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• W2079850068 abstract "We have previously shown that Xenopusrabaptin-5 is cleaved in apoptotic extracts, with a concomitant reduction in the ability of these extracts to support endosomal membrane fusion (Cosulich, S. C., Horiuchi, H., Zerial, M., Clarke, P. R., and Woodman, P. G. (1997) EMBO J.16, 6182–6191). In this report we demonstrate that caspase-dependent cleavage is a conserved feature of rabaptin-5. Human rabaptin-5 is cleaved at two sites (HSLD379 and DESD438) in apoptotic HeLa extracts. Cleavage is effected by caspase-3, since it is prevented when caspase-3 activity is either inhibited by Ac-DEVD-CHO or removed by immunodepletion. Moreover, an identical pattern of cleavage is observed using recombinant caspase-3. The action of caspase-3 is highly selective; neither caspase-2 nor caspase-7 are able to cleave recombinant or cytosolic rabaptin-5. Caspase-dependent cleavage of rabaptin-5 generates two physically separated coiled coil-forming domains, the C-terminal of which retains the ability to bind the Rab5 exchange factor rabex-5. We have previously shown that Xenopusrabaptin-5 is cleaved in apoptotic extracts, with a concomitant reduction in the ability of these extracts to support endosomal membrane fusion (Cosulich, S. C., Horiuchi, H., Zerial, M., Clarke, P. R., and Woodman, P. G. (1997) EMBO J.16, 6182–6191). In this report we demonstrate that caspase-dependent cleavage is a conserved feature of rabaptin-5. Human rabaptin-5 is cleaved at two sites (HSLD379 and DESD438) in apoptotic HeLa extracts. Cleavage is effected by caspase-3, since it is prevented when caspase-3 activity is either inhibited by Ac-DEVD-CHO or removed by immunodepletion. Moreover, an identical pattern of cleavage is observed using recombinant caspase-3. The action of caspase-3 is highly selective; neither caspase-2 nor caspase-7 are able to cleave recombinant or cytosolic rabaptin-5. Caspase-dependent cleavage of rabaptin-5 generates two physically separated coiled coil-forming domains, the C-terminal of which retains the ability to bind the Rab5 exchange factor rabex-5. Chinese hamster ovary aminomethylcoumarin polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid poly(ADP)-ribose polymerase Programmed cell death (apoptosis) plays a fundamental role in the development and homeostasis of multicellular organisms (1Chinnaiyan A.M. Dixit V.M. Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar, 2Raff M.C. Barres B.A. Burne J.F. Coles H.S.R. Ishizaki Y. Jacobson M.D. Phil. Trans. R. Soc. Lond. B. 1994; 345: 265-268Crossref PubMed Scopus (116) Google Scholar). The primary feature of apoptosis is rapid engulfment and degradation of dying cells by their neighbors, so that an inflammatory response can be avoided. Since in many cases the engulfing cells are not specialized for phagocytic uptake (3Savill J. Nature. 1998; 392: 442-443Crossref PubMed Scopus (207) Google Scholar), signals that expedite engulfment and degradation are likely to arise from the apoptotic cell. A critical event during apoptosis is therefore the expression of surface receptors that permit the specific recognition of a dying cell. One such receptor is probably phosphatidylserine, which is translocated from the inner leaflet to the outer leaflet of the plasma membrane during apoptosis (4Martin S.J. Reutelingsperger C.P.M. McGahon A.J. Rader J.A. van Schie R.C.A.A. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2562) Google Scholar). There is considerable evidence, however, that other surface moieties, including carbohydrate, form part of the recognition signal (5Savill J. Dransfield I. Hogg N. Haslett C. Nature. 