast, physical association of the Drosophila IAP DIAP1 with caspases is necessary but not sufficient to inhibit caspases in vivo (Wilson et al., 2002; Ditzel et al., 2003). In addition to caspase binding, DIAP1 requires the E3 ubiquitin protein ligase activity provided by its RING finger domain to effectively neutralize caspases. The RING finger domain of DIAP1 mediates ubiquitylation and inactivation of the Drosophila caspase DRONC (Wilson et al., 2002). In addition to neutralizing DRONC, DIAP1 also potently inhibits the caspases drICE and DCP-1 (Kaiser et al., 1998; Hawkins et al., 1999). While DRONC is an initiator caspase that is most homologous to the mammalian initiator caspase-9, drICE and DCP-1 are effector caspases with sequence and enzymological properties very similar to those of the mammalian effector caspases-3 and -7 (Fraser and Evan, 1997; Song et al., 1997). In Drosophila, DIAP1 represents the last line of defence against caspase-mediated damage since loss-of-function mutations in diap1 cause spontaneous and unrestrained cell death (Wang et al., 1999; Goyal et al., 2000; Lisi et al., 2000; Rodriguez et al., 2002). Hence, the caspase-neutralizing activity of DIAP1 is essential to maintain cell viability. In cells fated to die, the anti-apoptotic function of DIAP1 is thwarted by a set of specialized IAP-binding proteins called IAP-antagonists. In Drosophila the IAP-antagonists Reaper (Rpr), Grim, Hid, Sickle and Jafrac2 are thought to promote cell death by disrupting DIAP1–caspase association thereby alleviating DIAP1's inhibition of caspases (White et al., 1994; Grether et al., 1995; Chen et al., 1996; Christich et al., 2002; Srinivasula et al., 2002; Tenev et al., 2002). In mammals, an identical mechanism operates through the IAP-antagonists Smac/DIABLO and HtrA2/Omi (Vaux and Silke, 2003). Common to all IAP-antagonists is the presence of a conserved motif that is critical for IAP binding and is known as IBM (IAP-binding motif). IBMs bear an N-terminal Ala1 that anchors this motif to the BIR surface of IAPs (Huang et al., 2001). The increasing number of Drosophila and mammalian members of the IAP-antagonist protein family invokes the question as to why there are so many distinct IAP-antagonists. Although in Drosophila, over-expression of Rpr, Grim, Hid or Jafrac2 is sufficient to induce apoptosis in a wide variety of cell types, genetic analyses of Rpr, Grim and Hid argue that these IAP-antagonists are not redundant but must act in combination with each other to induce apoptosis (Robinow et al., 1997; Zhou et al., 1997; Wing et al., 1998). Thus, embryos with deletions that remove various combinations of rpr, grim and/or hid indicate that developmental cell death in the embryonic central nervous system (CNS) requires the cooperative actions of Rpr, Grim and Hid. Further, simultaneous ectopic expression of Rpr and Hid in embryonic CNS midline cells induces substantial apoptosis, while expression of two copies of either gene alone has little or no effect on midline cell viability (Zhou et al., 1997). Currently, little is known about the underlying coordinated mode of action through which IAP-antagonists synergistically oppose IAPs. Here we provide biochemical evidence for the non-redundant mode of action of Rpr, Grim and Hid. We find that Rpr, Grim and Hid display differential and selective binding to specific DIAP1 BIR domains. Further, we show that each BIR domain of DIAP1 associates with distinct caspases. Consistent with the notion that different IAP-antagonists compete with distinct sets of caspases for DIAP1 binding we show that Rpr but not Hid blocks the binding of drICE to DIAP1. We also provide evidence indicating that Rpr, Grim and Hid induce cell death predominantly, if not exclusively, in an IAP-binding-dependent manner. Finally, our