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J. Biol. Chem., Vol. 282, Issue 3, 2056-2068, January 19, 2007
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1
From the
Division of Biology, MC 156-29, California Institute of Technology, Pasadena, California 91125, the Department of Biochemistry, Seoul National University, Seoul, Korea, the ¶Department of Biology, College of Sciences, Kyung Hee University, Seoul 130-701, Korea, the ||Department of Neurology, Brain Research Institute, The David Geffen School of Medicine, UCLA, Los Angeles, California 90095, and **Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
Received for publication, August 22, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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70-amino acid motif known as a baculovirus IAP repeat (BIR), which mediates interactions with multiple death activators and plays an essential role in the ability of these proteins to inhibit cell death. IAPs also contain a C-terminal RING E3 ubiquitin ligase domain that can target bound proteins, as well as the IAP itself, for ubiquitination and in some cases degradation (1). The Drosophila genome encodes two BIR and RING domain-containing IAP family members, DIAP1 and DIAP2, and ectopic expression of either protein inhibits apoptosis (2-4). DIAP1 is required continuously in many cells to inhibit the apical caspase Dronc and effector caspases activated by Dronc, such as Drice (5, 6). Critical interactions between caspases and DIAP1 are mediated by a surface groove within each DIAP1 BIR domain and short IAP-binding motifs (IBM) present in Dronc or Drice (7-9). Apoptosis in the fly can be induced by expression of proteins such as Reaper, Hid, Grim, Sickle, and Jafrac2 (the RHG proteins). Each of these proteins contains an N-terminal IBM that mediates competitive binding to DIAP1 through the same BIR surface grooves that are required for DIAP1-caspase interactions (8, 10). RHG proteins can also promote ubiquitin-dependent degradation of DIAP1. Both activities have the effect of liberating active caspases, resulting in apoptosis (6). IAPs that inhibit apoptosis, as well as inhibitory RHG counterparts, are also found in mammals (1, 3).
Several observations have suggested that DIAP2 might also be an important apoptosis inhibitor. DIAP2 can bind Rpr and Hid (11, 12) and the caspases Drice (4) and Strica (13). Overexpression of DIAP2 can also inhibit Rpr- and Hid-dependent apoptosis, developmental apoptosis in the eye (2), as well as apoptosis associated with decreased levels of diap1 (4). In addition, RNAi-mediated knockdown of DIAP2 in the S2 cell line has been reported to result in increased susceptibility to stress-induced apoptosis (14). RNAi of diap2 in larvae and pupae has generated conflicting results. One group reported no effect of heat shock-induced expression of diap2 dsRNA on developmental cell death or viability (15), and second group reported organismal lethality in response to ubiquitous diap2 dsRNA expression (16). Finally, several RNAi-based studies in S2 cells have also provided evidence that DIAP2 is required for the innate immune response to Gram-negative bacteria infection (16, 17). However, these studies came to conflicting conclusions regarding the site of action for DIAP2 and involved primarily use of a single long time immortalized cell line.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Tyr at amino acid 472, alternative primer pairs (5'-CGCTCTAGATAAGCAACGCTCG-3' and 5'-CGCGTTAACCCGACAGCCACCTACAATC-3') were used for the synthesis of the 1.2-kb fragment. GMR-Rpr, GMR-Hid, GMR-Dronc, GMR-Dream/Strica, GMR-DIAP2, UAS-Rpr, UAS-Hid, UAS-P35, engrailed-Gal4, diptericin-GFP, UAS-dTak1, c564-Gal4, hs-diap1-rnai, dreddB118, and imd1 flies have been described previously (2, 15, 20, 21, 24-27). GMR-Drice flies, carrying both the GMR-Drice P20 and GMR-Drice P10 subunits, were generated by introducing Drice large (P20) or small (P10) subunit coding regions into pGMR separately. Transformants carrying both constructs were generated by recombination. Hs-RelishRHD flies were generated by introducing the RelishRHD coding region into pCaSpeR-hs-act vector (15, 28). UAS-diap1-miRNA and UAS-diap2-miRNA flies were generated by replacing 21-bp sequences within the mir-6 precursor backbone that are loaded into the RISC complex with sequences complementary to diap1 or diap2.3
Fly Immune Challenge—All Drosophila stocks were raised at 25 °C. One- to 3-day-old adult male flies were pricked with a fine glass needle dipped in an overnight live culture of Enterobacter cloacae or Staphylococcus aureus, resuspended at 20x concentration in sterile S2 cell medium.
