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dFADD, a Novel Death Domain-containing Adapter Protein for theDrosophila Caspase DREDD

2000; Elsevier BV; Volume: 275; Issue: 40 Linguagem: Inglês

10.1074/jbc.c000341200

1083-351X

Shimin Hu, Xiaolu Yang,

Bacillus and Francisella bacterial research

Apoptotic cell death occurs through activation of procaspases, the precursors of a group of aspartate-specific cysteine proteases known as caspases. Procaspase activation is mediated by death adapter proteins such as the mammalian proteins FADD and Apaf-1 and theCaenorhabditis elegans protein CED-4. These adapters bind to procaspases and facilitate oligomerization and subsequent auto-proteolytic processing of the zymogens. Here we report cloning and characterization of dFADD, a FADD homologue inDrosophila. dFADD contains a death domain that is highly homologous to the FADD death domain, and it also shares a novel domain with a Drosophila caspase DREDD, which we call death-inducing domain. dFADD binds to DREDD through the death-inducing domain and enhances the cell death activity and proteolytic processing of DREDD. dFADD and DREDD are stabilized by their interaction. The structural and functional similarities between dFADD and FADD suggest the existence of a FADD-like apoptosis pathway in Drosophila. Apoptotic cell death occurs through activation of procaspases, the precursors of a group of aspartate-specific cysteine proteases known as caspases. Procaspase activation is mediated by death adapter proteins such as the mammalian proteins FADD and Apaf-1 and theCaenorhabditis elegans protein CED-4. These adapters bind to procaspases and facilitate oligomerization and subsequent auto-proteolytic processing of the zymogens. Here we report cloning and characterization of dFADD, a FADD homologue inDrosophila. dFADD contains a death domain that is highly homologous to the FADD death domain, and it also shares a novel domain with a Drosophila caspase DREDD, which we call death-inducing domain. dFADD binds to DREDD through the death-inducing domain and enhances the cell death activity and proteolytic processing of DREDD. dFADD and DREDD are stabilized by their interaction. The structural and functional similarities between dFADD and FADD suggest the existence of a FADD-like apoptosis pathway in Drosophila. caspase recruitment domain cell death abnormal death effector domain death-inducing domain death domain death-related ced-3/Nedd2-like protein DrosophilaNedd-2-like caspase Fas-associated death domain protein Aequorea victoria green fluorescence protein the hemagglutinin epitope tag monoclonal antibody Apoptosis is a physiological process of cell auto-destruction that eliminates unwanted or severely damaged cells in multicellular organisms. It plays critical roles in development, maintenance of homeostasis, and host defense (1Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2436) Google Scholar, 2Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2415) Google Scholar). Apoptosis is executed by a proteolytic system involving caspases. Caspases are produced as latent precursors (procaspases) and during apoptosis are activated sequentially in cascades through proteolytic processing (3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar, 4Cryns V. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1160) Google Scholar, 5Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1943) Google Scholar). A key step in determining cell life or death is the activation of the apical procaspase (initiator caspase) in such a caspase cascade. This activation is mediated by death adapter proteins including the mammalian apoptotic proteins FADD (6Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2165) Google Scholar) and Apaf-1 (7Zou H. Henzel W.I. Liu X. Luschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar) and theCaenorhabditis elegans protein CED-4 (8Metzstein M.M. Stanfield G.M. Horvitz H.R. Trends Genet. 1998; 14: 410-416Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). An activated apical caspase processes downstream procaspases (effector caspases), which then cleave key cellular substrates to dismantle cells. An evolutionarily conserved apoptosis pathway is initiated by cell-intrinsic developmental cues or cytotoxic reagents and is found in the nematode C. elegans and mammals (9Horvitz H.R. Shaham S. Hengartner M.O. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 377-385Crossref PubMed Scopus (170) Google Scholar, 10Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Upon apoptosis activation, the death adapter proteins CED-4 and Apaf-1 form homo-oligomers that subsequently aggregate the CED-3 caspase and procaspase-9, respectively (11Yang X. Chang H.Y. Baltimore D. Science. 