The Drosophila Caspase DRONC Cleaves following Glutamate or Aspartate and Is Regulated by DIAP1, HID, and GRIM

Abstract

The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death. All caspases studied to date cleave substrates COOH-terminal to an aspartate. Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate. DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate. DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate. Expression of a dominant negative form of DRONC blocks cell death induced by theDrosophila cell death activators reaper,hid, and grim, and DRONC overexpression in flies promotes cell death. Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM. These observations suggest that DRONC acts to promote cell death. However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35. We discuss possible models for DRONC function as a cell death inhibitor. The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death. All caspases studied to date cleave substrates COOH-terminal to an aspartate. Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate. DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate. DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate. Expression of a dominant negative form of DRONC blocks cell death induced by theDrosophila cell death activators reaper,hid, and grim, and DRONC overexpression in flies promotes cell death. Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM. These observations suggest that DRONC acts to promote cell death. However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35. We discuss possible models for DRONC function as a cell death inhibitor. polyacrylamide gel electrophoresis poly(ADP-ribosyl)transferase Programmed cell death, or apoptosis, is a process by which organisms remove unwanted or damaged cells during development and in the adult (reviewed in Ref. 1Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2403) Google Scholar). 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This has suggested that caspases can function in a cascade in which initiator caspases, activated by upstream death signals, cleave and activate a set of executioner caspases that carry out proteolytic cleavages of cellular proteins (12Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1936) Google Scholar, 13Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4124) Google Scholar). Seven caspases, DCP-1 (14Song Z. McCall K. Steller H. Science. 1997; 275: 536-540Crossref PubMed Scopus (251) Google Scholar), drICE (15Fraser A.G. Evan G.I. EMBO J. 1997; 16: 2805-2813Crossref PubMed Scopus (171) Google Scholar), DCP-2/DREDD (16Inohara N. Koseki T. Hu Y. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10717-10722Crossref PubMed Scopus (278) Google Scholar, 17Chen P. Rodriguez A. Erskine R. Thach T. Abrams J.M. Dev. Biol. 1998; 201: 202-216Crossref PubMed Scopus (182) Google Scholar), DRONC (18Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (239) Google Scholar), DECAY (19Dorstyn L. Read S.H. Quinn L.M. Richardson H. Kumar S. J. Biol. Chem. 1999; 274: 30778-30783Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), and two caspases predicted on the basis of genomic sequence (20Rubin G.M. Yandell M.D. Wortman J.R. Miklos G.L.G. Nelson C.R. Hariharan I.K. et al.Science. 2000; 287: 2204-2215Crossref PubMed Scopus (1368) Google Scholar) have been identified in Drosophila. Evidence that caspases are important for cell death in Drosophilacomes from several sets of observations. Expression of the baculovirus caspase inhibitor p35 or the Drosophila caspase inhibitor DIAP1 blocks cell death in the fly in a number of different contexts (reviewed in Ref. 21Bergmann A. Agapite J. Steller H. Oncogene. 1998; 17: 3215-3223Crossref PubMed Scopus (109) Google Scholar), including normally occurring cell death and death induced by overexpression of the cell death activatorsreaper (rpr), head involution defective (hid), and grim (22Hay B.A. Wolff T. Rubin G.M. Development. 