Irradiation-induced Stress—Forty wild type or diap2E151 3rd instar larvae were placed in 1.5-ml tubes and X-irradiated with 2000 or 4000 rads. Subsequently, they were moved to fresh vials and scored for hatching efficiency. Each experiment was repeated three times. Error bars on figures indicate the standard deviation.
Oxidative Stress—One- to 3-day-old adult males were put in vials (20 flies to a vial, total five vials), with eight filter paper disks soaked with 500 µl of 5% sucrose and 20 mM methyl viologen (paraquat). Each experiment was repeated twice.
Salt Stress Tolerance—Solid medium that contained 2.5% sucrose, 1% agar, 0.15% methyl paraben, 0.003% methylene blue, and different concentrations of NaCl (0.4 or 0.8 M) was prepared in vials. Fifty young adult males of each genotype were introduced into these vials (25 flies per vial) and scored for survival over the next 4 days. Each experiment was repeated three times.
Heat Shock Induction—Wild type and diap2E151 mutant 3rd instar larvae carrying an hs-diap1-dsRNA construct were transferred to 1.5-ml microcentrifuge tubes and subjected to 10, 20, or 30 min of heat treatment in a 37 °C water bath. Each experiment was repeated in triplicate. For the induction of RelishRHD, adult wild type or diap2 mutant male flies carrying hs-RelishRHD were subjected to a 30-min heat shock at 37 °C and allowed to recover for 6 h at 25 °C before total RNA was isolated to quantify levels of antimicrobial peptide gene induction.
RNA Analysis—Total RNA was isolated from whole flies using the TRIzol reagent (Invitrogen). For real time PCR analysis, cDNAs were synthesized with the Superscript III reverse transcriptase system (Invitrogen). An amount of cDNA equivalent to 0.3 µg of total RNA was subjected to 40 cycles of PCR amplification for 15 s at 95 °C and a 1-min incubation at 60 °C. The output was monitored using SYBR Green core reagents and the ABI Prism 7900 system (Applied Biosystems). All results were normalized to the levels of rp49 mRNA. Individual primer sequences are available upon request.
Protein Analysis—For immunoblot analysis, 20 adult flies were homogenized in lysis buffer (20 mM HEPES-KOH (pH 7.6), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, and 1x protease inhibitor (Roche Applied Science)) and then subjected to SDS-PAGE. After transfer to a nitrocellulose membrane, proteins were probed with mouse antibodies raised against DIAP2 (1:50), DIAP1 (25) (1:100), the C-terminal domain of Relish (29) (1:5; a generous gift from Svenja Stöven and Dan Hultmark, Umeå University, Umeå, Sweden), and tubulin (1:1,000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City). DIAP2 antibodies were generated in mouse using a GST-DIAP2 fusion protein as the immunogen. Conditions for immunohistochemistry and confocal microscopy were described previously (25). Antibodies were used at the following concentrations: mouse anti-P35 (25) (1:100), mouse anti-DIAP2 (1:50), and rat anti-Relish (30) (1:1000, a generous gift from Young-Joon Kim, Yonsei University, Seoul, Korea).
Caspase Activity Assay—Fifteen wild type and diap2E151 mutant adult flies were homogenized in 100 µl of lysis buffer. 30 µg of each extract was added into 100 µl of caspase activity buffer (70.4 mM HEPES (pH 7.5), 140.8 mM NaCl, 1.4 mM EDTA, 0.14% CHAPS, 14.1% sucrose, 3.5 mM dithiothreitol, 5.6% glycerol, and 0.7% Triton X-100) in the presence of 100 µM Ac-DEVD-AFC (EMD Biosciences). Caspase activity was measured at 27 °C using a fluorometric plate reader (Fmax; Molecular Devices) in the kinetic mode with excitation and emission wavelengths of 405 and 510 nm, respectively. Assays were performed in triplicate.
