Membrane Oligomerization and Cleavage Activates the Caspase-8 (FLICE/MACHα1) Death Signal
1998; Elsevier BV; Volume: 273; Issue: 8 Linguagem: Inglês
10.1074/jbc.273.8.4345
ISSN1083-351X
AutoresRoland Martinꝉ, Richard M. Siegel, Lixin Zheng, Michael J. Lenardo,
Tópico(s)RNA Interference and Gene Delivery
ResumoMany forms of apoptosis, including that caused by the death receptor CD95/Fas/APO-1, depend on the activation of caspases, which are proteases that cleave specific intracellular proteins to cause orderly cellular disintegration. The requirements for activating these crucial enzymatic mediators of death are not well understood. Using molecular chimeras with either CD8 or Tac, we find that oligomerization at the cell membrane powerfully induces caspase-8 autoactivation and apoptosis. Death induction was abrogated by the z-VAD-fmk, z-IETD-fmk, or p35 enzyme inhibitors or by a mutation in the active site cysteine but was surprisingly unaffected by death inhibitor Bcl-2. Amino acid substitutions that prevent the proteolytic separation of the caspase from its membrane-associated domain completely blocked apoptosis. Thus, oligomerization at the membrane is sufficient for caspase-8 autoactivation, but apoptosis could involve a death signal conveyed by the proteolytic release of the enzyme into the cytoplasm. Many forms of apoptosis, including that caused by the death receptor CD95/Fas/APO-1, depend on the activation of caspases, which are proteases that cleave specific intracellular proteins to cause orderly cellular disintegration. The requirements for activating these crucial enzymatic mediators of death are not well understood. Using molecular chimeras with either CD8 or Tac, we find that oligomerization at the cell membrane powerfully induces caspase-8 autoactivation and apoptosis. Death induction was abrogated by the z-VAD-fmk, z-IETD-fmk, or p35 enzyme inhibitors or by a mutation in the active site cysteine but was surprisingly unaffected by death inhibitor Bcl-2. Amino acid substitutions that prevent the proteolytic separation of the caspase from its membrane-associated domain completely blocked apoptosis. Thus, oligomerization at the membrane is sufficient for caspase-8 autoactivation, but apoptosis could involve a death signal conveyed by the proteolytic release of the enzyme into the cytoplasm. A pivotal biochemical event of programmed cell death or apoptosis is the activation of cysteinyl, aspartate-specific proteases or caspases (1Nicholson D. Thornberry N. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2182) Google Scholar, 2Miller D.K. Semin. Immunol. 1997; 9: 35-49Crossref PubMed Scopus (194) Google Scholar). The caspase gene family in mammals includes at least 10 members that share protein sequence similarity to theced3 cell death gene from Caenorhabditis elegans(3Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.E. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2146) Google Scholar). The participation of caspases in programmed cell death is conserved widely in phylogeny from the nematode, C. elegansto humans (4Xue D. Horvitz H.R. Nature. 1995; 377: 248-251Crossref PubMed Scopus (438) Google Scholar). The essential role of caspases is to endoproteolytically cleave a select group of cellular proteins at aspartate residues, thereby causing the nuclear and cytoplasmic alterations that typify apoptosis. The principal regulation of caspases is post-translational. They reside in the cell as inactive zymogens, which must be proteolytically processed at internal aspartates to generate the subunits of the active enzyme. How caspases are activated is a critical question in the immune system, since normal lymphocyte homeostasis and immune tolerance involves CD95-induced apoptosis that depends on caspase activation (5Chinnaiyan A.M. Dixit V.M. Semin. Immunol. 1997; 9: 69-76Crossref PubMed Scopus (94) Google Scholar). Caspase activation is defective in patients that have inherited mutations in CD95 and suffer from the autoimmune/lymphoproliferative syndrome (ALPS) 1The abbreviations used are: ALPS, autoimmune/lymphoproliferative syndrome; DISC, death-inducing signal complex; PCR, polymerase chain reaction; FADD, Fas-associated death domain protein; GFP, green fluorescent protein; DED, death effector domain; C, caspase; Myr-C, myristoylated caspase; mAb, monoclonal antibody. , 2D. A. Martin and M. J. Lenardo, unpublished observations. (6Fisher G.H. Rosenberg F.J. Straus S.E. Dale J.K. Middleton L.A. Lin A.Y. Strober W. Lenardo M.J. Puck J.M. Cell. 1995; 81: 935-946Abstract Full Text PDF PubMed Scopus (1300) Google Scholar, 7Rieux-Laucat F. Le Deist F. Hivroz C. Roberts I.A. Debatin K.M. Fischer A. de Villartay J.P. Science. 