Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling
2012; Springer Nature; Volume: 31; Issue: 12 Linguagem: Inglês
10.1038/emboj.2012.121
ISSN1460-2075
AutoresAnnika Meinander, Christopher Runchel, Tencho Tenev, Li Chen, Chan‐Hee Kim, Paulo S. Ribeiro, Meike Broemer, François Leulier, Marketa Zvelebil, Neal Silverman, Pascal Meier,
Tópico(s)Antimicrobial Peptides and Activities
ResumoArticle1 May 2012free access Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling Annika Meinander Annika Meinander The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UKPresent address: Department of Biosciences, Åbo Akademi University, BioCity, FIN-20520 Turku, Finland Search for more papers by this author Christopher Runchel Christopher Runchel The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UK Search for more papers by this author Tencho Tenev Tencho Tenev The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UK Search for more papers by this author Li Chen Li Chen Division of Infectious Disease, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Chan-Hee Kim Chan-Hee Kim Division of Infectious Disease, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Paulo S Ribeiro Paulo S Ribeiro The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UKPresent address: Apoptosis and Proliferation Control Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields, London WC2A 3PX, UK Search for more papers by this author Meike Broemer Meike Broemer The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UKPresent address: Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Life and Medical Sciences (LiMES), Bonn, Germany Search for more papers by this author Francois Leulier Francois Leulier The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UKPresent address: IBDML—Institute de Biologie du Développement de Marseille Luminy, UMR 6216, Case 907—Parc Scientifique de Luminy, 13288 Marseille Cedex 9, France Search for more papers by this author Marketa Zvelebil Marketa Zvelebil The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UK Search for more papers by this author Neal Silverman Neal Silverman Division of Infectious Disease, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, 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, Chester Beatty Laboratories, London, UK Search for more papers by this author Annika Meinander Annika Meinander The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UKPresent address: Department of Biosciences, Åbo Akademi University, BioCity, FIN-20520 Turku, Finland Search for more papers by this author Christopher Runchel Christopher Runchel The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UK Search for more papers by this author Tencho Tenev Tencho Tenev The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UK Search for more papers by this author Li Chen Li Chen Division of Infectious Disease, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Chan-Hee Kim Chan-Hee Kim Division of Infectious Disease, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Paulo S Ribeiro Paulo S Ribeiro The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UKPresent address: Apoptosis and Proliferation Control Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields, London WC2A 3PX, UK Search for more papers by this author Meike Broemer Meike Broemer The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UKPresent address: Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Life and Medical Sciences (LiMES), Bonn, Germany Search for more papers by this author Francois Leulier Francois Leulier The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UKPresent address: IBDML—Institute de Biologie du Développement de Marseille Luminy, UMR 6216, Case 907—Parc Scientifique de Luminy, 13288 Marseille Cedex 9, France Search for more papers by this author Marketa Zvelebil Marketa Zvelebil The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UK Search for more papers by this author Neal Silverman Neal Silverman Division of Infectious Disease, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, 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, Chester Beatty Laboratories, London, UK Search for more papers by this author Author Information Annika Meinander1,‡, Christopher Runchel1,‡, Tencho Tenev1, Li Chen2, Chan-Hee Kim2, Paulo S Ribeiro1, Meike Broemer1, Francois Leulier1, Marketa Zvelebil1, Neal Silverman2 and Pascal Meier 1 1The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, UK 2Division of Infectious Disease, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA ‡These authors contributed equally to this work *Corresponding author. The