1998; 281: 1355-1357Crossref PubMed Scopus (237) Google Scholar, 12Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar, 13Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar), through homotypic interactions mediated by the caspase recruitment domain (CARD)1 (14Hofmann K. Bucher P. Tschopp J. Trends Biochem. Sci. 1997; 22: 155-156Abstract Full Text PDF PubMed Scopus (450) Google Scholar). Mammals have also evolved an extrinsic or instructive apoptosis pathway, mediated by small death adapters such as FADD, that allows a cell to instruct another to undergo self-destruction (15Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar, 16Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5169) Google Scholar). This pathway is engaged by a unique group of death receptors in the tumor necrosis factor receptor superfamily (e.g. TNFR1 and Fas) and plays important roles in the regulation of immune responses and the maintenance of homeostasis. Upon binding to their trimeric ligands, these receptors recruit the adapter protein FADD via death domain (DD)-DD interaction (6Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2165) Google Scholar). FADD then binds to procaspase-8 through another homotypic interaction involving death effector domain (DED), a motif present in the N-terminal region of both proteins (17Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar, 18Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar). Recruitment of procaspase-8 to the ligand-aggregated death receptors leads to its oligomerization and subsequent activation (19Yang X. Chang H.Y. Baltimore D. Mol. Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 20Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar, 21Martin D.A. Siegel R.M. Zheng L. Lenardo M.J. J. Biol. Chem. 1998; 273: 4345-4349Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). An Apaf-1/CED-4 homologue, Drosophila Apaf-1-related killer (DARK)/homolog of Apaf-1 and CED-4 (HAC)-1/Dapaf-1, was recently identified in the model genetic organism Drosophila(22Zhou L. Song Z. Tittel J. Steller H. Mol. Cell. 1999; 4: 745-755Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 23Rodriguez A. Oliver H. Zou H. Chen P. Wang X. Abrams J.M. Nat. Cell Biol. 1999; 1: 272-279Crossref PubMed Scopus (293) Google Scholar, 24Kanuka H. Sawamoto K. Inohara N. Matsuno K. Okano H. Miura M. Mol. Cell. 1999; 4: 757-769Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). However, it remains unclear whether a FADD-like pathway also exists in Drosophila. Here we describe the identification and characterization of a Drosophila FADD homologue, dFADD, and present evidence that dFADD is an adapter that activates theDrosophila apical caspase DREDD. Human embryonic kidney 293 cells, human cervical carcinoma HeLa cells, and murine fibroblast 3T3 cells were cultured in complete Dulbecco's modified Eagle's medium, and human breast carcinoma MCF7 cells were maintain in RPMI 1640. Expression constructs were based on pRK5 (a gift from Dr. D. Goeddel), pcDNA3 (Invitrogen), pEGFP-C1 (CLONTECH; for Aequorea victoria green fluorescence protein (GFP) fusion constructs), and pCaSpeR-hs (a gift of Dr. M. Fortini; for SL2 cell expression constructs). FLAG and HA-tags were placed at the N termini of the fusion proteins. Anti-FLAG and anti-HA polyclonal antibodies (Santa Cruz) and anti-FLAG mAb M2 conjugated on agarose beads (Sigma) were purchased from the indicated sources. A Drosophila expressed sequence tag clone (GenBankTM accession numberAI294992) was found to contain an open reading frame with significant sequence homology to mammalian death domain-containing proteins. The sequence of the open reading frame was confirmed on both strands with an automated sequencer (Applied Biosystems). A Northern blot membrane containing mRNA from different developmental stages of Drosophilaembryos was hybridized with a 32P-labeled cDNA probe corresponding to the DNA sequence of the prodomain (residue 1–141) of dFADD or a probe specific for ribosomal RNA RP49 as a loading control. Transient transfection of 293 cells was performed as described previously (25Hu S. Vincenz C. Ni J. Gentz R. Dixit V.M. J. Biol. Chem. 1997; 272: 17255-17257Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Unless indicated otherwise, cells were transfected with 1–10 μg of indicated plasmids to yield equivalent protein expression levels. 20–24 h after transfection, cell lysates were prepared and immunoprecipitated with anti-FLAG mAb M2 beads. The precipitates were resolved by SDS polyacrylamide gel electrophoresis and analyzed by immunoblotting with polyclonal anti-FLAG and anti-HA antibodies. HeLa, 3T3, and MCF7 cells were transiently transfected with each of the test plasmids plus the reporter plasmid pCMV-lacZ (1 and 0.2 μg, respectively, unless indicated otherwise). 22–24 h after transfection, cells were fixed in 0.5% glutaraldehyde and stained with X-Gal. The percentage of apoptotic cells was determined by the number of membrane-blebbing cells divided by the total number of blue cells. Data presented were obtained from representative experiments performed in duplicates, and the mean and standard deviation were calculated. Drosophila Schneider L2 cells were transfected with dFADD and DREDD expression plasmids together with a GFP expression plasmid. 