Gene Switch System—Flies carrying the adult fat body gene switch driver S106 in different genetic backgrounds were placed in empty vials with eight 3MM filter paper disks overnight (20 h) (31). To induce gene expression, disks were wetted with 800 µl of 500 µM RU486 (Sigma) in 2% sucrose solution. Following immune challenge, flies were allowed to recover in normal fly food vials for 4 h prior to RNA preparation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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840 bp and diap2E151 1,460 bp downstream of the original P element insertion site, without affecting neighboring genes (Fig. 1b). RT-PCR and Western blot analysis failed to detect full-length diap2 mRNA or protein in either mutant (Fig. 1, c and d, and Fig. 5a). Truncated diap2 transcripts are detectable in diap2E151 and diap2E119 animals. However, these transcripts are unlikely to generate functional proteins because multiple in-frame start and stop codons precede the remaining C-terminal diap2 coding sequences. Together these results suggest that diap2E151 and diap2E119 represent null alleles. Introduction of a 7.4-kb genomic fragment encoding wild type diap2 (diap27.4Wt) or a point mutant of diap2 with a mutation in the RING domain (diap27.4C472Y) into the diap2E151 mutant background resulted in expression of a full-length diap2 transcript (Fig. 1, a and c) and protein (Fig. 5a). In contrast to mutations in diap1 (alleles of th), which are lethal early in embryogenesis, animals homozygous for diap2E151 and diap2E119 are viable, healthy, and fertile. Therefore, diap2 is dispensable for most, if not all, developmental cell death.
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DIAP2 Is Required to Defend against Gram-negative Bacterial Infection—Bacterial infection, and the response to bacterial infection, constitutes a form of organismal and tissue stress that channels limited resources into a range of new tasks (19). Most notably, infection results in the activation of a multipronged immune response that involves phagocytosis, a melanin-producing phenol oxidase reaction, and a humoral response that leads to the expression of anti-microbial peptides and activation of the JNK signaling pathway (38-40). Two pathways, the Toll and IMD, mediate induction of the humoral response. In the Toll pathway, infection by Gram-positive bacteria or fungi triggers activation of the Toll receptor, which leads ultimately to the expression of AMPs such as drosomycin (38, 41, 42). The IMD pathway is activated in response to infection by Gram-negative bacteria when receptors such as PGRP-LC/ird7 bind Gram-negative peptidoglycan (26, 39, 43). PGRP-LC recruits IMD, which activates two downstream pathways. In one branch, IMD binds to dFADD (44), which recruits the caspase DREDD (45, 46). DREDD promotes (directly or indirectly) the cleavage of the REL/NF-B family member Relish in the cytoplasm (27, 47, 48). Once liberated from REL-49, the C-terminal IB-like inhibitory domain, the N-terminal REL-68 fragment, which contains the DNA binding Rel homology domain, translocates to the nucleus where it drives the expression of AMPs such as diptericin, attacin, cecropin, and defensin (49). IMD also activates, through unknown mechanisms, the MAPK kinase kinase dTAK1 (50-52). dTAK1 is required to activate (perhaps through direct phosphorylation) the Drosophila signalosome equivalent (50, 52), which consists of homologs of the kinase IKK
(ird5), and an accessory subunit IKK
(kenny) (53-55). The activated IKK complex phosphorylates Relish (48, 53), and this phosphorylation is required for Dredd-dependent cleavage of Relish (48).