Breakthrough Toby Robins Breast, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK Tel.:+44 20 7153 5326; Fax:+44 20 7153 5340; E-mail: [email protected] The EMBO Journal (2012)31:2770-2783https://doi.org/10.1038/emboj.2012.121 Present address: Department of Biosciences, Åbo Akademi University, BioCity, FIN-20520 Turku, Finland There is a Have you seen? (June 2012) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Caspases have been extensively studied as critical initiators and executioners of cell death pathways. However, caspases also take part in non-apoptotic signalling events such as the regulation of innate immunity and activation of nuclear factor-κB (NF-κB). How caspases are activated under these conditions and process a selective set of substrates to allow NF-κB signalling without killing the cell remains largely unknown. Here, we show that stimulation of the Drosophila pattern recognition protein PGRP-LCx induces DIAP2-dependent polyubiquitylation of the initiator caspase DREDD. Signal-dependent ubiquitylation of DREDD is required for full processing of IMD, NF-κB/Relish and expression of antimicrobial peptide genes in response to infection with Gram-negative bacteria. Our results identify a mechanism that positively controls NF-κB signalling via ubiquitin-mediated activation of DREDD. The direct involvement of ubiquitylation in caspase activation represents a novel mechanism for non-apoptotic caspase-mediated signalling. Introduction The conjugation and deconjugation of ubiquitin (Ub) to target proteins influence diverse biological processes that can contribute to tumour formation when deregulated (Hoeller and Dikic, 2009). The conjugation of Ub, thereby, can stimulate the assembly of transient signalling hubs in which the Ub adduct forms part of a new docking site for proteins with specialized binding motifs that help to build reversible, short-lived signalling centres (Grabbe and Dikic, 2009). Ub-mediated signalling is particularly important for activation of nuclear factor-κB (NF-κB) during innate immune responses (Skaug et al, 2009). Deregulated activation of NF-κB is recognized as one of the major underlying causes for chronic, cancer-related inflammation that drives tumour development, and is seen in most tumour types including leukaemia, lymphomas and solid tumours (Karin and Greten, 2005; Grivennikov et al, 2010; Nathan and Ding, 2010). Hence, a better understanding of the processes that regulate NF-κB and innate immunity is critically important. Metazoans live in close contact with a multitude of microbes with which they establish complex reciprocal interactions. Drosophila relies on innate immunity responses to combat microbial challenges (Ferrandon et al, 2007; Lemaitre and Hoffmann, 2007). Depending on the invading microorganism, Drosophila activate either the Toll or immune deficiency (imd) pathway (Lemaitre et al, 1995, 1996). Infection with fungi or certain Gram-positive bacteria activates the Toll pathway, which initiates an intracellular signalling cascade that culminates in the nuclear translocation of the NF-κB-like transcription factors Dif and Dorsal (Lemaitre et al, 1996; Manfruelli et al, 1999; Meng et al, 1999; Rutschmann et al, 2000a, 2002). On the other hand, the IMD pathway is activated by Gram-negative bacteria, which in turn stimulates activation of the NF-κB-like transcription factor Relish (also known as IRD4) (Hedengren et al, 1999). Activation of both pathways induces the expression of a plethora of NF-κB responsive antimicrobial peptide (AMP) genes, which are important for fending off invading microorganisms (Tzou et al, 2000). The IMD signal transduction pathway shows striking similarities to the ones stimulated by members of the mammalian TNF-receptor super-family (Tanji and Ip, 2005). These pathways are Ub-dependent signal transduction cascades in which Ub ligases, Ub receptors and deubiquitylating enzymes (DUBs) build up, recognize and remove Ub signals, allowing the temporally controlled assembly of protein complexes that lead to the activation of kinases that regulate NF-κB. Ub, thereby, functions as a binding surface for proteins with Ub-binding domains (UBD), also referred to as Ub receptors (Dikic et al, 2009). The conjugation of Ub requires a stepwise process that involves Ub-activating enzymes (E1), Ub-conjugating enzymes (E2) and Ub protein ligases (E3) (Dikic et al, 2009). E3s confer substrate specificity and enable the formation of an isopeptide linkage between the carboxyl-terminus of Ub (glycine (G)76) and the amino group of a reactive side chain of the substrate. Ub can be conjugated either as a single moiety or as