20 h after transfection, cells were heat shocked for 20 min three times at 37 °C. 24 h after the heat shock, cells were examined under a fluorescence microscope to determine the number and morphology of GFP-positive cells. HeLa cells were transfected with the GFP fusion plasmids. 20–24 h later, the cells were examined under a fluorescence microscope to determine the distribution and the relative intensity of fluorescence. In a search for Drosophila death domain-containing proteins in the expressed sequence tag data base, we identified a full-length cDNA predicted to encode a protein with a death domain at its C terminus (Fig. 1). This protein has an overall structure similar to that of FADD, and its death domain is highly homologous to the FADD death domain (28% identity and 49% similarity) (Fig. 1 B). We hence designated this first Drosophila death domain-containing protein dFADD.Figure 1Sequence and expression of dFADD. A, deduced amino acid sequence of dFADD. B, sequence alignment of the dFADD DD domain with the DD domains present in the mammalian proteins FADD (6Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2165) Google Scholar), RIP (28Stanger B.Z. Leder O. Lee T.-O. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (869) Google Scholar), RAIDD (29Duan H. Dixit V.M. Nature. 1997; 385: 86-89Crossref PubMed Scopus (469) Google Scholar), p84 (30Durfee T. Mancini M.A. Jones D. Elledge S.J. Lee W.H. J. Cell Biol. 1994; 127: 609-622Crossref PubMed Scopus (144) Google Scholar), and TRADD (31Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1749) Google Scholar). C, comparison of amino acid sequences of the death-inducing domains in dFADD and DREDD. D, expression of dFADD during Drosophila development. A Drosophilaembryonic mRNA blot was hybridized with radioisotope-labeled dFADD or control RP49 probe as described under "Materials and Methods."View Large Image Figure ViewerDownload Hi-res image Download (PPT) No homology was found between the N-terminal region of dFADD and any other proteins in the data base using the BLAST and SMART programs. However, a careful comparison of the dFADD sequence with the sequence of DREDD, a Drosophila apical caspase, revealed significant homology between the dFADD N-terminal region and a region in the DREDD prodomain (19% identity and 39% similarity) (Fig. 1 C). Although DREDD was thought to contain two DED domains (26Chen P. Rodriguez A. Erskine R. Thach T. Abrams J.M. Dev. Biol. 1998; 201: 202-216Crossref PubMed Scopus (183) Google Scholar), we found no homology between DREDD prodomain and any DED-containing proteins using BLAST or SMART. The conserved domain in dFADD and DREDD is distinct from DED and CARD (two domains involved in death adapter-procaspase interaction), but its function is similar to those of DED and CARD (see below). We therefore named this novel domain DID (death-inducing domain). Analysis of the dFADD genomic sequence revealed that it contains a single exon on chromosome 3. To examine the mRNA expression of dFADD, we hybridized mRNAs sampled from different stages of Drosophila embryonic development with a dFADD cDNA probe. Only the 3- to 12-h embryos contained a single 1.7-kilobase dFADD transcript (Fig. 1 D). Thus, the expression of dFADD is tightly regulated during Drosophiladevelopment. Because death adapter proteins associate with caspases through homotypic DED-DED or CARD-CARD interaction, we investigated whether dFADD interacted with DREDD through their homologous DIDs. Co-immunoprecipitation assays confirmed that dFADD specifically associated with DREDD but not with DRONC, a CARD domain-containing Drosophila apical caspase (27Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (240) Google Scholar) (Fig. 2 B). The specificity of the dFADD-DREDD interaction was further underlined by the observation that dFADD did not interact with various mammalian caspases except for its weak interaction with caspase-10 (Fig.2 C). The interaction domains in dFADD and DREDD were mapped using a panel of deletion mutants (Fig. 2 A). The N-terminal region of dFADD containing the DID domain was both necessary and sufficient for the interaction with DREDD (Fig. 2 D). dFADD interacted strongly with the DREDD prodomain but weakly with the caspase domain (Fig. 2 E). Further mutagenesis analysis of the DREDD prodomain region revealed that the DID domain was required for the interaction with dFADD (Fig. 2 F). Therefore, the DID domains mediate the interaction between DREDD and dFADD. To examine the effect of dFADD on DREDD-induced apoptosis, we transfectedDrosophila SL2 cells with dFADD and DREDD expression plasmids either alone or together. No apoptosis was observed (data not shown), consistent with previous results that ectopic expression of DREDD alone in SL2 cells did not lead to activation of the caspase (26Chen P. Rodriguez A. Erskine R. Thach T. Abrams J.M. Dev. Biol. 1998; 201: 