When exposed to the Gram-positive bacteria S. aureus, wild type, diap2E151, and diap2E119 flies died at similar rates (Fig. 3a). This suggests that diap2 does not function as a general inhibitor of organismal death in response to bacterial infection or as an essential component of the Toll pathway. In contrast, when exposed to the Gram-negative bacteria E. cloacae animals lacking diap2 were much more sensitive than wild type flies. In fact, diap2E151 and diap2E119 adults died at a rate similar to that of animals lacking IMD(Fig.3b), which is essential for the Relish-dependent production of Gram-negative-specific AMPs. Importantly, these phenotypes were completely suppressed in the presence of the wild type diap27.4Wt rescue transgene, demonstrating that they are due to loss of diap2. diap2E151 and diap27.4Wt flies actually showed greater resistance to infection than wild type flies (Fig. 3b), although they had decreased longevity in the absence of immune stimulation.4 The basis for this increased resistance is unknown, but it is interesting to speculate that it might reflect the fact that endogenous diap2 levels are set so as to optimize an evolutionary compromise between immune resistance and longevity, a feature not retained with our transgene insertion. Finally, survival was not rescued in the presence of the diap2 rescue transgene containing a point mutation in the RING domain, diap27.4C472Y, even though expression levels of wild type and mutant transgene mRNA (Fig. 1c) and protein (Fig. 3c and Fig. 5a) were similar.
To determine whether diap2 regulates the production of AMPs, we infected wandering third instar larvae carrying a diptericin-GFP reporter construct (27) with E. cloacae. Wild type animals, as well as diap2E151 animals carrying a copy of the diap27.4Wt rescue transgene, showed strong GFP expression in response to infection (Fig. 3d). In contrast, GFP was undetectable in diap2E151 and diap2E119 animals, suggesting that diap2 mutants are unable to mount an effective immune response. We also examined AMP expression using quantitative RT-PCR. When challenged with E. cloacae, wild type adult flies exhibited robust induction of attacin and diptericin. In contrast, the increases in attacin and diptericin were dramatically suppressed in diap2E151 and diap2E119 flies, even after 12 h (Fig. 3e). Importantly, these phenotypes were significantly suppressed in the presence of diap27.4Wt but not diap27.4C472Y (Fig. 4a). We note that the levels of the diap27.4Wt transgene were lower than those of endogenous diap2 (Fig. 1c) and that diptericin expression levels were rescued to a somewhat lesser extent than those of attacin (Fig. 4a). This may simply indicate that the attacin promoter contains cis elements that buffer changes in the concentration of processed Relish whereas the diptericin promoter drives transcription in a manner directly proportional to the level of Relish expression, a question that requires further exploration. In any case, animals lacking diap2 also failed to express two other Gram-negative-specific AMPs, cecropin and defensin, in response to bacterial challenge, suggesting a general failure to induce IMD-dependent antimicrobial peptide expression (Fig. 4b). In contrast to these observations with diap2 mutants, when the number of copies of wild type diap2 was increased from two (the endogenous chromosomal copies) to six (+ four copies of diap27.4Wt), the levels of antimicrobial peptide expression in response to immune challenge underwent a modest increase (supplemental Fig. 2d). This observation is potentially interesting because it suggests that regulation of diap2 levels may serve to control the intensity and kinetics of the immune response. To localize the site of diap2 action, we used micro-RNA-based RNAi5 to reduce diap2 levels specifically in the adult fat body, the insect analog of the mammalian liver, and the major site of AMP synthesis. As shown in Fig. 4c, this treatment phenocopied the immune defects observed in diap2E151 and diap2E119 animals, thereby identifying the fat body as a major site of the diap2 function.
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B-like transcription factor Relish (27, 47, 48). The resulting cleaved N-terminal product, RelishRHD (Rel homology domain), translocates into the nucleus and functions as a transcription activator for the induction of various AMP genes (49). Since diap2E151 animals lack IMD-dependent immune responses but have normal Toll-dependent signaling, we examined the fate of Relish in response to immune activation using an antibody that recognizes the C-terminal half of Relish (29). In unchallenged wild type animals, Relish was found mostly in the precursor form, although small amounts of processed Relish could also be observed (Fig. 5a, left panel). Relish processing was greatly increased post-challenge in wild type flies but not in flies lacking Dredd or Imd, which are required for challenge-dependent Relish cleavage. Flies lacking diap2 showed a low level of challenge-independent Relish processing, similar to that observed in unchallenged wild type flies. However, they showed only a modest increase in Relish cleavage in response to immune challenge. Importantly, high levels of challenge-dependent Relish cleavage were restored in the presence of the diap2 wild type rescue transgene (diap27.4Wt) but not in the presence of the Ring mutant form (diap27.4C472Y). Together, these observations indicate that diap2 is required to bring about normal levels of Relish cleavage and that this requires DIAP2 RING function. However, they also indicate that some challenge-independent, as well as challenge-dependent Relish cleavage (or stabilization of the C-terminal Relish cleavage product), occurs in the absence of diap2. Nuclear translocation of RelishRHD, which occurs rapidly following immune stimulation in wild type animals, was undetectable in diap2E151 animals (Fig. 5b). As with Relish cleavage, the defect in Relish nuclear translocation was eliminated in the presence of the diap2 genomic rescue transgene (Fig. 5, a and b).
