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A Novel Activation Mechanism of Caspase-activated DNase fromDrosophila melanogaster*

Open AccessPublished:April 28, 2000DOI:https://doi.org/10.1074/jbc.275.17.12978
      Caspase-activated DNase (CAD) is an enzyme that cleaves chromosomal DNA in apoptotic cells. Here, we identified a DNase in Drosophila Schneider cells that can be activated by caspase 3, and purified it as a complex of two subunits (p32 and p20). Using primers based on the amino acid sequence of the purified proteins, a cDNA coding for Drosophila CAD (dCAD) was cloned. The polypeptide encoded by the cDNA contained 450 amino acids with a calculated M r of 52,057, and showed significant homology with human and mouse CAD (22% identity). Mammalian CADs carry a nuclear localization signal at the C terminus. In contrast, dCAD lacked the corresponding sequence, and the purified dCAD did not cause DNA fragmentation in nuclei in a cell-free system. When dCAD was co-expressed in COS cells with Drosophilainhibitor of CAD (dICAD), a 52-kDa dCAD was produced as a heterotetrameric complex with dICAD. When the complex was treated with human caspase 3 or Drosophila caspase (drICE), the dICAD was cleaved, and released from dCAD. In addition, dCAD was also cleaved by these caspases, and behaved as a (p32)2(p20)2 complex in gel filtration. When aDrosophila neuronal cell line was induced to apoptosis by treatment with a kinase inhibitor, both dCAD and dICAD were cleaved. These results indicated that unlike mammalian CAD,Drosophila CAD must be cleaved by caspases to be activated.
      CAD
      caspase-activated DNase
      ICAD
      inhibitor of CAD
      dCAD
      Drosophila CAD
      dICAD
      Drosophila ICAD
      mCAD
      mouse CAD
      mICAD
      mouse ICAD
      GST
      glutathioneS-transferase
      PCR
      polymerase chain reaction
      H-7
      1-(5-isoquinolinesulfonyl)-2-methylpiperazine
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      Apoptosis, or programmed cell death, is a cell-autonomous process that removes surplus or harmful cells during development or aging in animals (
      • Vaux D.L.
      • Korsmeyer S.J.
      ,
      • Jacobson M.D.
      • Weil M.
      • Raff M.C.
      ). This process is triggered by the transcriptional activation of genes involved in apoptosis, cytotoxic cells, anti-cancer drugs, and death factors (
      • Green D.R.
      • Reed J.C.
      ,
      • Raff M.
      , ). Studies in Caenorhabditis elegans have identified several gene products that regulate programmed cell death (
      • Ellis R.E.
      • Yuan J.
      • Horvitz H.R.
      ). Ced-3 (cell-death abnormal) andCed-4 are required for cell death (
      • Ellis H.M.
      • Horvitz H.
      ). Ced-9blocks the action of Ced-4 and Ced-3 to inhibit the process (
      • Hengartner M.O.
      • Ellis R.E.
      • Horvitz H.R.
      ), while Egl-1 (egg laying defective) inhibits the anti-apoptotic function of Ced-9, thus promoting the cell death process (
      • Conradt B.
      • Horvitz H.R.
      ). Mammalian homologues for Ced-3 and Ced-4 have been identified as caspases (cysteine proteases) (
      • Thornberry N.A.
      • Lazebnik Y.
      ) and Apaf-1 (
      • Zou H.
      • Henzel W.J.
      • Liu X.
      • Lutschg A.
      • Wang X.
      ), respectively. The anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL are mammalian homologues of Ced-9, while pro-apoptotic Bcl-2 family members such as Bid and Bad, which carry only the BH3 domain, are homologues of Egl-1 (
      • Adams J.M.
      • Cory S.
      ). These observations indicate that the central molecular components required for apoptosis are well conserved among different species.
      At least 13 caspase family members have been identified in mammals, and these can be divided into three groups (
      • Thornberry N.A.
      • Lazebnik Y.