1990; 343: 170-173Crossref PubMed Scopus (702) Google Scholar). In addition to changes at the surface, the changes in cellular function that occur in an apoptotic cell are characterized by a variety of striking morphological and biochemical alterations. These include fragmentation of the nucleus and activation of endonuclease(s) (6Wood E.R. Earnshaw W.C. J. Cell Biol. 1980; 111: 2839-2850Crossref Scopus (141) Google Scholar, 7Earnshaw W.C. Curr. Opin. Cell Biol. 1995; 7: 337-343Crossref PubMed Scopus (510) Google Scholar), cell shrinkage and fragmentation, and plasma membrane blebbing (8Mills J. Stone N.L. Erhardt J. Pittman R.N. J. Cell Biol. 1998; 140: 627-636Crossref PubMed Scopus (410) Google Scholar). A further distinguishing feature of apoptotic cells is a loss of organized endomembrane structure; the nuclear envelope is frequently lost, and other recognizable membrane structures such as the Golgi complex are replaced by a disorganized array of vacuoles and vesicles (9Kerr J.F.R. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12874) Google Scholar, 10Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6725) Google Scholar). The so-called execution phase of apoptosis is evolutionarily conserved (11Jacobson M.D. Weil M. Raff M. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar), underlining its importance. It is now widely believed that many (although not all) apoptotic changes are linked to activation of a number of conserved cysteine proteases (caspases), which cleave specific substrates involved in key cellular processes (for review see Ref. 12Kidd V.J. Annu. Rev. Physiol. 1998; 60: 533-573Crossref PubMed Scopus (263) Google Scholar). Caspases themselves are present as proenzymes that are readily cleaved, either autocatalytically (13Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2111) Google Scholar, 14Yang X. Chang H.Y. Baltimore D. Mol. Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar) or by upstream “activator” caspases (15Muzio M. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1997; 272: 2952-2956Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar,16Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-490Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar), during apoptosis. This provides the cell with a means to rapidly amplify its apoptotic response. Caspases can be divided into groups based on their sequence-selective protease activity toward peptide substrates. Thus, caspases-2, -3, and -7 (group II caspases) all cleave preferentially after the sequence DEXD, whereas caspases-6, -8, and -9 (group III) prefer the sequence (V/L)E(H/T)D (17Thornberry N.A. Rano T.O. 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 (1849) Google Scholar). A major question is whether such overlapping substrate specificity within each group indicates that these enzymes represent tissue-specific isoforms or redundant isoforms within the same cell, or whether caspases exhibit far greater specificity toward polypeptide substrates in vivo. Given the profound morphological changes occurring to membranes within apoptotic cells, and evidence for alterations in the expression of surface receptors, we anticipated that apoptosis would be associated with changes in the dynamics of the endocytic/recycling pathways. On this basis, we examined whether endosomal membrane fusion, an event that is essential for endosomal organization and for transport of receptors through the endocytic recycling pathway, is affected in apoptotic extracts. Endosomal fusion was indeed substantially reduced during apoptosis in Xenopus extracts, and this reduction was associated with specific cleavage of the Rab5 effector rabaptin-5 (18Cosulich S.C. Horiuchi H. Zerial M. Clarke P.R. Woodman P.G. EMBO J. 1997; 16: 6182-6191Crossref PubMed Scopus (70) Google Scholar). Cleavage of rabaptin-5 was also apparent in cellular models of apoptosis, and was accompanied by reduced endocytic capacity. Hence, rabaptin-5 cleavage appears to be an important determinant in the abrogation of normal cellular function during apoptosis. In this study, we have examined in detail the activity that cleaves rabaptin-5. We have also mapped the site of rabaptin-5 cleavage, in order to understand how it might interfere with rabaptin-5 function and thus contribute to impairment of normal endocytic transport. Our previous data suggested that rabaptin-5 is cleaved by a caspase-related activity (18Cosulich S.C. Horiuchi H. Zerial M. Clarke P.R. Woodman P.G. EMBO J. 1997; 16: 