1994; 120: 2121-2129Crossref PubMed Google Scholar, 23Grether M.E. Abrams J.M. Agapite J. White K. Steller H. Genes Dev. 1995; 9: 1694-1708Crossref PubMed Scopus (592) Google Scholar, 24Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (643) Google Scholar, 25White K. Tahaoglu E. Steller H. Science. 1996; 271: 805-807Crossref PubMed Scopus (337) Google Scholar, 26Chen P. Nordstrom W. Gish B. Abrams J.M. Genes Dev. 1996; 10: 1773-1782Crossref PubMed Scopus (362) Google Scholar). Dominant negative forms of DCP-2/DREDD (27Rodriguez A. Oliver H. Zou H. Chen P. Wang X. Abrams J.M. Nat. Cell Biol. 1999; 1: 272-279Crossref PubMed Scopus (293) Google Scholar) and DRONC (28Meier P. Silke J. Leevers S.J. Evan G.I. EMBO J. 2000; 19: 598-611Crossref PubMed Scopus (275) Google Scholar) (this work) inhibitrpr-, hid-, andgrim-dependent cell death. Mutations indcp-1 (29McCall K. Steller H. Science. 1998; 279: 230-234Crossref PubMed Scopus (143) Google Scholar), the Drosophila homolog of the caspase-activating adaptor Apaf-1 (27Rodriguez A. Oliver H. Zou H. Chen P. Wang X. Abrams J.M. Nat. Cell Biol. 1999; 1: 272-279Crossref PubMed Scopus (293) Google Scholar, 30Kanuka 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, 31Zhou L. Song Z. Tittel J. Steller H. Mol. Cell. 1999; 4: 745-755Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), or heterozygosity for deficiencies that remove the dcp-2/dredd (17Chen P. Rodriguez A. Erskine R. Thach T. Abrams J.M. Dev. Biol. 1998; 201: 202-216Crossref PubMed Scopus (182) Google Scholar) ordronc loci (28Meier P. Silke J. Leevers S.J. Evan G.I. EMBO J. 2000; 19: 598-611Crossref PubMed Scopus (275) Google Scholar), suppress apoptosis in specific contexts. Also, immunodepletion of drICE prevents apoptotic events in cell extracts (32Fraser A.G. McCarthy N.J. Evan G.I. EMBO J. 1997; 16: 6192-6199Crossref PubMed Scopus (125) Google Scholar). Finally, mutants that eliminate the function of aDrosophila caspase inhibitor, DIAP1, result in massive cell death (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 34Goyal L. McCall K. Agapite J. Hartwieg E. Steller H. EMBO J. 2000; 19: 589-597Crossref PubMed Scopus (379) Google Scholar, 35Lisi S. Mazzon L. White W. Genetics. 2000; 154: 669-678PubMed Google Scholar), which is associated with an increase in caspase activity (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). How do the Drosophila caspases function to bring about cell death in the fly? DCP-1, drICE, DECAY, and one caspase predicted by genomic sequence (daydream; GenBank™ accession numberAF281077) have short prodomains characteristic of executioner caspases. In contrast, DCP-2/DREDD and DRONC have large NH2-terminal prodomains with homology to mammalian death effector and caspase recruitment domains, respectively. A second caspase predicted by genomic sequence (dream; GenBank™ accession number AF275814) has a long prodomain that lacks homology with any known death regulators. Death effector and caspase recruitment domains in caspases are thought to mediate their recruitment to death signal-dependent complexes in which activation occurs in response to oligomerization (reviewed in Ref. 36Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6151) Google Scholar). Thus DCP-2/DREDD and DRONC may act as initiator caspases in apoptotic signaling. In most caspases the catalytic site cysteine (C) is present in the pentapeptide sequence QAC(R/Q/G)(G/E), in which the QAC motif is invariant. DRONC is unique among caspases in that the sequence surrounding the active site is PFCRG (Fig. 1 A) (18Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (239) Google Scholar). Caspase crystal structures indicate that the glutamine at the first position of the canonical caspase pentapeptide QACRG is part of the substrate binding pocket. The fact that DRONC has a proline at this position suggests that it has a novel substrate specificity. Several Caenorhabditis elegans caspases, CSP-1a and CSP-2a, with pentapeptide sequences that differ in the first two pentapeptide positions, SACRG and VCCRG, respectively, have also been described (37Shaham S. J. Biol. Chem. 1998; 273: 35109-35117Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Here we