      ,
      • Nicholson D.W.
      • Thornberry N.A.
      ). Caspases in the first group are mainly involved in the production of cytokines, whereas caspases in the second and third groups play a direct role in apoptosis. Among the caspases involved in apoptosis, caspases 8, 9, and 10 carry a long prodomain at the N terminus, and are activated by oligomerization at the plasma membrane or by apoptosomes at mitochondria, and thus function as initiator elements. Caspases 3, 6, and 7 carry a short prodomain, are activated by the initiator caspases, and are responsible for cleaving at least 60 death-related substrates (
      • Stroh C.
      • Schulze-Osthoff K.
      ). The cleavage of these substrates is assumed to bring about the morphological changes of cells and nuclei that characterize apoptosis.
      Developmental programmed cell death plays an important role in embryogenesis, morphogenesis, and oogenesis in insects. InDrosophila, it begins at stage 11–12 of embryogenesis and becomes widespread in many tissues (
      • Abrams J.M.
      • White K.
      • Fessler L.I.
      • Steller H.
      ,
      • Jiang C.
      • Baehrecke E.H.
      • Thummel C.S.
      ). Deleterious stimuli such as γ-irradiation also induce apoptosis in Drosophilaembryos (
      • Nordstrom W.
      • Chen P.
      • Steller H.
      • Abrams J.
      ). Apoptosis in Drosophila is triggered by the expression of a set of genes: reaper, grim, andhead involution defective (hid), which are tightly linked at the H99 region of the third chromosome (
      • Bergmann A.
      • Agapite J.
      • Steller H.
      ). As found in C. elegans and mammals, the apoptotic process inDrosophila is mediated by caspase-like proteases (
      • Fraser A.G.
      • McCarthy N.J.
      • Evan G.I.
      ,
      • Song Z.
      • McCall K.
      • Steller H.
      ,
      • Chen P.
      • Rodriguez A.
      • Erskine R.
      • Thach T.
      • Abrams J.M.
      ,
      • Dorstyn L.
      • Colussi P.A.
      • Quinn L.M.
      • Richardson H.
      • Kumar S.
      ) and a Ced-4/Apaf-1-like molecule (Dark) (
      • Rodriguez A.
      • Oliver H.
      • Zou H.
      • Chen P.
      • Wang X.
      • Abrams J.
      ). Five caspases have been identified in Drosophila so far. Among them, DCP-1, drICE, and DECAY carry a short prodomain, and share properties with Ced-3 and human caspase 3 (
      • Fraser A.G.
      • McCarthy N.J.
      • Evan G.I.
      ,
      • Song Z.
      • McCall K.
      • Steller H.
      ,
      • Dorstyn L.
      • Read S.H.
      • Quinn L.M.
      • Richardson H.
      • Kumar S.
      ), and Dredd/DCP-2 and DRONC have a long prodomain (
      • Chen P.
      • Rodriguez A.
      • Erskine R.
      • Thach T.
      • Abrams J.M.
      ,
      • Dorstyn L.
      • Colussi P.A.
      • Quinn L.M.
      • Richardson H.
      • Kumar S.
      ) and may work as initiators.
      One of the biochemical hallmarks of apoptosis is the degradation of chromosomal DNA (
      • Wyllie A.H.
      ,
      • Earnshaw W.C.
      ), which is observed in C. elegans,Drosophila, and mammals. We and others have identified a DNase in humans and mice (caspase-activated DNase, CAD) that can be activated by human caspase 3 (
      • Enari M.
      • Sakahira H.
      • Yokoyama H.
      • Okawa K.
      • Iwamatsu A.
      • Nagata S.
      ,
      • Halenbeck R.
      • MacDonald H.
      • Roulston A.
      • Chen T.T.
      • Conroy L.
      • Williams L.T.
      ,
      • Liu X.
      • Li P.
      • Widlak P.
      • Zou H.
      • Luo X.
      • Garrard W.T.
      • Wang X.