6182-6191Crossref PubMed Scopus (70) Google Scholar). We now show that caspase-3 cleaves human rabaptin-5 at two closely positioned and conserved sites to generate physically separated N- and C-terminal domains. The activity of caspase-3 toward rabaptin-5 is surprisingly selective, since neither caspase-2 nor caspase-7 effect cleavage. Cytochrome c was obtained from Roche Molecular Biochemicals Ltd., Lewes, Sussex, United Kingdom (UK). Ac-DEVD-CHO1 and Ac-DEVD-AMC were bought from Calbiochem-Novabiochem (UK) Ltd. (Nottingham, UK) and stored as 10 mm stocks in Me2SO at −20 °C. Ac-LDESD-AMC was synthesized by SNPE Ltd. (Croyden, Surrey, UK). Antibodies to caspase-3 (N-19, H-277) were from Santa Cruz Inc. (Autogenbioclear, Calne, Wilts, UK). Antiserum to EEA1 was a kind gift from Harald Stenmark, The Norwegian Radium Hospital, Oslo, Norway. Antibodies and reagents to rabaptin-5 and rabex-5 were generously provided by Marino Zerial, Max Planck Institute for Molecular Cell Biology and Genetics, c/o EMBL, Heidelberg, Germany. Recombinant active caspases were generous gifts from Donald Nicholson and Sophie Roy, Merck Frosst Center for Therapeutic Research, Quebec, Canada. Horseradish peroxidase-conjugated secondary antibodies used for ECL Western blotting were obtained from Dako, Glostrup, Denmark. All other reagents were obtained from Sigma. Suspension HeLa cells were grown to a density of 106 cells/ml in minimal essential medium modified for suspension cultures (Life Technologies, Inc., Paisley, Scotland) supplemented with 5% fetal calf serum. Cells were harvested from 2 liters of culture, washed twice in KEHM buffer (50 mm KCl, 10 mm EGTA, 50 mm Hepes, pH 7.4, 2 mm MgOAc), and resuspended in 2 volumes of the same buffer. After addition of dithiothreitol (1 mm) and protease inhibitors (1 μg/ml apopain, 1 μg/ml pepstatin A, 5 μg/ml E64, 1 μg/ml chymostatin, 40 μg/ml phenylmethylsulfonyl fluoride from a 1000x stock in Me2SO), cells were homogenized by passing through a 8.02-mm bore in a stainless steel block containing a 8.004-mm diameter ball (19Balch W.E. Dunphy W.G. Braell W.A. Rothman J.E. Cell. 1984; 39: 405-416Abstract Full Text PDF PubMed Scopus (480) Google Scholar). A cytosol fraction was produced by centrifuging the homogenate at 300,000 ×g av for 45 min. The cytosol was snap-frozen and stored at −80 °C. To generate apoptotic extracts, cytosol (20 mg of protein/ml) was incubated for the indicated times at 30 °C with an ATP regenerating mixture (1 mm ATP, 5 mmcreatine phosphate, 10 μg/ml creatine kinase final) and 10 μm cytochrome c. HL-60 cells (grown in RPMI 1640 medium with 5% fetal calf serum) were induced to undergo apoptosis by treatment with 50 μmetoposide (20Beere H.M. Chresta C.M. Hickman J.A. Mol. Pharmacol. 1996; 49: 842-851PubMed Google Scholar) or with 1 μg/ml anisomycin (21Polverino A.J. Patterson S.D. J. Biol. Chem. 1997; 272: 7013-7021Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). To generate apoptotic HL60 extracts, 1–2-liter cultures of cells were treated with etoposide, then harvested by centrifugation, washed in KEHM buffer, and homogenized as for HeLa cells. For immunodepletion of caspase-3, cytosol (4 mg of protein) was incubated overnight with gentle rotation in a tube containing 40 μl of protein A-Sepharose beads to which had been pre-bound 8 μg of anti-caspase-3 antibody. As a control, cytosol was incubated with beads pre-treated with a non-relevant antibody. Xenopus egg extracts were prepared exactly as described (18Cosulich S.C. Horiuchi H. Zerial M. Clarke P.R. Woodman P.G. EMBO J. 1997; 16: 6182-6191Crossref PubMed Scopus (70) Google Scholar). Recombinant active caspases (17Thornberry N.A. Rano T.O. 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 (1849) Google Scholar) were diluted into caspase cleavage buffer (50 mm Hepes-KOH, pH 7.4, 2 mm EDTA, 0.1% (w/v) CHAPS, 10% (w/v) sucrose, 5 mm dithiothreitol; with the exception of caspase-2, which was diluted in the same buffer except with 50 mm NaOAc, pH 5.5, to provide a pH optimum), and duplicate samples (5 μl) were incubated with 0–100 μm Ac-DEVD-AMC or Ac-LDESD-AMC for 10 min at 30 °C. Samples were diluted to 2.5 ml in water and the generation of fluorescent product determined (22Thornberry N.A. Methods Enzymol. 