1995; 268: 1347-1349Crossref PubMed Scopus (1181) Google Scholar). The activation of caspase-8 (FLICE/MACH) appears to be the first step in the cascade of apoptotic events induced by CD95 (5Chinnaiyan A.M. Dixit V.M. Semin. Immunol. 1997; 9: 69-76Crossref PubMed Scopus (94) Google Scholar,8Muzio M. Chinnaiyan A.M. Kishchekel 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 (2741) Google Scholar). Caspase-8 is recruited to the "death-inducing signal complex" (DISC), a multiprotein complex that forms rapidly on the cytoplasmic portion of the Fas/APO-1/CD95 receptor after ligand engagement, by the adapter protein FADD/MORT1 (8Muzio M. Chinnaiyan A.M. Kishchekel 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 (2741) Google Scholar, 9Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1787) Google Scholar, 10Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1995; 85: 803-815Abstract Full Text Full Text PDF Scopus (2111) Google Scholar, 11Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar, 12Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1041) Google Scholar, 13Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2161) Google Scholar). The caspase-8 precursor protein is cleaved at 3 aspartate residues to become active, but processing has only been demonstrated by exposing the caspase-8 precursor to an active DISC complex, raising the important question of how activation is initiated (12Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1041) Google Scholar). We therefore investigated the requirements of caspase-8 activation and apoptosis induction. The PCR3-uni vector and TA cloning kit were from Invitrogen, San Diego, CA. The vectors pCEFL, pCEFL-CD8-EMPTY, and pCEFL-Myr containing the src myristoylation sequence were gifts from Dr. J. Silvio Gutkind, NIDR, NIH. The FADD-AU-1 pcDNA3 was provided by Dr. Vishva Dixit (13Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2161) Google Scholar), and p35-pCI and 3LacZ plasmids were provided by Dr. John Bertin, NIAID, NIH. The Taq and Pwopolymerases and the rapid DNA ligation kit were from Boehringer Mannheim. z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) was from Enzyme Systems Products. PE-labeled anti-human CD8 and anti-Tac antisera were obtained from Pharmingen, San Diego, CA, anti-GFP mAb was from CLONTECH, and horseradish peroxidase-conjugated goat-anti-mouse IgG was from Jackson ImmunoResearch. SuperSignal horseradish peroxidase substrate was from Pierce. The parental and Bcl-2-overexpressing stable MCF-7 lines were kindly provided by Drs. Ulrich Brinkmann and Ira Pastan, National Cancer Institute, NIH. The caspase-8 (MACH-α1/FLICE) coding sequence was cloned using reverse transcription PCR into the pCR3-Uni vector using the TA cloning kit per manufacturer's protocols. The full-length cDNA was sequenced and then subcloned into a modified pcDNA3 vector, pCEFL, in which the cytomegalovirus promoter was replaced by the promoter for elongation factor 2 (EF-2). High-fidelity PCR products of caspase-8 (98–479 and 209–479) were subcloned asHindIII-NotI fragments into digested pCEFL. The vector pCEFL-CD8-EMPTY vector and the pCEFL-Myr vector containing thesrc myristoylation sequence were used for the in-frame cloning of the caspase-8 protease domain. The Tac-C construct was made by amplifying the extracellular and transmembrane domains (Tac EX-TM) of the Tac cDNA using high-fidelity PCR. CD8 was removed from the CD8-C construct using HindIII and BamHI, and the digested PCR product of Tac EX-TM was ligated in frame with caspase-8 Δ209. Point mutations were made in the pCEFL-CD8-C using the altered sites mutagenesis kit (Promega, Madison, WI) and the Quik-Change kits (Stratagene), according to the instructions of the manufacturers. For all Jurkat transfections, plasmid constructions (pCEFL, pCEFL-caspase-8, pCEFL-caspase-8 Δ98, pCEFL-caspase-8 Δ209, pCEFL-Myr-C, pCEFL-CD8-C, pCEFL-Tac-C, pcDNA3-FADD, p35-pCI, CD8-C, and the Tac-C mutants as outlined in the figures), along with pCEFL-GFP, were electroporated into 4–8 × 106 Jurkat cells in 0.4 ml of complete medium in an Electrocell Manipulator 600 (BTX Corp., San Diego, CA). Plasmids were added to the cells at a 3:1 mass excess of the caspase constructions to GFP