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Why might apoptosing cells choose to down-regulate DIAP2? One possibility is that this helps to prevent inappropriate immune activation in response to the presence of proinflammatory molecules released by dying cells. To explore this possibility, we created cells in which apoptosis effectors were activated at modest levels in the fat body (thereby inducing low levels of death) but in which this activation occurred independent of expression of RHG proteins (thus preventing down-regulation of diap2 that would occur in response to their expression). We did this by targeting diap1 mRNA for degradation specifically in the adult fat body, using a drug-inducible, fat body Gal4 driver (31), and a UAS-diap1-targeted micro-RNA (UAS-diap1-miRNA) that is known to promote the loss of diap1 when expressed in other tissues (supplemental Fig. 1, d and e). As illustrated in supplemental Fig. 2e, a 20-h induction of UAS-diap1-miRNA had no negative effect on expression of the Gram-positive immune response gene drosomycin, indicating that the fat body was still largely intact. In contrast, with diptericin, a Gram-negative response gene, a modest 20% increase in expression was seen. Most importantly, however, this increase in diptericin was suppressed by coexpression of a micro-RNA targeting diap2 (UAS-diap2-miRNA) that is known to promote the loss of diap2 when expressed in other tissues (supplemental Fig. 1, b and c). No significant change in drosomycin expression was observed. Together, these observations suggest a model in which activation of apoptosis signaling pathways (which often involves decreasing diap1 levels) can lead (through unknown mechanisms) to hyperactivation of immune responses unless diap2 is also removed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Our observations, derived from in vivo studies of deletion mutants, stand in contrast to several recent reports, in which down-regulation of diap2 was brought about by using long dsRNA to induce RNAi-dependent degradation of diap2 mRNA. RNAi of diap2 was reported to sensitize S2 cells to several cell death activators (14) and to result in pupal lethality when expressed ubiquitously (16). Differences between these results and our own are most simply explained as a result of RNAi off-targeting of unknown transcripts required for cell health and organismal viability in the earlier works. The 500-bp dsRNAs used in the above-mentioned studies have the potential to be diced into many different 21-bp fragments for loading into the RISC complex and subsequent use as guide sequences. A number of recent reports have shown that off-targeting is a frequent occurrence (59-61) and can result in the production of toxic phenotypes (62). The basis for this has been made clear through experiments demonstrating that a single RISC-bound guide sequence can target many mRNA sequences, which need be only partially complementary to the guide strand (59, 63, 64).
Two groups, again using long dsRNA to target diap2 in S2 cells, have also recently reported that diap2 is required for the IMD response. One group concluded that diap2 acted upstream of or at the level of TAK1 (17). This conclusion was based, in part, on the observation that reduction of diap2 resulted in decreased expression of the dTAK1- and JNK-dependent early response gene puckered following immune challenge. In contrast, we found that expression of puckered, and a second early response gene, CG13482, were induced normally in animal mutants for diap2 (Fig. 4d, lower panel, and supplemental Fig. 2c), making this site of action unlikely (see also below). How can these different observations be reconciled? One possibility is that differences in the systems are important. We measured immune responses in the context of the entire organism. In doing this our observations reflect the summed action of (and potentially cross-talk between) multiple cell types and signaling pathways. In contrast, cell line-based studies are, by their nature, much more focused. Thus, it seems not unreasonable to postulate that while Tak-dependent activation of JNK and/or JNK target genes may be sensitive to levels of diap2 in one cell type, a different pathway may dominate in the intact organism. Alternatively, the effects of RNAi-mediated knockdown of diap2 on puckered expression in S2 cells may again reflect off-targeting of unknown transcripts by long dsRNA.