1994; 244: 615-631Crossref PubMed Scopus (203) Google Scholar). Cleavage of polypeptide substrates by recombinant caspases was carried out in the appropriate buffer for 2 h at 30 °C, in a final volume of 10 μl. Bacterially expressed His6-rabaptin-5 was purified as described by Stenmarket al. (23Stenmark H. Vitale G. Ullrich O. Zerial M. Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google Scholar). For some experiments, His6-rabaptin-5 containing a C-terminal protein C peptide tag was expressed and purified as above. This reagent was generated by inserting the protein C coding sequence (GAA GAT CAG GTA GAT CCA CGG TTA ATC GAT GGT AAG TAA) immediately downstream from the rabaptin-5 coding sequence. The protein C coding sequence was followed by an in-frame stop codon (underlined) and was inserted using polymerase chain reaction-based site-directed mutagenesis (ExSite; Stratagene) according to the manufacturer's instructions. PARP, caspase-3, and caspase-2 cDNAs were present in pcDNA3 vectors (24Swanton E. Savory P. Cosulich S. Clarke P. Woodman P. Oncogene. 1999; 18: 1781-1787Crossref PubMed Scopus (117) Google Scholar). [35S]Methionine-labeled proteins were generated by incubating 1 μg of cDNA with a combined transcription/translation kit (Promega) supplemented with T7 RNA polymerase and 60 μCi of [35S]methionine. Rabaptin-5 cleavage site mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene). To examine cleavage of in vitro translated His6-rabaptin-5 in extracts, translation product was diluted 10-fold into Xenopus or HeLa extracts and incubated at 25 °C or 30 °C respectively. Samples were denatured by boiling for 5 min in 1% SDS, then diluted to 200 μl with immunoprecipitation buffer (10 mm Tris-HCl, pH 7.5, 140 mm NaCl, 1 mm EDTA, 1% Triton X-100) and precipitated with 0.5 μl of appropriate antibody with protein G- or protein A-agarose. Gel filtration chromatography to separate rabaptin-5 from procaspases 2 and 3 was performed using a 24-ml Sepharose-6 column (Amersham Pharmacia Biotech) on a Beckman BioSys 510 HPLC system. Analytical fractionation of apoptotic cytosol was achieved using a Superose 6 column on a Smart System (Amersham Pharmacia Biotech). For immunoprecipitation experiments, polyclonal anti-rabaptin-5 serum (50 μl) or monoclonal anti-rabaptin-5 (50 μl of ascites) were preincubated with 100 μl of protein A- or protein G-agarose, respectively, and the antibody was covalently attached to the beads using dimethylpimelimidate according to published methods (25Harlow E. Lane D. Antibodies: A Laboratory Approach. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). These beads were then used to immunoprecipitate rabaptin-5 or its fragments from 2 mg of cytosol. For determination of N-terminal sequences, protein C-tagged rabaptin-5 was incubated with 10 nm caspase-3 for 4 h at 37 °C, then precipitated in the presence of 1 mm CaCl2 onto protein G-agarose beads to which anti-protein C antibody (HPC4; Roche Molecular Biochemicals, Lewes, UK) had been covalently attached. Residual full-length rabaptin-5 and C-terminal fragments were eluted with 5 mm EDTA. The products were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and subjected to N-terminal sequence analysis. Endosome fusion was assayed as described previously (18Cosulich S.C. Horiuchi H. Zerial M. Clarke P.R. Woodman P.G. EMBO J. 1997; 16: 6182-6191Crossref PubMed Scopus (70) Google Scholar), using for each sample 5 μl of donor membranes, 7 μl of acceptor membranes, 10 μl of cytosol in a total volume of 40 μl. Our previous work had demonstrated that Xenopus rabaptin-5 is cleaved in egg extracts to yield a C-terminal fragment of 45–50 kDa (18Cosulich S.C. Horiuchi H. Zerial M. Clarke P.R. Woodman P.G. EMBO J. 1997; 16: 6182-6191Crossref PubMed Scopus (70) Google Scholar). Similar extracts have been shown to undergo several apoptotic events, including activation of caspases and endonucleases (26Cosulich S.C. Green S. Clarke P.R. Curr. Biol. 1996; 6: 997-1005Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 27Newmeyer D.D. Farschon D.M. Reed J.C. Cell. 1994; 79: 353-364Abstract Full Text PDF PubMed Scopus (492) Google Scholar). To establish whether cleavage by apoptotic proteases is a conserved feature of rabaptin-5, human His6-rabaptin-5 was translated in vitroand then combined with a Xenopus egg extract. After appropriate incubation, rabaptin-5 cleavage products of approximately 62 (Fig. 1 A) and 47 kDa (Fig. 1 B) were produced, which could be immunoprecipitated by anti-His antibody (Fig. 1 A) or an antibody recognizing the C-terminal portion of rabaptin-5 (Fig. 1 B), respectively. The time course of cleavage was similar to that previously reported for cleavage of endogenous Xenopus rabaptin-5 (18Cosulich S.C. Horiuchi H. Zerial M. Clarke P.R. Woodman P.G. EMBO J. 1997; 16: 6182-6191Crossref PubMed Scopus (70) Google Scholar) (data not shown), and was prevented by inclusion of the specific caspase inhibitor Ac-DEVD-CHO (Fig. 1). Hence, human His6-rabaptin-5 behaves in apoptotic Xenopusextracts in the same way as Xenopus rabaptin-5, confirming that cleavage is a conserved function. Recent work from Wang and colleagues (16Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-490Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar, 28Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-158Abstract Full Text Full Text PDF PubMed Scopus (4463) Google Scholar, 29Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar) has shown that cytosolic extracts from mammalian cells can be triggered to enter apoptosis by addition of cytochrome c. This process can be sensitized by, although it is not strictly dependent upon, inclusion of dATP (24Swanton E. Savory P. Cosulich S. Clarke P. Woodman P. Oncogene. 1999; 18: 1781-1787Crossref PubMed Scopus (117) Google Scholar, 28Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-158Abstract Full Text Full Text PDF PubMed Scopus (4463) Google Scholar). We examined whether this system would be convenient for studying the apoptotic cleavage of rabaptin-5. Previous work has shown that activation of caspase-3 in HeLa extracts occurs via cytochromec-dependent activation of caspase-9 (16Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-490Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar), and is maximal within 30 min of addition of cytochrome c (24Swanton E. Savory P. Cosulich S. Clarke P. Woodman P. Oncogene. 1999; 18: 1781-1787Crossref PubMed Scopus (117) Google Scholar). Substrates for caspase-3 and/or downstream caspases are cleaved more slowly. For example, caspase-2, whose cleavage requires caspase-3 activity (24Swanton E. Savory P. Cosulich S. Clarke P. Woodman P. Oncogene. 1999; 18: 1781-1787Crossref PubMed Scopus (117) Google Scholar, 30Li H. Bergeron L. Cryns V. Pasternack M.S. Zhu H. Shi L. Greenberg A. Yuan J. J. Biol. Chem. 1997; 272: 21010-21017Crossref PubMed Scopus (168) Google Scholar), is cleaved in these extracts between 1 and 3 h after addition of cytochrome c to yield an immunoreactive 12/13-kDa fragment (24Swanton E. Savory P. Cosulich S. Clarke P. Woodman P. Oncogene. 1999; 18: 1781-1787Crossref PubMed Scopus (117) Google Scholar). Rabaptin-5 is also cleaved, although somewhat more slowly than caspase-2, to yield a C-terminal fragment similar in size to that generated within apoptotic Xenopus extracts. Using a monoclonal antibody that recognizes an epitope within the N-terminal portion of rabaptin-5, the 62-kDa N-terminal fragment was also identified (Fig. 2 B). An additional minor product of approximately 53 kDa (N-53) was also seen with this antibody, indicating the presence of a further cleavage site. This was confirmed by inclusion of in vitro translated rabaptin-5 (see Fig. 3). Prolonged incubation of apoptotic extracts resulted in the disappearance of the N-62 fragment, while the N-53 fragment remained resistant to further proteolysis (Fig. 2 C, left panel), consistent with it being the result of a second, slower, cleavage event. Importantly, appearance