202-216Crossref PubMed Scopus (183) Google Scholar). However, expression of DREDD in several mammalian cell lines caused significant apoptosis (Fig. 3,A, B, and C). We examined the effect of dFADD on DREDD-mediated apoptosis in these cells and found that although dFADD did not induce apoptosis, it potently enhanced DREDD-mediated apoptosis in a dose-dependent manner (Fig.3, A, B, and C). The dFADD DID was both necessary and sufficient for this enhancement. dFADD did not enhance DRONC-induced apoptosis, consistent with the lack of interaction between these two proteins (Fig. 3 D). Initiator caspases are activated by adapter-mediated oligomerization and subsequent auto-processing. Because dFADD associated with DREDD and enhanced DREDD-induced apoptosis, we investigated whether dFADD promoted DREDD processing. Overexpression of DREDD in human 293 cells led to zymogen processing (Fig. 4 A). DREDD associated with itself at high expression levels either directly or indirectly via an endogenous adapter protein (Fig. 4 B), and such self-association could facilitate its auto-cleavage. The DREDD processing was not inhibited by p35 and crmA, two active site-specific caspase inhibitors that can inhibit most caspases (Fig. 4 Aand data not shown), and DREDD may possess unique substrate specificity. In the presence of dFADD, DREDD processing went to completion (Fig. 4 A), whereas DRONC was not processed upon overexpression either in the presence or absence of dFADD (Fig.4 A). It appears that the enhancement of DREDD processing by dFADD is mediated by direct protein-protein interaction. DREDD was previously reported to associate with theDrosophila Apaf-1 homologue DARK (23Rodriguez A. Oliver H. Zou H. Chen P. Wang X. Abrams J.M. Nat. Cell Biol. 1999; 1: 272-279Crossref PubMed Scopus (293) Google Scholar). OtherDrosophila caspases such as DRONC were also reported to be targets for DARK (22Zhou L. Song Z. Tittel J. Steller H. Mol. Cell. 1999; 4: 745-755Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 24Kanuka H. Sawamoto K. Inohara N. Matsuno K. Okano H. Miura M. Mol. Cell. 1999; 4: 757-769Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). We compared the dFADD-DREDD and DARK-DREDD interactions using co-immunoprecipitation assay and found that the former was much stronger than the latter. Unlike the dFADD-DREDD interaction that was mainly mediated by the DREDD prodomain (Fig.2 E), both the DREDD prodomain and the caspase domain interacted weakly with DARK (Fig. 4 D). The strong dFADD-DREDD interaction suggests that DREDD may mainly serve as an apical caspase for dFADD. To examine the cellular localization of dFADD and DREDD, we fused them to GFP. When expressed in HeLa cells, both GFP-dFADD and GFP-DREDD proteins were localized in the cytoplasm (Fig.5 A, a andc). This localization of dFADD was mediated by its N-terminal region (Fig. 5 A, e and f). dFADD and DREDD appeared to stabilize each other, because expression of DREDD significantly increased fluorescence intensity in the GFP-dFADD-transfected cells (Fig. 5 A, b versus a), and similarly, expression of dFADD enhanced fluorescence intensity in the GFP-DREDD-transfected cells (Fig. 5 A, d versus c). We also compared the expression levels of dFADD in 293 cells in the presence and absence of DREDD. Co-transfection of dFADD with DREDD led to a higher level of dFADD in both the soluble and insoluble fractions of the cell extracts compared with transfection of FADD alone (Fig.5 B). In contrast, DRONC did not enhance dFADD protein expression (Fig. 5 B). The stabilization of dFADD by DREDD was mediated by the DREDD prodomain (Fig. 5 C and data not shown). Reciprocally, dFADD stabilized DREDD but not DRONC in 293 cells (data not shown). The mutual stabilization of dFADD and DREDD suggests that the expression of these two proteins may be co-regulated in vivo. In summary, we describe here the molecular cloning and partial characterization of the first Drosophila death domain-containing protein dFADD. dFADD contains a bipartite structure highly homologous to that of the mammalian adapter FADD. It functions similarly to FADD and physically interacts with and activates theDrosophila caspase DREDD. The interaction between dFADD and DREDD stabilizes both proteins. We conclude that Drosophilaalso contains a FADD-like apoptotic pathway. To date, no death receptors have been identified in Drosophila, and despite the high homology between the death domains of FADD and dFADD, we did not detect interaction between dFADD and any of the mammalian death receptors (data not shown). Identification of components upstream of dFADD should help determine the regulation and function of this evolutionarily conserved apoptosis pathway. We thank Lara Gardner for technical assistance, Judith Leatherman and Dr. Thomas Jongens for providingDrosophila Northern blots, and Drs. J. Abram, S. Kumar, H. Steller, and E. Alnemri for reagents. We also thank Yihong Ye and Drs. Mark Fortini and Morris Birnbaum for advice.