In contrast to Gesellchen et al. (17), Kleino et al. (16) proposed DIAP2 functions in the IMD pathway downstream of Relish cleavage because they saw no effect of diap2 RNAi on Relish cleavage in S2 cells. This difference from our observations, in which loss of diap2 resulted in a significant decrease in Relish cleavage, may indicate differences in systems. However, we think it more likely to reflect the difficulty in generating null mutant phenotypes for diap2 using RNAi. These authors did find that expression of pre-cleaved Relish was able to activate the immune response (and thus presumably able to translocate into the nucleus) in cells treated with diap2 dsRNA. Importantly, we observed a similar ability of precleaved Relish to activate transcription of AMP in the complete absence of diap2 in flies.
What do these observations tell us about the site(s) of DIAP2 action? Our observations demonstrate that DIAP2 plays a role in bringing about Relish cleavage, which is required for Relish nuclear translocation and function (29). Does DIAP2 function at other steps as well? The fact that loss of DIAP2 had a greater effect on nuclear translocation of endogenous Relish (Fig. 5b) and AMP production (Figs. 3 and 4) than it did on Relish cleavage (Fig. 5) is consistent with the hypothesis that it may. Expression of pre-cleaved Relish bypassed the requirement for DIAP2 in AMP production (Fig. 5c) (16), suggesting that DIAP2 is not absolutely required for Relish nuclear translocation or function. In addition, we have not observed nuclear translocation of DIAP2 in response to immune challenge.6 However, these observations do not exclude the possibility that the levels of pre-cleaved Relish generated in the fat body by heat shock are so high that Relish enters the nucleus through unphysiological pathways. For example, perhaps overexpression of pre-cleaved Relish titrates out an inhibitor of Relish nuclear translocation/function that is normally removed by DIAP2. It is also worth considering that pre-cleaved Relish expressed from a transgene may not be the same as processed Relish generated in response to IMD signaling. For example, endogenous Relish is phosphorylated by the IKK complex in response to IMD signaling (48, 52) and cleaved by Dredd. Processed endogenous Relish (but perhaps not transgene-expressed pre-cleaved Relish) may exist as a part of a complex and/or interact with factors that inhibit nuclear import unless DIAP2 promotes their removal. These hypotheses can best be explored through the identification of proteins that bind endogenous cleaved Relish and DIAP2. Finally, what is the significance of DIAP2-independent cleavage of Relish? Perhaps DIAP2 is not absolutely essential, mechanistically, for Relish cleavage (for example, if it acts as an inhibitor of an inhibitor of cleavage, some Relish may escape the inhibitor and thus be cleaved). Alternatively, other related E3s such as DIAP1 may be able to partially substitute for DIAP2 function (at least when DIAP2 is completely missing), an issue that requires further exploration. Regardless, our observation that loss of DIAP2 prevents a significant fraction of Relish cleavage and that DIAP2 is required for Relish-dependent AMP expression demonstrates that DIAP2 is a critical regulator of Relish function.