to ensure that all cells expressing GFP simultaneously expressed the cotransfected DNA of interest. The total amount of DNA/cuvette ranged from 15 to 20 μg. After pulse discharge at settings 260 V, 1050 μF, and 720Ω, the cells were immediately placed in 7 ml of fresh RPMI medium containing 10% fetal calf serum, incubated for 16–20 h at 37 °C, and analyzed using flow cytometry. Where indicated 50 μm of the caspase inhibitor z-VAD-fmk was added to the cells/media immediately following electroporation. For the chimeric caspase-8 constructs, the cells were stained for surface expression of either CD8 or Tac (CD25) prior to analysis using flow cytometry. Dead cells were gated out using forward and side scatter profiles, and the percent cell death was calculated from the loss of live GFP-positive cells in treated samples compared with the control vector. For the inhibition of CD8-C apoptosis in Jurkat T-cells, 4 μg of CD8-C (or pCEFL control plasmid) was cotransfected with 2 μg of the GFP vector and either 20 μg of pCEFL with or without 50 μm z-VAD-fmk, 50 μm z-IETD-fmk (Enzyme Systems Products, Livermore, CA) or 20 μg of p35-pCI. After transfection, the cells were analyzed for CD8 surface expression using flow cytometry. For 293T cells, subconfluent cells were transfected in six-well plates using the calcium phosphate method (Stratagene) according to manufacturer's instructions, with the modification that 25 μm chloroquine was added to the medium to facilitate DNA uptake. Cells were transfected with the plasmid combinations described in the figures with a 2.5:1 excess of the DNA of interest to the 3Lac-z construct (total DNA: 1.5–2.0 μg). After 24–30 h, the cells were fixed in 2% formaldehyde, 0.2% glutaraldehyde in phosphate-buffered saline for 15 min at room temperature and then stained in phosphate-buffered saline containing 5 mm each K3Fe(CN)6 and K4Fe(CN)6·3H2O, 2 mmMgCl2, 0.01% SDS, 0.02% Nonidet P-40, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside until a suitable color developed, usually for 3–4 h at 37 °C. To enumerate the fraction of blue cells that had undergone apoptotic changes, a minimum of 200 blue-staining cells/well were counted using light microscopy and unambiguously scored as being apoptotic or nonapoptotic by morphological assessment. The Bcl-2 stable MCF-7 line showed approximately 20-fold excess Bcl-2 protein over the parental line by Western blot and had a demonstrable defect in tumor necrosis factor-induced apoptosis (data not shown). Transfections were done using the calcium phosphate method as described above. For the CD8-C inhibition experiments in 293T cells, 250 ng of CD8-C was transfected along with 1–5 μg of either CD8-empty or CD8-C D210A/D216A or 5 μg of pCEFL using Superfect (Qiagen) according to manufacturer's protocol. Cells were fixed at 24 h, stained, and enumerated with light microscopy as described above. Surface expression of Jurkat T-cells transfected with CD8 and Tac fusion proteins was done using PE-labeled anti-human CD8 and anti-Tac antisera (Pharmingen, San Diego, CA). Flow cytometry was carried out on a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA) using CellQuest software. GFP fluorescence was analyzed using the FL1 channel. Twenty-four hours after transfection (as described above) with the indicated constructs (see figure legends), 293T cells were lysed in buffer containing 140 mm NaCl, 10 mm Tris (pH 7.2), 2 mm EDTA, 1% Nonidet P-40, complete protease inhibitor mix (Boerhinger Mannheim), and 10 mm iodoacetamide. After lysis for 30 min on ice, supernatants were diluted in SDS sample buffer with or without 40 mm dithiothreitol, boiled, and electrophoresed on 4–20% Tris/glycine/SDS gels. Proteins were blotted onto nitrocellulose using a semidry transfer apparatus. The blots were then probed with 1:1000 dilution of anti-GFP mAb (Fig. 3) followed by 1:10,000 dilution of goat anti-mouse horseradish peroxidase (Jackson ImmunoResearch) or 1:20 dilution of a p18-specific anti-caspase-8 mAb (a kind gift of Dr. Peter Krammer, German Cancer Research Center) followed by 1:2500 dilution of isotype-specific goat anti-mouse horseradish peroxidase (Caltag) (Fig. 4). Bands were imaged with SuperSignal horseradish peroxidase substrate (Pierce). Equivalent cell numbers were loaded onto each lane.Figure 4Mutations reveal amino acids that are essential for apoptosis induction by CD8-C. A, expression plasmids containing single nucleotide changes that cause the substitution