biochemical data on the interaction between DIAP1 and caspases expose significant differences between DIAP1 and XIAP. Intriguingly, DIAP1 does not contain sequence homology to the caspase-binding residues of XIAP, which are predicted to be strictly conserved in IAPs capable of binding caspases; yet, DIAP1 specifically interacts with activated caspases such as drICE and DCP-1. Our data indicate that residue Asn117, located immediately C-terminal to the BIR1 domain of DIAP1, is indispensable for caspase association arguing that DIAP1 interacts with activated caspases through a mechanism that is distinct from the one with which XIAP binds to caspase-3 and -7. Results The DIAP1 BIR1 region binds to activated effector caspases To determine the region of DIAP1 that binds to effector caspases, we used wild-type and mutant DIAP1–GST fusion proteins (Figure 1A) as affinity reagents to purify drICE or DCP-1 from cellular extracts. DIAP1–GST was expressed in Drosophila Schneider (S2) cells and affinity purified from cell lysates using glutathione resin. The resin-purified DIAP1–GST was subsequently incubated with total extracts of human 293 cells expressing either drICE or DCP-1. After binding the extracts to the DIAP1–GST proteins, the bound proteins were eluted and analysed by immunoblotting. As expected, DIAP1 exclusively interacted with the processed, activated forms of drICE and DCP-1 but failed to bind to their zymogens (Figure 1B, top right panel, lanes 3 and 7). Significantly, the gain-of-function mutant DIAP16–3s, which suppresses Rpr- and Hid-mediated cell death (Goyal et al., 2000), displayed greatly enhanced binding to activated effector caspases when compared with wild-type DIAP1 (Figure 1B, compare lane 4 with 3, and 8 with 7). The BIR1 region was required for the binding of DIAP1 to activated effector caspases as DIAP1 deletion mutants lacking this region (ΔBIR1) completely failed to associate with drICE and DCP-1. Similarly, DIAP1 fragments consisting of only the DIAP1 RING finger region (RING) also failed to interact with activated caspases. Thus, loss of the BIR1 region abrogated caspase binding indicating that this domain is required for caspase association. Figure 1.Co-purification of drICE-V5 or DCP-1-V5 with DIAP1–GST. (A) Schematic representation of the DIAP1 mutants used in this study. (B) The gain-of-function mutant DIAP16–3s binds more efficiently to caspases than wild-type (wt) DIAP1. Top right panel: DIAP1–caspase co-purification; affinity-purified DIAP1–GST was used to precipitate drICE-V5 (lanes 3–6) or DCP-1-V5 (lanes 7–10) from cellular extracts. Top left panel: total extracts of 293 cells expressing the indicated caspases. Caspase expression (top left panel) and caspase–DIAP1 binding (top right panel) were detected by immunoblot analysis using anti-V5 antibody. Bottom right panel: the purification of DIAP1–GST was confirmed by western blot analysis of the eluate using anti-DIAP1 RING antibody. (C and D) The BIR1 region of DIAP1 is necessary and sufficient for caspase binding. (E and F) DIAP111–3e fails to bind to drICE and DCP-1. (C–F) Experiments were conducted as in (B). Top panels: expression (lane 1) and co-purification (lanes 2–end) of drICE-V5 (C and E) and DCP-1-V5 (D and F) with the indicated DIAP1–GST fragments. Bottom panel: purification of the DIAP1–GST fragments was confirmed by immunoblot analysis of the eluate using anti-GST antibody. Note, the purified wild-type and 6–3s mutant DIAP1 BIR1 fragments (E and F; lanes 2 and 3) are cleaved at position 20 by drICE and DCP-1 while BIR111–3e that failed to bind to caspases was only partially processed. Download figure Download PowerPoint Next, we examined whether the DIAP1 BIR1 region on its own was sufficient for caspase binding. Various DIAP1 deletion mutants (BIR1, Linker1, BIR2 and Linker2; see Figure 1A) were tested for their ability to interact with drICE and DCP-1. drICE exclusively co-purified with the BIR1 fragment (Figure 1C, lane 2). The BIR2, Linker1 or Linker2 regions did not interact with drICE, indicating that the BIR1 region mediates caspase binding. Similarly, activated DCP-1 also preferentially bound to the BIR1 region (Figure 1D, lane 2). However, DCP-1 also interacted with the BIR2 region of DIAP1, albeit only very weakly (lane 3). Taken together, our results indicate that the BIR1 region of DIAP1 is required for efficient binding to activated effector caspases. A segment immediately C-terminal to the BIR1 domain is indispensable for caspase association Embryos homozygous for the loss-of-function allele diap111–3e die early during embryogenesis due to unrestrained caspase-dependent cell death (Goyal et al., 2000). Surprisingly, the mutation in diap111–3e was associated with a single amino acid substitution mutation (Asn117Lys) downstream of the DIAP1 BIR1 domain (Table I; Figure 1A). To determine the molecular consequences of the loss-of-function mutation in DIAP111–3e, we compared wild-type and mutant DIAP1 BIR1 regions (1–146) for their ability to bind to activated caspases (Figure 1E and F). Both wild-type and BIR16–3s fragments co-purified with drICE and DCP-1, whereby the BIR16–3s fragment showed enhanced binding to drICE and DCP-1 (compare lanes 2 with 3). In contrast, the BIR111–3e region completely failed to bind to activated drICE and DCP-1 (compare lanes 2 with 4). Thus, amino acid substitution of Asn to Lys at position 117 of DIAP111–3e abrogated DIAP1's interaction with effector caspases. Although Asn117 in the linker region between the BIR1 and BIR2 domain is critical for caspase binding, the Linker1 segment on its own failed to bind to caspases, indicating that regions preceding Linker1 are also required for caspase association. Our results indicate that diap111–3e is a loss-of-function allele because DIAP111–3e fails to bind to activated effector caspases causing unrestrained caspase- mediated cell death. On the other hand, the 6–3s mutation, which significantly enhances the ability of DIAP to bind activated effector caspases causes a gain-of-function phenotype whereby apoptosis is suppressed. Table 1. Phenotypes and molecular changes associated with the diap1 alleles used in this study diap1 allele Modifier phenotype Recessive phenotype Mutation reaper hid 6-3s suppressor suppressor viable G88S 23-4s suppressor suppressor lethal G269S 21-4s enhancer suppressor lethal C406Y th4 enhancer enhancer lethal H283Y 11-3e enhancer enhancer lethal N117K DIAP1 simultaneously binds to DRONC and activated drICE or DCP-1 Since the BIR1 region of DIAP1 interacts with effector caspases and the BIR2 region binds to initiator caspases (Meier et al., 2000b), we tested whether DIAP1 simultaneously bound to initiator and effector caspases. To this end, we examined whether drICE or DCP-1 interacted with DIAP1 that was pre-bound to the initiator caspase DRONC. DRONC-FLAG was co-expressed with either DIAP1 or controls in S2 cells and anti-FLAG antibody-coupled agarose resin was used to immunoprecipitate DRONC-FLAG from cellular extracts. Resin-purified DRONC-FLAG was subsequently incubated with extracts of 293 cells expressing either drICE-V5 or DCP-1-V5. After binding the extracts to the DRONC-FLAG resin, the bound proteins were eluted and analysed by immunoblotting (Figure 2). In the presence of DIAP1 activated drICE and DCP-1 co-immunoprecipitated with DRONC-FLAG (lanes 1 and 2). In the absence of DIAP1 however, purified DRONC-FLAG on its own failed to interact with either drICE or DCP-1 (lanes 3 and 4). Our results indicate that one molecule of DIAP1 can interact with DRONC and either