show that, unique among caspases characterized to date, DRONC cleaves tetrapeptide and protein substrates COOH-terminal to glutamate as well as aspartate residues. A role for DRONC as a cell death caspase is suggested by the observations that expression of a dominant negative form of DRONC blocks cell death, that DRONC expression induces cell death, and that DRONC interacts with other Drosophila cell death regulators, including DIAP1, drICE, hid, andgrim. Interestingly, however, DRONC is not inhibited by baculovirus p35, which inhibits cell death in flies. The substrate specificity of recombinant DRONC was tested using positional scanning synthetic combinatorial libraries, as described previously (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1844) Google Scholar, 38Rano T.A. Timkey T. Peterson E.P. Rotunda J. Nicholson D.W. Becker J.W. Chapman K.T. Thornberry N.A. Chem. Biol. 1997; 4: 149-155Abstract Full Text PDF PubMed Scopus (238) Google Scholar). The DRONC coding region was amplified by polymerase chain reaction and introduced into pET23(a) (Novagen) to produce pDRONC-His6. This plasmid was used to prepare active DRONC from Escherichia coli as described for DCP-131-His6 (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar). The subunits were resolved on a 15% SDS-PAGE1 gel and transferred to polyvinylidene difluoride (Millipore). The membrane was stained with Coomassie and the smaller band excised. Microsequencing was carried out at the Caltech Protein Microanalytical Laboratory under the direction of Gary M. Hathaway. Fluorogenic peptide cleavage assays were carried out as described (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar), using DRONC or DCP-1 at 0.2 μm or 0.2 nm final concentration as specified in the text. AFC-tetrapeptide substrates were purchased from Enzyme Systems (Livermore, CA), including the custom made Ac-TQTE-AFC and AC-TQTD-AFC. Bacterially produced DCP-1-His6 and drICE-His6 have been described (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar). The coding regions for DRONC, drICE, and DCP-1 were cloned from the yeast expression constructs into Bluescript KS+ (Stratagene). The coding regions of mutant versions of these proteins were generated using polymerase chain reaction and the QuickChange site-directed mutagenesis kit (Stratagene). Sequencing verified the existence of the desired mutation and the absence of other polymerase chain reaction-generated mutations. A transcription-coupled rabbit reticulocyte translation system (TNT, Promega) with Redivue [35S]methionine (Amersham Pharmacia Biotech) was used to generate 35S-labeled proteins. 2 μl of each 35S-labeled product was incubated with 2 μm of active caspase or buffer alone in caspase activity buffer (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar) in a total 10-μl volume. The products were resolved by 15% SDS-PAGE, the proteins transferred to Hybond ECL membrane (Amersham Pharmacia Biotech). Bio-Max MR-1 film (KODAK) was used to visualize the labeled fragments. The concentration of active DRONC was determined by active site titration using the active site inhibitor carbobenzoxy-VAD-fluoromethyl ketone (z-VAD-fmk), as described in Ref. 40Garcia-Calvo M. Peterson E.P. Rasper D.M. Vaillancourt J.P. Zamboni R. Nicholson D.W. Thornberry N.A. Cell Death Diff. 1999; 6: 362-369Crossref PubMed Scopus (194) Google Scholar. The activity of DRONC was measured in triplicate for each substrate using continuous fluorometric assays as described in Ref. 33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar. Appropriate dilutions of enzyme were added to reaction mixtures containing substrate and caspase activity buffer (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) in a total volume of 100 μl.k cat/K m values were calculated as described in Ref. 41Stennicke H.R. Salvesen G.S. Methods. 