of either serine for the active site cysteine (CD8-C:C360S) or alanines for aspartates required for membrane release (CD8-C:D210A/D216A) were cotransfected with a GFP expression plasmid into Jurkat cells. Cell death was measured as the fraction of transfected (GFP-positive) cells that were lost compared with control. B, photomicrographs of 293T cells cotransfected with pCMV-β-galactosidase along with either CD8-fusion constructs as indicated. Viable cells have a large fibroblastoid morphology, whereas apoptotic cells are shrunken, circular, and extensively blebbed. Magnification is approximately × 200. C, flow cytometry profiles of Jurkat T-cells transfected with 10 μg of the indicated expression constructs with or without 50 μmz-VAD-fmk. The cells were stained with PE-labeled anti-CD25 (Pharmingen) prior to analysis, and only live cells as measured by forward scatter versus side scatter were analyzed for GFP and CD25 (Tac) expression. Viable transfected cells are the double positive cells in the right upper quadrant. D, polyacrylamide gel electrophoresis of protein extracts from 293T cells that were transiently transfected with CD8-C (lane 1) or CD8-C D210/216A double mutant (lane 2) and probed with anti-caspase-8-specific (p18) mAb. Molecular mass markers in kilodaltons are shown at the right.View Large Image Figure ViewerDownload (PPT) We first studied caspase-8 by transfecting expression constructs containing either full-length or truncated versions into Jurkat T-cells (Fig. 1). We found that the full-length protein inefficiently induced apoptosis compared with the death-signaling protein, FADD/MORT1 (10Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1995; 85: 803-815Abstract Full Text Full Text PDF Scopus (2111) Google Scholar, 13Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2161) Google Scholar) (Fig. 2 A). Removal of one or both death effector domains (DEDs) decreased rather than increased apoptosis demonstrating that the caspase prodomain does not inhibit formation of the active protease. Protein blots confirmed that equivalent protein expression was obtained with each of the constructs (data not shown). Thus, simple overexpression of this caspase did not efficiently induce activation or apoptosis in Jurkat T-cells. We therefore tested the concept that membrane localization and/or oligomerization could initiate caspase autoactivation and apoptosis induction as might be envisioned based on the observation that caspase-8 can be recruited to the DISC (8Muzio M. Chinnaiyan A.M. Kishchekel 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 (2741) Google Scholar, 9Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1787) Google Scholar, 10Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1995; 85: 803-815Abstract Full Text Full Text PDF Scopus (2111) Google Scholar, 11Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar). The caspase domain was genetically fused to the transmembrane and extracellular portion of the human CD8α chain, which is known to form disulfide-linked homodimers (14Baur A.S. Sawai E.T. Dazin P. Fantl W.J. Cheng-Mayer C. Peterlin B.M. Immunity. 1994; 1: 373-384Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 15Irving B.A. Weiss A. Cell. 1991; 64: 891-901Abstract Full Text PDF PubMed Scopus (630) Google Scholar, 16Leahy D.J. Axel R. Hendrickson W.A. Cell. 1992; 68: 1145-1162Abstract Full Text PDF PubMed Scopus (241) Google Scholar), and this expression construct, CD8-C, was transfected into Jurkat cells (Figs. 1and 2 B). CD8-C dramatically induced apoptosis, implying extremely efficient protease activation. Control experiments using the expression vector pCEFL, CD8-empty, or CD8-nef caused little or no apoptosis (Fig. 2 and data not shown). Therefore, the CD8-C chimera, either by membrane targeting, spontaneous oligomerization, or both, strongly induced apoptosis without further cross-linking.Figure 2Chimeric caspase-8 molecules have dramatically enhanced apoptotic activity that can be blocked by caspase inhibitors. A–C, in three separate experiments, Jurkat T-cells were transfected with 10 μg of the indicated constructs along with 2.5 μg of the pCEFL-GFP vector to track the fate of the transfected cells. The cells were analyzed flow cytometrically 24 h after transfection, and percent kill was calculated by measuring the deficit of GFP-positive viable cells in transfections of caspase or FADD compared with control vector alone. Under these conditions, Fas