drICE or DCP-1 at the same time, suggesting that DIAP1 can simultaneously block the activity of both initiator and effector caspases. Figure 2.DIAP1 simultaneously binds to initiator and effector caspases. DRONC-FLAG was co-expressed with DIAP1 or controls in S2 cells. DRONC-FLAG was purified by immunoprecipitation using anti-FLAG antibody-coupled agarose beads. Resin-bound DRONC-FLAG was subsequently incubated with extracts of 293 cells expressing the indicated caspases. Following incubation of the DRONC-FLAG beads with the extracts, the bound proteins were eluted with FLAG peptides. Co-immunoprecipitation (lanes 1–4) and expression (lanes 5 and 6) of the indicated proteins were determined by immunoblot analysis using the indicated antibodies. Download figure Download PowerPoint IAP-antagonists display differential binding to specific BIR domains Next, we assessed whether IAP-antagonists differentially interact with distinct BIR domains of DIAP1 and hence may compete with specific caspases for DIAP1 binding. To identify the region of DIAP1 that is required for the binding to Rpr, Grim, Hid and Jafrac2, we tested various DIAP1 fragments (Figure 1A) for their interaction with IAP-antagonists. S2 cells expressing the baculovirus, pan-caspase inhibitor p35 were used to avoid Rpr-, Hid- and Grim-induced apoptosis and concomitant loss of cellular material. Importantly, Rpr, Grim and Hid specifically co-purified with both the BIR1 as well as the BIR2 regions of DIAP1 (Figure 3). Rpr associated with the BIR1 and BIR2 regions with equal efficiency (Figure 3A, compare lane 2 with 3). Likewise, Grim also bound equally to both BIR regions (Figure 3B, compare lane 2 with 3). However, Hid, Sickle and Jafrac2 interacted preferentially with the BIR2 and associated only weakly with the BIR1 region (Figure 3C and D and Supplementary figure 1 available at The EMBO Journal Online). Under the same conditions, Rpr, Grim, Hid and Jafrac2 did not interact with the Linker regions of DIAP1 or GST alone (lanes 4 and 5). Collectively, our data indicate that the BIR1 and BIR2 regions of DIAP1 are required for caspase binding and that the IAP-antagonists Rpr and Grim target both these BIR domains equally, while Hid, Sickle and Jafrac2 show preferential binding to BIR2. Figure 3.IAP-antagonists bind to distinct regions of DIAP1. DIAP1–GST fragments were used to purify Rpr-V5 (A), Grim-TAP (B), Hid-V5 (C) or Jafrac2 (D) from S2/p35 cellular extracts. Top panel: DIAP1–GST deletion mutants were affinity purified from cellular extracts using glutathione–Sepharose beads and associated IAP-antagonists were detected by immunoblotting using anti-V5 (A and C), anti-protein A (B) or anti-Jafrac2 (D) antibodies, respectively. Middle panel: purification of the DIAP1–GST fragments was verified by western blot analysis using anti-GST antibody. Bottom panel: equal expression of the indicated IAP-antagonist was examined by immunoblotting the S2 extracts using the indicated antibodies. The asterisk denotes a cross-reactive band. Download figure Download PowerPoint To reveal differences in the binding of IAP-antagonists to DIAP1 we tested various DIAP1 mutants with single point mutations for their ability to bind to IAP-antagonists [Table I; Figure 1A; Goyal et al. (2000)]. Since Rpr, Grim and Hid bind to both BIR domains of DIAP1, we used each BIR region in isolation to study the ability of mutant BIR regions to bind to IAP-antagonists. DIAP16–3s with a gain-of-function mutation in the BIR1 domain showed greatly diminished binding to Rpr and Hid (Figure 4A and C, compare lane 1 with 2). Surprisingly, the 6–3s mutation did not abrogate Grim binding as Grim co-purified with wild-type or DIAP16–3s with equal efficiency (Figure 4B, compare lane 1 with 2). The observation that BIR16–3s showed impaired