1999; 17: 313-319Crossref PubMed Scopus (160) Google Scholar. Yeast expression plasmids for galactose inducible expression of DCP-1, DIAP1, DIAP2, and P35 have been previously described (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar). pCUP-DIAP1, for copper inducible expression of DIAP1, pGALL-RPR, pGALL-GRIM, and pGALL-HID have been previously described (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). The coding region of DRONC was amplified from a clone (LD12627) obtained from the BerkleyDrosophila Genome project, and cloned into pGALL-(LEU2) (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar) to produce pGALL-DRONC. A DRONC active site mutant, DRONCC318S, was generated by polymerase chain reaction using a mutagenic oligonucleotide encompassing the internalSacII site of DRONC, and cloned to replace the corresponding section of wild-type DRONC in pGALL-(LEU2), generating pGALL-DRONCC318S. A section of human PARP encoding amino acids 1–337, spanning the caspase cleavage site, was amplified from a HeLa cell Matchmaker library (CLONTECH) and cloned into pADH-(TRP1) (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) in front of a Myc epitope tag (encoding MEQKLISEEDLAS), to generate pADH-mycPARP337. A fragment encoding p35 was excised from pEF-p35 (42Hawkins C.J. Uren A.G. Hacker G. Medcalf R.L. Vaux D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13786-13790Crossref PubMed Scopus (96) Google Scholar) and cloned into pADH-(TRP1) (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) to give pADH-p35. W303α yeast were transformed as described previously (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar). For survival assays, transformants were grown in selective liquid medium to saturation, then pelleted, and resuspended in TE toA 600 = 0.02. Five-fold dilutions were prepared, and 2-μl drops of each solution were spotted onto selective plates with galactose and 0, 10, or 100 μm CuSO4 as specified. For Western blotting to analyze p35 and PARP cleavage, transformants were grown in glucose-containing selective medium to stationary phase, washed in TE and grown in YP medium with galactose for 8 h. Yeast were pelleted, lysed by 2 cycles of boiling, and vortexing with glass beads in cracking buffer (8 m urea, 5% SDS, 40 mm Tris, pH 6.8, 0.1 mm EDTA, 1% β-mercaptoethanol, 0.4 mg/ml bromphenol blue). Samples were subjected to 12% SDS-PAGE, transferred to Hybond ECL membrane (Amersham Pharmacia Biotech), and probed with antibodies recognizing the Myc epitope or p35. Washes were followed by incubation with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2000), followed by detection with ECL (Pierce). The coding regions for full-length wild type DRONC, DRONCC318S, GRIM, and HID were cloned into pGMR (22Hay B.A. Wolff T. Rubin G.M. Development. 1994; 120: 2121-2129Crossref PubMed Google Scholar), and introduced into theDrosophila germline using standard techniques (43Spradling A.C. Rubin G.M. Science. 1982; 218: 341-347Crossref PubMed Scopus (1169) Google Scholar), generating GMR-DRONC GMR-DRONCC318S, GMR-grim,and GMR-hid flies, respectively. GMR-rpr (24Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (643) Google Scholar) and GMR-p35 (22Hay B.A. Wolff T. Rubin G.M. Development. 1994; 120: 2121-2129Crossref PubMed Google Scholar) flies have been described previously. Fixation, embedding and sectioning of adult fly heads, and were carried out as described in Ref. T. Development. 1991; Google Scholar. caspases cleave and activate and DRONC cleaves itself we and coli a COOH-terminal of The protein of two of the large and small subunits not this protein is active as a the site of cleavage the large and small was on the smaller The NH2-terminal sequence determined A) occurs COOH-terminal to the sequence that DRONC cleaves itself following a glutamate an aspartate. this we DRONC to wild type DRONC and DRONC generated by in translation and incubated with produced DRONC or in 1 DRONC cleaved itself to generate a product corresponding in to the prodomain and large This band was not DRONC was the with the that DRONC processes itself following DCP-1 and drICE not cleaved DRONC at several sites. This cleavage was by the of the that these caspases cleaved in DRONC, in the DRONC the of cleavage the DRONC prodomain we generated a form of DRONC, in which the P1 