      ). CAD1 is complexed with its inhibitor protein (inhibitor of CAD, ICAD, also called DNA fragmentation factor-45) in growing cells (
      • Enari M.
      • Sakahira H.
      • Yokoyama H.
      • Okawa K.
      • Iwamatsu A.
      • Nagata S.
      ,
      • Liu X.
      • Zou H.
      • Slaughter C.
      • Wang X.
      ). When apoptotic stimuli activate caspase 3, it then cleaves ICAD at two positions, causing ICAD to lose its ability to bind CAD (
      • Sakahira H.
      • Enari M.
      • Nagata S.
      ). CAD, thus released from ICAD, cleaves the chromosomal DNA in the nucleus. To examine whether this mechanism of apoptotic DNA fragmentation is conserved among different species, and to study its role in developmental programmed cell death, we sought to identify CAD and ICAD inDrosophila cells. In this report, we identified aDrosophila CAD (dCAD) in Drosophila cells. The purification and molecular cloning of dCAD revealed a novel activation mechanism for dCAD.

      DISCUSSION

      The signal transduction machinery for apoptosis appears to be conserved among metazoans. Drosophila melanogaster is well studied model system in which massive cell death occurs during embryogenesis and metamorphosis, and this cell death process is accompanied by condensation and fragmentation of nuclei, as well as degradation of chromosomal DNA (
      • Abrams J.M.
      • White K.
      • Fessler L.I.
      • Steller H.
      ,
      • Bergmann A.
      • Agapite J.
      • Steller H.
      ). In this report, we identified a DNase in Drosophila that can be activated by human caspase 3, as well as Drosophila caspase drICE. Accordingly, dCAD and its inhibitor dICAD were cleaved during the apoptotic process in aDrosophila neuronal cells. Among five caspases identified inDrosophila, three of them have properties similar to human caspase 3 (
      • Fraser A.G.
      • McCarthy N.J.
      • Evan G.I.
      ,
      • Song Z.
      • McCall K.
      • Steller H.
      ,
      • Dorstyn L.
      • Read S.H.
      • Quinn L.M.
      • Richardson H.
      • Kumar S.
      ). It is therefore likely that the dCAD/dICAD system identified in this report is responsible for the DNA degradation during the apoptotic process of Drosophila cells.
      In mice and humans, the proform of CAD (CAD·ICAD complex) is activated by the cleavage of ICAD by caspase 3 (
      • Enari M.
      • Sakahira H.
      • Yokoyama H.
      • Okawa K.
      • Iwamatsu A.
      • Nagata S.
      ,
      • Sakahira H.
      • Enari M.
      • Nagata S.
      ). On the other hand, purification and molecular cloning of dCAD indicated that the active dCAD is a heterotetrameric complex consisting of two subunits (p32 and p20) that are generated by proteolytic cleavage from a single polypeptide of 52 kDa. This structure differs from that of mammalian CAD, which consists of a single polypeptide of 40 kDa. However, this structure of dCAD is similar to that of human caspases that are heterotetrameric complexes consisting of two subunits generated from a precursor protein (
      • Thornberry N.A.
      • Bull H.G.
      • Calaycay J.R.
      • Chapman K.T.
      • Howard A.D.
      • Kostura M.J.
      • Miller D.K.
      • Molineaux S.M.
      • Weidner J.R.
      • Aunins J.
      • Elliston K.O.
      • Ayala J.M.
      • Casano F.J.
      • Chin J.
      • Ding G.J.-F.
      • Egger L.A.
      • Gaffney E.P.
      • Limjuco G.
      • Palyha O.C.
      • Raju S.M.
      • Rolando A.M.
      • Salley J.P.
      • Yamin T.-T.
      • Lee T.D.
      • Shively J.E.
      • MacCross M.
      • Mumford R.A.
      • Schmidt J.A.
      • Tocci M.J.
      ,
      • Rotonda J.
      • Nicholson D.W.