Rpr and Hid but not Grim binding indicates that Grim interacts with DIAP1 in a manner that is distinct from Rpr and Hid. Importantly, the loss-of-function mutation in DIAP111–3e that completely disrupts binding to activated caspases did not impair the association of DIAP1 to Rpr, Grim or Hid, indicating that the 11–3e mutation does not grossly disrupt the structure of the BIR fold. The DIAP1th4 and DIAP123–4s mutants, which carry single amino acid mutations in the BIR2 domains, showed impaired binding to Rpr, Grim and Hid (Figure 4A–C, lanes 4–6). Furthermore, the th4 BIR2 mutation (His283Tyr) completely abolished the interaction with Jafrac2 and Sickle (Figure 4D, compare lane 4 with 5, and see Supplementary figure 1). Interestingly, the same th4 mutation similarly abrogates DRONC binding resulting in a loss-of-function phenotype (Tenev et al., 2002). Thus, physical association between DIAP1 and caspases is essential for DIAP1 function. Figure 4.Rpr, Grim, Hid and Jafrac2 differentially interact with DIAP1. (A–D) Co-purification of Rpr, Grim, Hid and Jafrac2 with wild-type or mutant BIR fragments. Expression and purification of the indicated constructs were determined as in Figure 3. The BIR1 region of DIAP16–3s shows impaired binding to Rpr (A) and Hid (C), while its association to Grim (B) remains unaffected. The 11–3e mutation does not affect the binding of Rpr, Grim or Hid to DIAP1 (A–C, top left panel). (A–D) The th4 and 23–4 BIR2 mutations impair DIAP1's ability to bind to Rpr (A), Grim (B), Hid (C) and Jafrac2 (D). Download figure Download PowerPoint IAP-antagonists compete with caspase for DIAP1 binding The differential interaction between DIAP1 and caspases or IAP-antagonists suggests that different IAP-antagonists may compete with distinct sets of caspases for DIAP1 binding. Therefore, we determined whether different IAP-antagonists displayed contrasting abilities to compete with drICE for DIAP1 binding. As expected, Rpr or Grim, both of which efficiently bind to the BIR1, significantly reduced the ability of DIAP1 to interact with drICE (Figure 5A and data not shown). In parallel experiments, Rpr or Grim also abrogated the association of DIAP1 with DCP-1 (data not shown). In complete contrast however, Hid, that only relatively weakly interacts with the BIR1 region, failed to compete with drICE for DIAP1 binding (Figure 5B). Thus, Rpr and Hid greatly differ in their competence to interfere with DIAP1–drICE interaction. Rpr's ability to compete with drICE for DIAP1 binding was contingent on Rpr's IBM. Thus, a Rpr mutant that lacked Ala1 and failed to bind to DIAP1 (see below) also failed to block the binding of DIAP1 to drICE (Figure 5C, compare lane 7 with 8). Figure 5.Rpr but not Hid directly competes with drICE for DIAP1 binding. (A and B) Co-purification of drICE with DIAP1–GST in the presence or absence of Rpr (A) or Hid (B). DIAP–GST was affinity purified from S2 cellular extracts. Resin-bound DIAP1 was subsequently incubated with a mixture of cellular extracts containing drICE-V5 and Rpr-V5 (A) or drICE-V5 and Hid-V5 (B). Bound proteins were eluted and analysed by immunoblotting using anti-V5 antibody (top right panel, lanes 4 and 5). Left panel: total extracts of cells expressing the indicated constructs; western blot analysis using anti-V5 antibody. Bottom right panel: effective DIAP1 purification was confirmed by immunoblotting the eluate with anti-DIAP1 antibody. (C) Rpr's ability to compete with drICE for DIAP1 binding is contingent on its association with the BIR1 domain. Co-purification of drICE with the indicated DIAP1–GST proteins in the presence of wild-type or mutant Rpr or Jafrac2. Top right panel: DIAP1–GST co-purification. Top left panel: total extracts expressing the indicated constructs; immunoblot analysis using