chains of variable length (Komander, 2009). Further complexity is provided by different linkage types, as Ub moieties can be conjugated to one another via different lysine (K) residues within Ub. At least eight different types of Ub chains exist. The different types of Ub chain linkages exert distinct functional outcomes (Hoeller and Dikic, 2009). This is predominantly due to the fact that the different chain types adopt distinct topologies, which in turn are recognized by specific Ub receptors (Komander, 2009). Signalling through the IMD pathway is activated by the Gram-negative cell wall component diaminopymelic-type peptidoglycan (DAP-PGN). DAP-PGN is recognized by the pattern recognition protein PGRP-LE and the transmembrane receptor PGRP-LCx (Choe et al, 2002; Gottar et al, 2002; Ramet et al, 2002; Leulier et al, 2003; Kaneko et al, 2004, 2006). Binding of DAP-PGN to PGRP-LCx causes receptor oligomerization and triggers recruitment of the adaptor protein IMD (Choe et al, 2005), which carries a C-terminal death domain (DD) that is similar to the one of the mammalian adaptor protein RIP1 (Georgel et al, 2001). Through its DD, IMD subsequently recruits dFADD (also known as BG4), which in turn binds and presumably activates the Drosophila caspase-8 orthologue Death-related ced-3/Nedd2-like protein (DREDD; also known as DCP2) (Leulier et al, 2000, 2002; Naitza et al, 2002). Following activation, DREDD cleaves off the amino (N)-terminal portion of IMD, thereby exposing an evolutionarily conserved inhibitor of apoptosis (IAP)-binding motif (IBM) at the neo-N-terminus of IMD (Paquette et al, 2010). This IBM is recognized by the E3 ligase Drosophila IAP protein 2 (DIAP2), which brings it into position for IMD ubiquitylation. In conjunction with the E2s Effete (UBC5) and UEV1a/Bendless (UEV1a/UBC13), DIAP2 targets IMD for K63-linked polyubiquitylation (Zhou et al, 2005; Paquette et al, 2010). According to the current model, the attached Ub chains function as scaffolds for the recruitment of the Drosophila MAP kinase kinase kinase dTAK1/TAB2 and the Relish kinase complex IRD5/Kenny (IKKβ/IKKγ) (Ferrandon et al, 2007). Ub-dependent recruitment of dTAK1/TAB2 and IRD5/Kenny is thought to be mediated by their respective Ub receptors TAB2 and Kenny (Rutschmann et al, 2000b; Silverman et al, 2000; Lu et al, 2001; Vidal et al, 2001; Silverman et al, 2003; Kanayama et al, 2004; Kleino et al, 2005; Zhuang et al, 2006). Ub-dependent complex formation is presumed to result in activation of dTAK1, which in turn phosphorylates and activates IRD5/Kenny and MKK4/7 (Silverman et al, 2003; Geuking et al, 2009). While MKK4/7 promotes JNK activation, IRD5/Kenny phosphorylate Relish (Rutschmann et al, 2000b; Silverman et al, 2000; Lu et al, 2001; Vidal et al, 2001; Geuking et al, 2009), which activates its transcriptional activity (Erturk-Hasdemir et al, 2009). In addition to IRD5/Kenny-mediated phosphorylation of Relish, activation also requires DREDD-dependent proteolytic processing of Relish (Elrod-Erickson et al, 2000; Leulier et al, 2000; Stoven et al, 2000, 2003; Erturk-Hasdemir et al, 2009). Activated DREDD cleaves off an inhibitory C-terminal ankyrin repeat domain of Relish, thereby allowing the translocation of the N-terminal portion to the nucleus where it induces expression of AMP genes (Silverman et al, 2000; Stoven et al, 2000, 2003; Erturk-Hasdemir et al, 2009). Loss-of-function mutations in most of the components of the IMD signalling cascade results in an immune deficiency phenotype, in which animals become acutely susceptible to infection by Gram-negative bacteria. Common to all these mutants is their failure to induce expression of antibacterial peptide genes and, therefore, to fend off bacterial infection (Ferrandon et al, 2007; Lemaitre and Hoffmann, 2007). While E3 ligases promote IMD signalling via ubiquitylation, pathway activation can be suppressed via Ub deconjugation by dUSP36 and dCYLD (Tsichritzis et al, 2007; Thevenon et al, 2009), reinforcing the importance of Ub in the regulation of innate immunity. At present, DIAP2 is the sole E3 ligase implicated in Ub-mediated IMD signalling (Gesellchen et al, 2005; Kleino et al, 2005; Leulier et al, 2006; Huh et al, 2007). DIAP2 is a member of the evolutionarily conserved IAP protein family, whose members are best known for their ability to regulate caspases and apoptosis (Gyrd-Hansen and Meier, 2010). The defining feature of an IAP protein is the presence of the baculovirus IAP repeat (BIR) domain(s), a zinc-binding fold of ∼70 amino-acid residues that mediates protein interactions. Most IAPs harbour additional domains such as the C-terminal