of a 53-kDa polypeptide reactive against this antibody was observed in apoptotic cells and correlated with the disappearance of full-length rabaptin-5 (Fig. 2 C,right panel). These results suggest that N-53 is a final cleavage product in apoptotic cells. Moreover, they demonstrate that the cleaving activity that is present in apoptotic cells is likely to be similar, if not identical, to that present in cytochromec-activated extracts.FIG. 3Rabaptin-5 is cleaved after aspartate-438. A. In vitro translated His6-rabaptin-5 was incubated in HeLa cytosol at 30 °C as indicated, then analyzed by SDS-PAGE and phosphorimaging.B, as A, but using Asp438 mutant His6-rabaptin-5. C, as A, but using Asp446 mutant His6-rabaptin-5.View Large Image Figure ViewerDownload (PPT) The amino acid sequence of rabaptin-5 (23Stenmark H. Vitale G. Ullrich O. Zerial M. Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google Scholar) and the size of the C-terminal cleavage product in Xenopus extracts (18Cosulich S.C. Horiuchi H. Zerial M. Clarke P.R. Woodman P.G. EMBO J. 1997; 16: 6182-6191Crossref PubMed Scopus (70) Google Scholar) had suggested to us that caspase-dependent cleavage was most likely to occur after aspartate 438 or aspartate 446. Indeed, inclusion of a 22-mer peptide that included both aspartate residues retarded cleavage in Xenopus extracts by 1–2 h (data not shown). To identify the exact cleavage site, rabaptin-5 mutants were prepared with either Asp438 or Asp446 replaced by alanine, and were incubated in apoptotic HeLa extracts. Compared with wild-type rabaptin-5 (Fig. 3 A), Asp438 rabaptin-5 did not produce N-62 or C-47 fragments (Fig. 3 B) while Asp446 rabaptin-5 behaved identically to the wild-type (Fig. 3 C). Hence, human rabaptin-5 is cleaved at the sequence DESD438F. Asp438 rabaptin-5 still gave rise to the N-53 fragment, confirming that it is the product of a second cleavage event that is not dependent on prior cleavage at Asp438. Systematic studies of caspase cleavage sites using synthetic modified tetrapeptides have identified the amino acid preferences at the P2–P4 positions for each caspase (17Thornberry N.A. Rano T.O. 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 (1849) Google Scholar). Based on these studies, caspases have been divided into several groups. The group II “effector” caspases, caspase-3 and caspase-7, both cleave effectively after the sequence DEVD. Although this is similar to the primary cleavage site within rabaptin-5 (DESD), the activity of both caspases toward peptides is reduced when serine is placed at P2. In contrast, DESD is a preferred cleavage site, second only to DEHD, for the group II “activator” caspase, caspase-2 (17Thornberry N.A. Rano T.O. 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 (1849) Google Scholar). Based on these studies, and our previous observation that concentrations of recombinant caspase-3 just sufficient to accelerate apoptotic changes in Xenopus egg extracts were unable to cleave rabaptin-5 directly (18Cosulich S.C. Horiuchi H. Zerial M. Clarke P.R. Woodman P.G. EMBO J. 1997; 16: 6182-6191Crossref PubMed Scopus (70) Google Scholar), it seemed likely that a caspase-2-like activity would be responsible for cleaving rabaptin-5. However, we undertook a detailed examination to establish the true identity of the cleaving activity. First, we examined whether cleavage of cytosolic rabaptin-5 in HeLa extracts is dependent on caspase-3 activity. We have already used this approach to establish that cleavage of caspase-2 occurs via caspase-3 (24Swanton E. Savory P. Cosulich S. Clarke P. Woodman P. Oncogene. 1999; 18: 1781-1787Crossref PubMed Scopus (117) Google Scholar). We first examined the sensitivity of cleavage to the specific caspase-3 inhibitor Ac-DEVD-CHO, and found that cleavage of rabaptin-5 was prevented by inclusion of 50 nm Ac-DEVD-CHO (data not shown), similar to those concentrations that prevent cleavage of the caspase-3 substrates PARP and caspase-2 (24Swanton E. Savory P. Cosulich S. Clarke P. Woodman P. Oncogene. 