of caspase target sites the and were to in 35S-labeled in translated was by wild type DRONC, but not by were obtained with cleavage by drICE not Thus DCP-1 and drICE can process DRONC the but not at the this processing occurs in it may as a of of DRONC scanning synthetic combinatorial have been a to cleavage site of other caspases (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1844) Google Scholar). The is of of In each one position is with one of amino acids the two a of amino acids present in of the a of the amino in the and We used this to for amino acids at each of these The positional scanning synthetic combinatorial all aspartate at the P1 DRONC cleaves itself it is also to cleave protein substrates aspartate Thus we that the about cleavage specificity. in DRONC a for or at the P2 A of amino acids was at the and This suggests that an DRONC P1 aspartate tetrapeptide cleavage The of the were by in which DRONC activity was tested with a number of used tetrapeptide activity DRONC of activity with the and and of activity with (Fig. 2 However, any activity was with or which are predicted to be 2 also that DRONC of activity with the pentapeptide with the tetrapeptide as well as other observations suggests that a residue is important for DRONC cleavage we carried out assays in which the cleavage of DRONC and DCP-1 were measured for two different peptide Ac-TQTE-AFC and (Fig. 2 Ac-TQTE-AFC is from the known DRONC site and is also predicted to correspond to a DRONC cleavage site on the obtained from is a tetrapeptide substrate for caspases as of apoptosis caspases (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1844) Google activity is in with However, DRONC a cleavage for the Ac-TQTE-AFC substrate (Fig. 2 DCP-1, which has a of the standard caspase active site pentapeptide has a for the tetrapeptide substrate with a P1 (Fig. 2 Z. A. Nicholson D.W. Thornberry N.A. Peterson E.P. Steller H. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). the fact that DRONC of activity with tetrapeptide substrates containing a P1 DRONC is inhibited by the caspase inhibitor carbobenzoxy-VAD-fluoromethyl ketone not We to P1 specificity with to aspartate and glutamate. do this we a second tetrapeptide that from Ac-TQTE-AFC by the P1 We used these substrates to activity cat/K as described under We calculated a cat/K of for and a cat/K of for Thus DRONC a for cleavage of tetrapeptide substrates with a P1 aspartate with a P1 glutamate. DRONC however, a of tetrapeptide The calculated cat/K values for DRONC are described for which itself is a enzyme in as with most other caspases M. Peterson E.P. Rasper D.M. Vaillancourt J.P. Zamboni R. Nicholson D.W. Thornberry N.A. Cell Death Diff. 1999; 6: 362-369Crossref PubMed Scopus (194) Google Scholar). This may the fact that DRONC has an or that we have not identified However, DRONC activity may also be with theDrosophila homolog of Apaf-1 known as 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), L. Song Z. Tittel J. Steller H. Mol. Cell. 1999; 4: 745-755Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), and (27Rodriguez A. Oliver H. Zou H. Chen P. Wang X. Abrams J.M. Nat. Cell Biol. 1999; 1: 272-279Crossref PubMed Scopus (293) Google in a to that of mammalian by Apaf-1 J. Lazebnik Y. Genes Dev. 1999; PubMed Scopus Google Scholar). DRONC of activity to DCP-1 on the protein substrate drICE DRONC cleavage may sequences surrounding the target DRONC is an cell death caspase, substrates other Drosophila caspases. drICE is a to be a target immunodepletion show that drICE is for apoptotic events in cell extracts (32Fraser A.G. McCarthy N.J. Evan G.I. EMBO J. 1997; 16: 6192-6199Crossref PubMed Scopus (125) Google Scholar), and suggest that DRONC to hid-, andgrim-dependent cell death Ref. P. Silke J. Leevers S.J. Evan G.I. EMBO J. 2000; 19: 598-611Crossref PubMed Scopus (275) Google and We generated 35S-labeled in drICE and incubated it with produced DRONC or We with the observations of (28Meier P. Silke J. Leevers S.J. Evan G.I. EMBO J. 2000; 19: 598-611Crossref PubMed Scopus (275) Google Scholar, Z. A.