cross-linking with 100 ng/ml CH11 antibody induced ∼90% cell death. Each experiment was replicated three or more times and a representative result is shown. D, flow cytometry profiles of Jurkat T-cells transfected with the indicated constructs combined with empty vector to preserve a constant amount of transfected DNA in each sample. Fifty μm z-VAD-fmk was added immediately after transfection to the cells in the fourth panel. The cells were stained with PE-labeled anti-CD8 (Pharmingen) prior to analysis, and gated live cells were analyzed for GFP and CD8 expression. Viable transfected cells are the double positive cells in the right upper quadrant. E, either no peptide or 50 μm IETD peptide inhibitor that is specifically recognized by caspase-8 was added immediately after transfection with either pCEFL or CD8-C as inA–D. F, to determine the effect of Bcl-2, conventional MCF-7 cells or MCF-7 cells stably overexpressing Bcl-2 (21Brinkmann U. Mansfield E. Pastan I. Apoptosis. 1997; 2: 192-198Crossref PubMed Scopus (20) Google Scholar) were transiently transfected using pCMV-β-galactosidase with a 4-fold excess of vector alone or the indicated expression constructs (total DNA = 2 μg). CD8-nef contains the human immunodeficiency virus nef gene in place of the caspase coding sequence in CD8-C. Percent kill is the percentage of dark (transfected) cells that have a characteristic apoptotic morphology (illustrated in Fig. 4 B). Data are representative of three independent experiments.View Large Image Figure ViewerDownload (PPT) We next tested whether membrane targeting alone could induce caspase activation, by appending the myristoylation sequence from thesrc kinase onto the amino terminus of the caspase domain in an expression construct, Myr-C (17Lacal P.M. Pennington C.Y. Lacal J.C. Oncogene. 1988; 2: 533-537PubMed Google Scholar). Overexpression of Myr-C was only modestly more active than unmodified caspase-8 in inducing apoptosis (Fig. 2 B). We also prepared a chimera, Tac-C, between the interleukin-2 receptor α chain (Tac) extracellular and transmembrane domains and the caspase domain (18Letourneur F. Klausner R.D. Science. 1992; 255: 79-82Crossref PubMed Scopus (348) Google Scholar). Overexpression of Tac has been recently shown to cause self-association in the absence of ligand. 3D. Eicher and T. A. Waldmann, personal communication. Tac-C, similar to CD8-C, greatly augmented apoptosis, even without antibody cross-linking of the Tac moiety (Fig. 2 C), suggesting that spontaneous oligomerization of the extracellular domains of CD8 or Tac is sufficient to powerfully induce caspase-8 autoactivation and apoptosis. The cell death caused by CD8-C involved caspase activation, because either the z-VAD-fmk peptide (19Tomita Y. Kawasaki T. Bilim V. Takeda M. Takahashi K. Int. J. Can. 1996; 68: 132-135Crossref PubMed Scopus (27) Google Scholar) or the baculovirus p35 caspase inhibitors (20Clem R.J. Fechheimer M. Miller L.K. Science. 1991; 254: 1388-1390Crossref PubMed Scopus (707) Google Scholar) inhibited apoptosis (Fig. 2 D). Under caspase-8-inhibited conditions, we observed abundant surface CD8 staining on viable cells, which confirmed that the CD8-C chimera was actually expressed following transfection (Fig. 2 D). Moreover, we also tested an inhibitor peptide, z-IETD-fmk, based on the optimal sequence recognized by caspase-8 and found that this peptide completely abrogated apoptosis induced by CD8-C (Fig. 2 E). By contrast, the overexpression of Bcl-2 (21Brinkmann U. Mansfield E. Pastan I. Apoptosis. 1997; 2: 192-198Crossref PubMed Scopus (20) Google Scholar), which could protect against apoptosis mediators such as staurosporine, was incapable of protecting from programmed death due to direct caspase-8 activation by CD8-C (Fig. 2 F). The viral inhibitory protein MC159, which disrupts normal DISC formation and blocks CD95-induced apoptosis by preventing caspase-8 from binding to FADD (22Bertin J. Armstrong R.C. Ottilie S. Martin D.A. Wang Y. Banks S. Wang G.H. Senkevich T.G. Alnemri E.S. Moss B. Lenardo M.J. Tomaselli K.J. Cohen J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1172-1176Crossref PubMed Scopus (383) Google Scholar, 23Hu S. Vincenz C. Buller M. Dixit V.M. J. Biol. Chem. 1997; 272: 9621-9624Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), also did not inhibit apoptosis by CD8-C (data not shown), implying that the autoactivation of caspase-8 in our system did not require the formation or participation of