RING finger domain that provides them with E3 Ub ligase activity by mediating the transfer of Ub from E2s to target substrate. Although it is clear that DIAP2 is required for Rel/NF-κB activation (Gesellchen et al, 2005; Kleino et al, 2005; Leulier et al, 2006; Huh et al, 2007), the precise mechanism through which DIAP2 mediates Ub-dependent activation of NF-κB remains ill defined. At present, the only known targets for DIAP2-induced ubiquitylation are the adaptor protein IMD and DIAP2 itself (Paquette et al, 2010). To gain a better understanding of Ub-dependent NF-κB activation, and to explore the possibility that DIAP2 mediates its effect through modulating caspases, we studied the molecular mechanism through which DIAP2 triggers Ub-dependent signalling. Here, we show that DIAP2 interacts with the initiator caspase DREDD and that DIAP2 targets this caspase for non-degradative ubiquitylation in a signal-dependent manner. Further, we find that impaired ubiquitylation of DREDD blocks IMD and Relish cleavage and renders mutant flies acutely sensitive to Gram-negative bacterial infection. Thus, the Ub chains on DREDD might function as anchor points for Ub receptors that help dimerization and full activation of DREDD. The mechanism of DREDD activation might parallel the one of caspase-8 in mammals, where the Ub receptor p62 reportedly promotes aggregation, activation and processing of CUL3-modified caspase-8 (Jin et al, 2009). Together, our data indicate that DIAP2-mediated ubiquitylation of DREDD is required for innate immune signalling. Results DIAP2 binds to DREDD and targets it for polyubiquitylation in a RING finger-dependent manner To determine the molecular mechanism through which DIAP2 controls innate immunity, we first determined the contribution of individual domains of DIAP2 that are required for IMD signalling. To this end, we used UAS-driven transgenes encoding specific DIAP2 mutants and assessed their ability to rescue the lethality of Diap2 mutant flies following septic injury by Erwinia carotovora carotovora 15 (Ecc15), a Gram-negative bacteria. Transgenes were expressed ubiquitously under the control of daughterless (Da)-GAL4. While expression of wild-type (WT) DIAP2 rescued the lethality of the Diap2 mutant flies (Diap27c) (Leulier et al, 2006) in response to Ecc15 septic injury, reconstitution with an E3-defective DIAP2 mutant (DIAP2RINGmut) completely failed to protect Diap2 mutant animals (Figure 1A), which is in agreement with an earlier report (Huh et al, 2007). Of note, DIAP2RINGmut, which carries a mutation that removes the zinc-coordinating cysteine required for the proper folding of the RING domain, is as stable as WT DIAP2 (Ribeiro et al, 2007). To evaluate the contribution of individual BIR domain of DIAP2, we generated transgenic flies using germ-line-specific phiC31 integration into pre-defined landing sites, which allows accurate comparison between mutants (Bischof et al, 2007). DIAP2 transgenes encoding DIAP2 mutants with amino-acid (aa) substitutions in either the BIR2 (D163A) or BIR3 (D263A) domain (Ribeiro et al, 2007) efficiently rescued the sensitivity to Ecc15 infection seen in Diap27c mutant flies. In contrast, double mutants carrying a substitution in BIR2 and BIR3 (D163A/D263A) failed to provide protection from Ecc15 septic injury (Figure 1B). This is consistent with the observation that either BIR2 or BIR3 can mediate IMD binding (Paquette et al, 2010). Given that expression of DIAP2RINGmut fails to rescue Ecc15-mediated lethality of diap27c mutant animals, this indicates that DIAP2-mediated ubiquitylation of IMD pathway components is indispensable for IMD signalling. Figure 1.DIAP2 binds and ubiquitylates the initiator caspase DREDD. (A, B) DIAP2's RING finger and both BIR domains are required to resist Ecc15 bacterial infection. Diap27c mutant animals were reconstituted with rescue constructs expressing the indicated DIAP2 proteins. To confirm the requirement of DIAP2 E3 ligase activity for the IMD pathway (A), DIAP2 constructs were ubiquitously expressed in transgenic flies under the control of daughterless (Da)-GAL4. Animals were pricked with a needle previously dipped into Ecc15 and the survival rates monitored. To evaluate the contribution of individual BIR domain of DIAP2 (B), transgenic flies were generated using germ-line-specific phiC31 integration (Bischof et al, 2007). Transgenes were expressed using the fat-body-specific c564-Gal4 driver line. Animals were injected with 13.8 nl of Ecc15 bacterial solution (optical density of 300) and the survival rates monitored. The experiments were performed using at least 20 flies for each