anti-V5 antibody. Bottom right panel: purification of DIAP1–GST was confirmed by western blot analysis of the eluate using anti-DIAP1 RING antibody. The asterisk denotes a cross-reactive band. Download figure Download PowerPoint Since Rpr binds to both BIR domains of DIAP1 we next examined whether Rpr needed to bind to the BIR1 domain in order to successfully compete with drICE for DIAP1 association. To this end, DIAP16–3s was used because the 6–3s mutation selectively impairs BIR1–Rpr binding while the interaction of Rpr to the BIR2 domain remains unaffected (Figure 5C, lane 9 and data not shown). Intriguingly, unlike wild-type DIAP1, DIAP16‒3s maintained drICE binding in the presence of Rpr. In contrast, the IAP-antagonist Jafrac2, which predominantly targets the BIR2 domain, eliminated drICE–DIAP1 association. Together these results indicate that Rpr must interact with the first BIR domain to thwart drICE binding. Conversely, Jafrac2 appears to antagonize DIAP1–drICE association from a BIR2 position. Rpr, Grim and Hid promote apoptosis exclusively in an IAP-binding-dependent manner Recent evidence suggest that some IAP-antagonists such as Rpr and Grim promote apoptosis not only through a mechanism that relies on IAP binding but also by a mechanism that is entirely independent of IAP association (Claveria et al., 1998; Holley et al., 2002; Yoo et al., 2002). To examine the contribution of the IBM in Rpr, Grim and Hid for IAP binding and induction of apoptosis, we tested wild-type and mutant IAP-antagonists, lacking Ala1, for their ability to bind to DIAP1 and induce cell death. Wild-type Rpr, Grim, Hid and their respective Ala1 mutants were expressed in S2/p35 cells through the ubiquitin (Ub) fusion technique in which a Ub reporter is fused to the test protein. Ubiquitin-specific proteases cleave these fusion proteins at the C-terminus of the Ub moiety, yielding the test proteins (Varshavsky, 2000). Wild-type AVA-Rpr, AIA-Grim and AVP-Hid were efficiently co-purified by DIAP1 (Figure 6A). Conversely, the Ala1 mutants VA-Rpr, IA-Grim and VP-Hid displayed greatly diminished binding to DIAP1. In parallel experiments neither Rpr nor Grim or Hid bound to GST alone (data not shown). Since the Ala1 mutant Rpr and Grim proteins still bound to DIAP1, albeit weakly, we examined whether the residual IBM of Rpr and Grim was responsible for the weak interaction. As shown in Figure 6B, Rpr and Grim lacking the entire IBM completely failed to interact with DIAP1. Figure 6.Ala1 of the IBM of Rpr, Grim and Hid is indispensable for their binding to DIAP1. (A) Co-purification of Rpr-V5, Grim-V5 or Hid-V5 with DIAP1–GST from cellular extracts. Wild-type but not mutant Rpr, Grim or Hid lacking Ala1 efficiently interacted with DIAP1. Rpr, Grim and Hid were expressed using the ubiquitin fusion technique. Protein expression (bottom panel) and co-purification (top panel) was examined by immunoblot analysis with the indicated antibodies. Purification of DIAP1–GST was verified by western blot analysis of the eluate using anti-DIAP1 RING antibody (middle panel). (B) Rpr and Grim proteins that lack their entire IBM completely failed to bind to DIAP1. Note, while the difference in mobility between Grim and ΔIBM-Grim is readily detectable, Rpr and ΔIBM-Rpr appear to migrate at the same level due to the SDS–acrylamide gel used. (C) Detection of rpr and grim mRNAs by RT" @default.
• W2040289871 created "2016-06-24" @default.
• W2040289871 creator A5000096044 @default.
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• W2040289871 date "2003-12-15" @default.
• W2040289871 modified "2023-10-18" @default.
• W2040289871 title "IAP-antagonists exhibit non-redundant modes of action through differential DIAP1 binding" @default.
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