the DISC. Also, brefeldin A was incapable of blocking CD8-C-mediated death, implying that oligomerization and caspase activation may occur in the membranes of early compartments prior to transport to the membrane at the cell surface (data not shown). An important biochemical feature of the CD8α extracellular domain is its ability to form disulfide-linked homodimers. Since no additional external cross-linking (such as by antibody) was required for the powerful apoptosis induction by CD8-C, we reasoned that dimerization was critical in promoting association and processing of the caspase precursors into an active form. To assess whether CD8-C had undergone dimerization, we analyzed detergent lysates from 293T cells that were transfected with a construct expressing a CD8-C linked to GFP (24Cormack B.P. Valdivia R.H. Falkow S. Gene (Amst.). 1996; 173: 33-38Crossref PubMed Scopus (2517) Google Scholar) (CD8-C-GFP), either alone or together with constructs expressing CD8-empty or an inactive CD8-C without the GFP tag (CD8-C:D210A/D216A, see below). Western blots with an anti-GFP mAb showed that CD8-C-GFP formed an apparent dimer complex (molecular mass = 170 kDa, lane 4) in nonreducing conditions, but only monomers (molecular mass = 85 kDa) in reducing conditions (Fig. 3 A). Coexpression of CD8-C-GFP and either an inactive CD8-C chain without the GFP tag or CD8-empty caused a decrease in the CD8-C-GFP homodimer and the appearance of apparent heterodimer complexes (molecular mass = 142 kDa, lanes 1, 2, and 5), which was absent without CD8-C-GFP. Similar results were obtained with coexpression of CD8-C-GFP and CD8-empty, with smaller heterodimeric complexes. Thus, consistent with the formation of disulfide-linked homodimers by native CD8α (15Irving B.A. Weiss A. Cell. 1991; 64: 891-901Abstract Full Text PDF PubMed Scopus (630) Google Scholar, 16Leahy D.J. Axel R. Hendrickson W.A. Cell. 1992; 68: 1145-1162Abstract Full Text PDF PubMed Scopus (241) Google Scholar), the CD8-C chimera formed dimers with itself and other CD8α expression proteins. To determine if dimerization was essential for caspase activation, we tested whether coexpression of CD8-empty or an inactive CD8-C chimera (D210A/D216A, see below) dominantly interfered with the ability of CD8-C to induce apoptosis. Cotransfection of these constructs confirmed this prediction (Fig. 3 B). We found that either CD8-empty or an inactive CD8-C chimera blocked the lethality of CD8-C in a dose-response fashion (Fig. 3 B). We next investigated how enzymatic processing of CD8-C could lead to apoptosis. With a single nucleotide change, we substituted serine for the catalytic site cysteine (CD8-C:C360S). This change completely abrogated apoptosis, indicating that the active site cysteine was indispensable (Fig. 4 A). We therefore assessed the functional importance of aspartate residues that reside at the site for cleaving the prodomain from the caspase domain (which have been preserved in both the CD8-C and Tac-C constructs) by mutations. We found that the substitution of alanines for the Asp210 and Asp216 residues unexpectedly blocked apoptosis by CD8-C (Figs. 1 and 4 C). The two aspartates were not equivalently important, since the D216A mutation only modestly reduced apoptosis, whereas mutation of Asp210 or both Asp210 and Asp216 completely inhibited apoptosis (Fig. 4 B). This striking effect was also observed with the corresponding mutations in the Tac-C chimera (Fig. 4 C and data not shown). Additionally we performed a Western blot of 293T cells transiently transfected with either CD8-C or the double mutant CD8-C D210/216A using a mAb specific for caspase-8 to examine processing of the caspase chimera. The wild-type CD8-C underwent cleavage into processed fragments, which were released into the soluble cytosolic fraction, whereas the double mutant chimera did not (Fig. 4 D). No cleavage products were found in the membrane-associated pellet for either the wild-type or mutant molecules (data not shown). Thus, even with an intact catalytic site cysteine, the lethality of the CD8-C or Tac-C chimeras is lost if the caspase cannot be proteolytically cleaved at the point of its association with the membrane. Caspase activation is a crucial biochemical event involved in most, if not all, forms of apoptosis, so it is of central importance to understand the activation requirements of these enzymes (1Nicholson D. Thornberry N. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2182) Google Scholar, 2Miller D.K. Semin. Immunol. 