genotype. Shown is a representative experiment of at least three repeats. C466Y represents a RING mutant of DIAP2, which lacks E3 ligase activity (Ribeiro et al, 2007), D163 and D263A disrupts the BIR pocket of BIR2 and BIR3, respectively. (C, D) DIAP2 binds to DREDD. Reciprocal binding assays with DIAP2 and DREDD. The indicated constructs were transfected into S2 cells and GST-tagged DIAP2 (C) and V5-tagged DREDD (D) were purified with Glutathione resin and α-V5 antibodies, respectively. The expression and the presence of the co-purified proteins were assessed by immunoblotting with the indicated antibodies. (E) DIAP2 ubiquitylates DREDD in a RING finger-dependent manner. HA–Ub was co-expressed with DREDD–V5 in S2 cells together with vector control, DIAP2WT or the DIAP2 RING mutants DIAP2C466Y and DIAP2F469A. Cells were lysed under denaturing conditions and ubiquitylated proteins isolated using α-HA antibodies. The presence and ubiquitylation of DREDD and DIAP2 was assessed with the indicated antibodies. (F) DREDD is transiently ubiquitylated in a signal-dependent manner. S2 cells were transfected with DREDD–V5. Subsequently, cells were treated with 1 μg/ml DAP-PGN for the indicated time points before being harvested. DREDD was immunoprecipitated using α-V5 resin and ubiquitylated DREDD analysed using α-Ub antibodies. (G) DIAP2 promotes the conjugation of K63-linked Ub chains on DREDD. The indicated constructs were transfected into S2 cells, and HA-tagged DREDD and IMD were purified under denaturing conditions. The presence of the purified proteins and identity of the respective Ub chains were detected by immunoblotting using the indicated antibodies (see Materials and methods for details). Download figure Download PowerPoint To identify components of the IMD pathway that are targeted by DIAP2, we first performed binding assays with DIAP2 and known members of this pathway. We co-expressed DIAP2 with tagged components of the IMD pathway in S2 cells and analysed their ability to interact with each another. The interaction between DIAP2 and PGRP-LCx was performed in the presence or absence of DAP-PGN to assess whether receptor activation is required for binding to DIAP2. PGRP-LCx failed to interact with DIAP2 under these conditions (data not shown). Cleaved IMD, which is truncated at aspartic acid residue 30 following pathway activation and exposes a bona fide IBM at the neo-amino-terminus of the C-terminal portion of IMD, readily bound to DIAP2 (data not shown; Paquette et al, 2010). While DIAP2 failed to associate with other IMD pathway components such as dFADD, IRD5, dTAB2 and dTAK1 (data not shown), DIAP2 tightly bound to DREDD (Figure 1C). DIAP2 also bound to the heterodimeric E2 UEV1A/Bendless that promotes the conjugation of K63-linked polyubiquitin chains, as previously reported (data not shown; Zhou et al, 2005; Paquette et al, 2010). Reciprocal pulldown experiments confirmed the interaction between DREDD and DIAP2 (Figure 1D). To assess whether DIAP2 can target DREDD for ubiquitylation, we next expressed non-tagged DIAP2 in S2 cells together with HA-tagged Ub and V5-tagged DREDD. Ubiquitylated proteins were affinity purified under denaturing conditions using α-HA columns (see Materials and methods for details), and the presence of ubiquitylated proteins was assessed by immunoblotting the eluate. Under these conditions, DIAP2 efficiently ubiquitylated DREDD (Figure 1E) and IMD (data not shown; Paquette et al, 2010). Ubiquitylation of DREDD was dependent on DIAP2's E3 ligase activity, as the RING mutants DIAP2F469A and DIAP2C466Y, which abrogate the Ub-E3 activity of IAPs (Ribeiro et al, 2007; Ditzel et al, 2008), failed to ubiquitylate DREDD (Figure 1E, top panel, compare lane 3 with lanes 4 and 5). Together, these data indicate that DIAP2 can bind to DREDD and promote its polyubiquitylation. Next, we determined whether ubiquitylation of DREDD occurred in a signal-dependent manner. For this purpose, we induced the IMD pathway in Drosophila S2 cells using bacterial-derived DAP-PGN. For this assay, we relied on endogenous DIAP2, but stably expressed DREDD–V5, because no antibody is currently available that detects endogenous DREDD. Interestingly, while ectopically expressed DREDD remained unmodified under these conditions, treatment with DAP-PGN induced prominent and transient ubiquitylation of DREDD (Figure 1F). DREDD ubiquitylation occurred as early as 1 min and was no longer detectable 40–60 min after DAP-PGN treatment. Consistent with the notion that DIAP2 can bind to the heterodimeric E2 UEV1 A/Bendless, we found that DIAP2 promoted the conjugation of K63-linked Ub chains