How does DIAP2 regulate Relish cleavage? It is unlikely that DIAP2 functions as an inhibitor of the caspase Dredd since DIAP2 does not inhibit other tested caspases, and Dredd, like DIAP2, is required for activation of the IMD pathway. However, it is worth noting that several mammalian IAPs that inhibit apoptosis when overexpressed, cIAP1 and cIAP2, bind active caspases even though they do not function as caspase inhibitors (65). Binding of DIAP2 to Drice and Strica has been reported (4). Binding between DIAP2 and Dredd has not been reported (it is not clear it has been searched for). Thus, it remains possible that interactions between Dredd and DIAP2, perhaps involving ubiquitination by DIAP2, positively regulate Dredd activation, activity, or substrate targeting. Roles for E3 ligase activity of DIAP2 in the IMD pathway activation (upstream and/or downstream of Relish cleavage) are suggested by several observations. Expression of a RING domain point mutant version of diap2, diap27.4C472Y, in the diap2E151 mutant background failed to rescue Relish cleavage and IMD signaling (Figs. 3, 4 and 5). In contrast, expression of this same mutant protein in a wild type background suppressed the IMD response, suggesting dominant negative activity (supplemental Fig. 2d). In mammals, ubiquitination exerts degradation-dependent effects on immunity through removal of IB and processing of NF-B. In contrast, activation of Tak1 and the IKK complex require ubiquitination in degradation-independent roles (66, 67). As discussed in the Introduction, Drosophila counterparts of Tak1 and the IKK complex are also essential for Relish processing. However, several observations suggest that diap2 does not act at the level of dTAK1. First, as noted above, expression of puckered and CG13482, immediate transcriptional targets of the Tak1-JNK signaling module (30), are not affected in diap2 mutant flies (Fig. 4d and supplemental Fig. 2c). Second, ectopic expression or down-regulation of diap2 in the fly eye fails to influence Tak1-dependent cell killing, which is JNK-dependent (20) (supplemental Fig. 3). Recent observations suggest that activation of the IKK complex requires the Drosophila ubiquitin-carrier proteins Bendless (Ubc13) and dUEV1a, along with a yet to be identified E3 ubiquitin ligase (68). Both Ubc13 (MAALTP) and dUEV1a (MANTSS) contain potential IBM motifs at their N termini. It will be interesting to determine whether DIAP2 binds one or both these proteins and participates in this process.
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Related, alternative models can also be considered. For example, caspase cleavage exposes IBM-like motifs in many proteins (71). If this occurs in response to Dredd activation in the insect fat body, it could serve as a form of negative feedback, creating proteins that bind DIAP2 BIRs, displacing bound proteins required (we speculate) for the immune response, and/or promoting changes in DIAP2 localization or stability. Target genes activated by the immune response might play a similar role (Fig. 7b). RNAi in S2 cells of several transcripts encoding proteins with N-terminal sequences similar to those of known IAP-binding proteins or peptides (supplemental Fig. 4) results in increased immune activation (72), suggesting these as candidate negative regulators of DIAP2. IBM domain proteins might also play positive roles in immune regulation. In the context of this possibility, it is interesting to note that binding of the mammalian IBM protein Smac/Diablo promotes XIAP stabilization rather than degradation (73, 74). The hypothesis that IBM proteins regulate DIAP2 activity, in its capacity as an essential component of the innate immune system, is speculative but is testable. Given that IBM domain proteins function as evolutionarily conserved regulators of cell death by displacing IAP-bound proteins and promoting IAP degradation and that DIAP2 is able to bind proteins with these same motifs, it would be surprising if similar regulatory mechanisms were not utilized in the immune system.
Addendum—A recent report also described the characterization of Drosophila IAP2 mutant flies (75). These authors placed DIAP2 function downstream or in parallel to Relish because diap2 mutant phenotypes were not rescued by ectopic expression of full-length Relish. However, since DIAP2 plays a significant role in promoting Relish cleavage (our study), a failure of full-length Relish (which requires cleavage) to rescue immune responsiveness in diap2 mutants is not surprising.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4.
1 To whom correspondence should be addressed: Division of Biology, MC 156-29, California Institute of Technology, 1200 East California Blvd., Pasadena, CA 91125. Tel.: 626-395-3399; Fax: 626-449-0756; E-mail: haybruce{at}caltech.edu.
2 The abbreviations used are: IAP, inhibitor of apoptosis; BIR, baculovirus IAP repeat; IBM, IAP-binding motif; E3, ubiquitin-protein isopeptide ligase; RT, reverse transcription; RNAi, RNA interference; dsRNA, double-stranded RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; IKK, IB kinase; GFP, green fluorescent protein.
3 C. Chen and B. Hay, manuscript in preparation.
4 J. R. Huh, unpublished observations.
5 C. Chen and B. Hay, manuscript in preparation.
6 J. R. Huh and B. A. Hay, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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