1997; 9: 35-49Crossref PubMed Scopus (194) Google Scholar). Our results show that membrane-associated oligomerization of caspase-8, the most proximal caspase in the CD95 signal cascade, is sufficient to powerfully induce apoptosis in several different cell types. Although previous studies have detected the cleaved caspase-8 prodomain in the "DISC" proteins aggregated with the cytoplasmic tail of CD95, these studies were limited by the fact that they did not determine the stoichiometry of the proteins to know if oligomerization of multiple caspase-8 molecules was likely to have occurred (8Muzio M. Chinnaiyan A.M. Kishchekel 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 (2741) Google Scholar, 9Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1787) Google Scholar, 10Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1995; 85: 803-815Abstract Full Text Full Text PDF Scopus (2111) Google Scholar, 11Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar). Furthermore, the previous two-dimensional gel analyses were descriptive and did not directly test the necessity of cleavage at various aspartate residues for apoptosis induction. Our data address these issues and suggest that there are two critical steps in the caspase-8 death pathway. First, we have shown that oligomerization is sufficient to activate the enzyme. Homodimers of the wild-type CD8-caspase induced death, whereas heterodimers between the wild-type and an inactive CD8 construct were unable to stimulate death. Second, we have shown that proteolytic cleavage of the active caspase at the point of its membrane association was also required. Death induced by the caspase chimera occurred spontaneously without cross-linking of the CD8 extracellular domain. Also our results suggest that apart from the ability of CD95 and FADD/MORT1 to bring the caspase-8 molecules together, the DISC complex is not required for autocatalytic activation. Thus our data suggest that dimerization of caspases favors the spontaneous generation of an unstable, but active, conformation that can initiate autoprocessing into a thermodynamically more stable caspase. Although our experiments demonstrate that membrane-linked oligomerization causes activation, it is very likely that oligomerization of the enzyme within the cytosol may also strongly activate caspase activity. The crystal structures of active caspase tetramers show that each partner in a pair of large or small subunit chains is positioned antiparallel with respect to the other (25Rotonda 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. Nat. Struct. 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Alternatively, dimers may enhance the cross-cleavage of precursor chains either by creating proximity, inducing a favorable orientation for processing, or by preventing the association of endogenous inhibitory proteins, such as cellular FLIP (28Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.L. Schroter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Crossref PubMed Scopus (2227) Google Scholar, 29Han D.K.M. Chaudhary P.M. Wright M.E. Friedman C. Trask B.J. Riedel R.T. Baskin D.G. Schwartz S.M. Hood L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11333-11338Crossref PubMed Scopus (222) Google Scholar). Importantly, we find that death programmed by caspase-8 dimerization is resistant to Bcl-2, which could explain the apparent resistance of CD95-induced apoptosis to Bcl-2 inhibition in many cell types (30Strasser A. Harris A.W. Huang D.C. Krammer P.H. Cory S. 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However, we have found that D374A/D384A mutants that prevent cleavage between p18 and p10 do not abrogate apoptosis (data not shown). Thus, proteolysis at D210/D216 plays a critical role that is different than other processing events. A leading possibility is that release of the active caspase from membrane association is important for apoptosis. Detachment of the caspase from the membrane may release the mature enzyme into the cytoplasm where it may catabolize apoptosis substrates that could be sequestered away from the cell membrane. Alternatively, cleavage away from the prodomain may also increase the enzymatic activity or stability; however, enzymatic activity has been found to be associated with the DISC complex (12Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1041) Google Scholar). 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