on DREDD (Figure 1G). Likewise, DIAP2 also triggered K63-ubiquitylation of IMD. Given that DIAP2 binds and ubiquitylates DREDD, and that DIAP2 is the key E3 ligase of the IMD pathway (Gesellchen et al, 2005; Kleino et al, 2005; Leulier et al, 2006; Huh et al, 2007), these data suggest that activation of the IMD pathway results in transient, DIAP2-mediated ubiquitylation of DREDD. DIAP2 binds to the DED1 of DREDD DIAP2 is the first IAP found to interact with a death effector domain (DED)-containing initiator caspase. To better understand the nature of this interaction, we generated various deletion mutants of DREDD and DIAP2 (Figure 2A) and determined the domains that are required for their association. Similar to caspase-8, DREDD contains a long pro-domain with two DEDs in tandem (Chen et al, 1998). This is followed by the large (p20) and small (p10) caspase subunits. Co-immunoprecipitation assays revealed that DIAP2 readily bound to the prodomain of DREDD (DED1/2, aa 1–264) (Figure 2B). In contrast, DIAP2 failed to associate with the C-terminal caspase domains (p20/p10, aa 265–517) of DREDD. Further, the binding of DIAP2 to DREDD appeared to depend on the presence of the DED1 because DIAP2 only weakly bound to DREDD–ΔDED1 (aa 143–517) that lacks DED1. Consistently, DED1 in isolation co-purified with DIAP2 (Figure 2C), while the DED2 did not interact with DIAP2. This indicates that the first DED is necessary and sufficient for DREDD to bind to DIAP2. Intriguingly, we found that the adaptor protein dFADD also selectively bound to DED1 (Figure 2D), which is in contrast to an earlier report (Hu and Yang, 2000). Although dFADD and DIAP2 both bound to DED1, they did not compete with one another for the binding to DREDD (Figure 2E). This is evident because co-expression of dFADD did not abrogate the interaction of DIAP2 with DREDD. Together, our data indicate that DIAP2 and dFADD bind to the DED1 of DREDD, forming a trimeric complex. Figure 2.DIAP2 binds to the DED1 of DREDD. (A) Schematic representation of the DIAP2 and DREDD constructs used in this study. (B) DIAP2 binds to the pro-domain of DREDD. Co-IP assays with DIAP2 and various fragments of DREDD. S2 cells were co-transfected with HA-tagged DIAP2 and V5-tagged DREDD. Expression and the presence of co-purified proteins were analysed by immunoblotting with the indicated antibodies. (C) DIAP2 associates with DED1 of DREDD. Co-IPs were performed with DIAP2 and individual DEDs or the prodomain of DREDD as in (B). (D) dFADD also binds to the DED1 of DREDD. Co-IPs were performed with dFADD and individual DEDs or the prodomain of DREDD as in (B). (E) Competition assay with DIAP2, dFADD and DREDD indicates that DIAP2 and dFADD can bind to DREDD simultaneously. Co-IPs were performed with dFADD, DIAP2 and DREDD. Expression and the presence of co-purified proteins were analysed as in (B). (F) The N-terminal portion of DIAP2 interacts with DREDD. Co-IPs were performed with DREDD and the indicated fragments of DIAP2. Expression and the presence of co-purified proteins were analysed as in (B). (G) The BIR2 and BIR3 domains mediate binding to DREDD. Co-IPs were performed with DREDD and individual BIR domains of DIAP2. Expression and the presence of co-purified proteins were analysed as in (B). An asterisk marks a cleavage product of BIR3. (H) The interaction between DIAP2 and DREDD does not require the hydrophobic pocket of the BIR2 and BIR3. Co-IPs were performed with DREDD and the indicated WT or mutant BIR domains. Expression and the presence of co-purified proteins were analysed as in (B). (I) In contrast, the binding of DIAP2 to cleaved IMD critically depends on the hydrophobic pockets of the BIR2 and BIR3. IMD corresponds to the DREDD-cleaved form of IMD (aa 31–273) and was expressed using the Ub-fusion technique (Varshavsky, 2000; Tenev et al, 2005). Co-IPs with DREDD and the indicated WT or mutant BIR domains were performed as in (H). BIR1/2/3 encompasses DIAP2's N-terminal segment that carries the BIR1, BIR2 and BIR3 domains. Download figure Download PowerPoint To identify the region of DIAP2 that interacts with DREDD, we first examined the requirement of the BIR domains, since BIRs generally function as protein interaction motifs (Eckelman et al, 2008). We found that the BIR domain-containing amino-terminal portion of DIAP2 (DIAP2-BIR1/2/3, aa 1–338) bound to DREDD as efficiently as WT DIAP2. In contrast, the carboxy-terminal portion of DIAP2 (DIAP2281–498), which lacks the BIR domains, failed to associate with DREDD (Figure 2F). Based on the presence of a deep peptide-binding groove
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