1999; 18: 1781-1787Crossref PubMed Scopus (117) Google Scholar). To further establish a dependence on caspase-3 activity, extracts were depleted of caspase-3 prior to addition of cytochrome c. Cytosols pre-treated with an antibody to caspase-3 were depleted of caspase-3 precursor by at least 90% compared with mock-depleted cytosols (data not shown). When incubated with cytochrome c, these extracts were unable to cleave rabaptin-5 (Fig. 4 A,left panel). In contrast, rabaptin-5 was cleaved almost to completion within 4 h when mock-depleted extracts were incubated with cytochrome c (Fig. 4 A,right panel). Although cleavage of cytosolic rabaptin-5 is dependent on caspase-3, it is possible that it is cleaved directly by a downstream effector of caspase-3, such as caspase-2. To address this question, HeLa extracts were preincubated for a period sufficient to activate caspase-3, as well as potential downstream caspases. They were then depleted of activated caspase-3. Western blotting of extracts confirmed that greater than 90% of activated caspase-3 had been removed (data not shown). When excess bacterially expressed rabaptin-5 was added to depleted extract, no cleavage product was detected before 3 h, and significant cleavage did not occur until after 5 h incubation (Fig. 4 B, top left). In contrast, cleavage of rabaptin-5 was observed within 1 h of its addition to mock-depleted extract, and the majority of recombinant rabaptin-5 was cleaved after 4–5 h (Fig. 4 B, top right). The rate at which caspase-3-depleted extract could cleave rabaptin-5 was increased significantly by inclusion of recombinant caspase-3 (Fig. 4 B, bottom left) or the beads isolated from the immunodepletion step (Fig. 4 B, bottom right), confirming that the major cleaving activity was caspase-3. To demonstrate that the caspase activated within apoptotic cells most likely to cleave rabaptin-5 is caspase-3, cytosol was prepared from apoptotic HL60 cells. These cytosols had substantial rabaptin-5 cleaving activity, since significant cleavage of recombinant rabaptin-5 was observed within 1–2 h (Fig. 4 C, left panel). Again, prior depletion of active caspase-3 from these extracts significantly reduced the rate at which rabaptin-5 was cleaved (Fig. 4 C, right panel). These results indicated that the major rabaptin-5 cleaving activity within apoptotic extracts is caspase-3. To confirm this, and to further demonstrate the selectivity of rabaptin-5 as a caspase-3 substrate, recombinant rabaptin-5 was incubated with purified caspases. As shown in Fig. 5 A (left panel), rabaptin-5 was cleaved in a Ac-DEVD-CHO-sensitive manner when incubated with immunoprecipitated activated caspase-3. Furthermore, recombinant caspase-3 cleaved rabaptin-5 to generate the same cleavage products as did apoptotic cytosol (Fig. 5 A,right panel). Cleavage of rabaptin-5 was first observed at caspase-3 concentrations of 0.5 nm or above (Fig. 5 B). In contrast, PARP cleavage was observed above 0.1 nm caspase-3 (Fig. 5 C) and caspase-2 cleavage was observed above 0.25 nm caspase-3 (Fig. 5 D). This somewhat lower activity of caspase-3 toward rabaptin-5 compared with caspase-2 correlates well with the slower rate of rabaptin-5 cleavage in apoptotic extracts. By analyzing caspase-3 cleavage of purified in vitro translated rabaptin-5 at 37 °C (data not shown) we obtained aK cat/K m of 1 × 105m−1 s−1(versus PARP; 20 × 105m−1 s−1). Caspase-2 has a preference for the cleavage site DESD over DEVD, so it was expected that rabaptin-5 would be a good substrate for caspase-2 (17Thornberry N.A. Rano T.O. 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 (1849) Google Scholar). Surprisingly, no cleavage of rabaptin-5 was observed at concentrations of recombinant caspase-2 as high as 2 μm(Fig. 5 E). The activity of the caspase-2 preparation was conf" @default.
• W2079850068 created "2016-06-24" @default.
• W2079850068 creator A5040867627 @default.
• W2079850068 creator A5048177703 @default.
• W2079850068 creator A5050986255 @default.
• W2079850068 date "1999-12-01" @default.
• W2079850068 modified "2023-09-29" @default.
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