      • Fazil K.M.
      • Gallant M.
      • Gareau Y.
      • Labelle M.
      • Peterson E.P.
      • Rasper D.M.
      • Ruel R.
      • Vaillancourt J.P.
      • Thornberry N.A.
      • Becker J.W.
      ,
      • Walker N.P.
      • Talanian R.V.
      • Brady K.D.
      • Dang L.C.
      • Bump N.J.
      • Ferenz C.R.
      • Franklin S.
      • Ghayur T.
      • Hackett M.C.
      • Hammill L.D.
      • Herzog L.
      • Hugunin M.
      • Houy W.
      • Mankovich J.A.
      • McGuiness L.
      • Orlewicz E.
      • Paskind M.
      • Pratt C.A.
      • Reis P.
      • Summani A.
      • Terranova M.
      • Welch J.P.
      • Xiong L.
      • Möller A.
      • Tracey D.E.
      • Kamen R.
      • Wong W.W.
      ). Recently, human caspase 3 was shown to contain a peptide called a “safety catch” near the large subunit/small subunit junction.
      D. Nicholson, personal communication.
      The removal of this peptide resulted in a proenzyme that is enzymatically active. The dCAD carries a stretch of 35 amino acids (amino acids 255–289) where the caspase 3-cleavage site is located (Fig. 3). This region is not present in mouse and human CADs, suggesting that this stretch of amino acids keeps dCAD in an inactive form. Cleavage of dCAD at Asp-278 may cause its conformational change to an active form. In this regard, it will be interesting to examine whether removal of the 35-amino acid stretch from dCAD generates an active form of dCAD. In any case, the requirement of cleavage by a caspase for dCAD to be activated indicates that the activation of Drosophila CAD is regulated in more elaborate manner than that of human and mouse CADs. Although both dCAD and DREP-1/dICAD are cleaved by drICE, different Drosophilacaspases may be responsible for their cleavage of dCAD and DREP-1/dICAD in Drosophila cells to allow more fine-tuned regulation of this potentially damaging DNase activity.
      The overall identity between dCAD and hCAD, and between dCAD and mCAD is about 22%, which agrees with the similarities found in caspases and Apaf-1 between Drosophila and human species (
      • Fraser A.G.
      • McCarthy N.J.
      • Evan G.I.
      ,
      • Song Z.
      • McCall K.
      • Steller H.
      ,
      • Chen P.
      • Rodriguez A.
      • Erskine R.
      • Thach T.
      • Abrams J.M.
      ,
      • Dorstyn L.
      • Colussi P.A.
      • Quinn L.M.
      • Richardson H.
      • Kumar S.
      ). CAD carries a domain (CAD or CIDE domain) in the N terminus that is also found in ICAD and other proteins of unknown function (
      • Mukae N.
      • Enari M.
      • Sakahira H.
      • Fukuda Y.
      • Inazawa J.
      • Toh H.
      • Nagata S.
      ,
      • Inohara N.
      • Koseki T.
      • Chen S.
      • Wu X.
      • Nunez G.
      ). The homology of the amino acid sequences between Drosophila and mammalian CADs in this domain is rather limited. In contrast, the C-terminal portion (amino acids 308–415), which contains five cysteines and four histidines, was highly conserved among dCAD, mCAD, and hCAD (Fig. 3 B). We recently found that most cysteine residues in mCAD are in the reduced form (
      • Sakahira H.
      • Iwamatsu A.
      • Nagata S.
      ). However, treatment of mCAD with a reagent that modifies free thiol groups did not inactivate mCAD, suggesting the cysteine residues are not in the catalytic active site of the enzyme. In some DNases such as DNase I and colicin, histidine residues form an active site (
      • Ito K.
      • Akiyama D.
      • Minamiura N.
      ,
      • Ko T.P.
      • Liao C.C.
      • Ku W.Y.
      • Chak K.F.
      • Yuan H.S.
      ). Although the sequence in CAD surrounding these histidine residues is not homologous to those found in DNase I or colicin, it is likely that the catalytic active site of CAD is composed of some of the histidine residues conserved among dCAD, mCAD, and hCAD.
      Biochemical fractionation of the cell lysates showed most of the proform of CAD is present in the cytoplasm of growing mouse (
      • Enari M.
      • Sakahira H.
      • Yokoyama H.
      • Okawa K.
      • Iwamatsu A.
      • Nagata S.
      ) andDrosophila cells (this report), whereas another group detected CAD/DFF-40 in nuclei by histochemical staining of the growing cells (
      • Liu X.
      • Li P.
      • Widlak P.
      • Zou H.
      • Luo X.
      • Garrard W.T.
      • Wang X.
      ). Irrespective of whether it is in the cytoplasm or nuclei of the growing cells, activated CAD must function in the nuclei of apoptotic cells to degrade the chromosomal DNA. Human and mouse CADs carry a stretch of basic amino acids at the C terminus, which is indispensable for the CAD-induced DNA fragmentation in vitroand in vivo.
      M. Enari, H. Sakahira, and S. Nagata, unpublished observation.
      Drosophila CAD lacked the corresponding sequence at the C terminus. Since dCAD did not cause DNA fragmentation in nuclei in vitro, it is unlikely that it contains a nuclear localization signal in some other part of the molecule. In the mouse CAD chromosomal gene, the nuclear localization signal is encoded by an independent exon (exon 7) (
      • Kawane K.
      • Fukuyama H.
      • Adachi M.
      • Sakahira H.
      • Copeland N.G.
      • Gilbert D.J.
      • Jenkins N.A.
      • Nagata S.
      ). Sequencing analysis of the isolated dCAD chromosomal gene and the 3′-rapid amplification of cDNA ends product with mRNA from adult Drosophila could not detect an exon coding for the putative nuclear localization signal.
      H. Yokoyama and S. Nagata, unpublished results.
      These results suggest that dCAD does not carry a nuclear localization signal. In dying Drosophila cells, the nuclei become permeable, which is followed by DNA degradation detected by the TUNEL procedure (
      • Jiang C.
      • Baehrecke E.H.
      • Thummel C.S.
      ,
      • McCall K.
      • Steller H.
      ). There are two possible mechanisms by which dCAD may cause DNA degradation. In one, dCAD that is activated in the cytoplasm enters the nucleus by passive transport through the permealized nuclear membrane. The other possibility is that the dCAD·dICAD complex enters the nucleus using an as yet unidentified nuclear localization signal in dICAD or dCAD, and remain in the nucleus. In this case, the activated caspase would have to enter the nucleus to activate dCAD.
      The apoptotic degradation of DNA in Drosophila cells can be detected by the TUNEL procedure (
      • McCall K.
      • Steller H.
      ), or by flow cytotometry after staining the cells with propidium iodine (
      • Nordstrom W.
      • Chen P.
      • Steller H.
      • Abrams J.
      ). However, no obvious DNA fragmentation was reported in Drosophila cells except for a neuronal cell line (
      • Nagano M.
      • Suzuki H.
      • Ui-Tei K.
      • Sato S.
      • Miyake T.
      • Miyata Y.
      ). Whether this is due to the low expression of dCAD in Drosophila cells remains to be studied. In any case, the identification of a caspase-activated DNase inDrosophila will be useful for studying the biochemical properties of the CAD/ICAD system as well as the physiological role of DNA fragmentation during developmental programmed cell death.

      Acknowledgments

      We thank Dr. T. Uemura (Kyoto University) for the Drosophila embryo cDNA library, and Dr. S. Kumar (Hanson Center for Cancer Research) for the drICE expression vector. We are grateful to Drs. Y. Miyata and K. Ui-Tei (Nippon Medical University) for supplying the BG2-c2 cell line and for instruction in culturing the cell line. We also thank Dr. M. Enari for help in the initial stage of this work.

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