From the Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1892
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ABSTRACT |
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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.
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INTRODUCTION |
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A pivotal biochemical event of programmed cell death or apoptosis is the activation of cysteinyl, aspartate-specific proteases or caspases (1, 2). The caspase gene family in mammals includes at least 10 members that share protein sequence similarity to the ced3 cell death gene from Caenorhabditis elegans (3). The participation of caspases in programmed cell death is conserved widely in phylogeny from the nematode, C. elegans to humans (4). 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 (5). Caspase activation is defective in patients that have inherited mutations in CD95 and suffer from the autoimmune/lymphoproliferative syndrome (ALPS)1, 2 (6, 7). The activation of caspase-8 (FLICE/MACH) appears to be the first step in the cascade of apoptotic events induced by CD95 (5, 8). 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 (8-13). 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 (12). We therefore investigated the requirements of caspase-8 activation and apoptosis induction.
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EXPERIMENTAL PROCEDURES |
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Materials
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 (13), and p35-pCI and 3LacZ plasmids were provided by Dr. John Bertin, NIAID, NIH. The Taq and Pwo polymerases 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.
Methods
DNA Constructions--
The caspase-8 (MACH-1/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 as
HindIII-NotI fragments into digested pCEFL. The
vector pCEFL-CD8-EMPTY vector and the pCEFL-Myr vector containing the
src 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
209.
Site-directed Mutagenesis-- 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.
Cell Death Assays--
For all Jurkat transfections, plasmid
constructions (pCEFL, pCEFL-caspase-8, pCEFL-caspase-8 98,
pCEFL-caspase-8
209, 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 720
, 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.
Flow Cytometry Analysis-- 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.
Western Blotting-- 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.
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RESULTS AND DISCUSSION |
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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 (10, 13) (Fig.
2A). 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 (8-11). The caspase domain was genetically fused to the
transmembrane and extracellular portion of the human CD8 chain,
which is known to form disulfide-linked homodimers (14-16), and this
expression construct, CD8-C, was transfected into Jurkat cells (Figs. 1
and 2B). 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.
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We next tested whether membrane targeting alone could induce caspase
activation, by appending the myristoylation sequence from the
src kinase onto the amino terminus of the caspase domain in
an expression construct, Myr-C (17). Overexpression of Myr-C was only
modestly more active than unmodified caspase-8 in inducing apoptosis
(Fig. 2B). We also prepared a chimera, Tac-C, between the
interleukin-2 receptor chain (Tac) extracellular and transmembrane domains and the caspase domain (18). Overexpression of Tac has been
recently shown to cause self-association in the absence of ligand.3 Tac-C, similar to
CD8-C, greatly augmented apoptosis, even without antibody cross-linking
of the Tac moiety (Fig. 2C), 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 (19) or the baculovirus p35 caspase inhibitors (20) inhibited apoptosis (Fig. 2D). 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. 2D). 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. 2E). By contrast, the overexpression of Bcl-2 (21), 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. 2F). The viral inhibitory protein MC159, which disrupts normal DISC formation and blocks CD95-induced apoptosis by preventing caspase-8 from binding to FADD (22, 23), 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 CD8 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 (24)
(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.
3A). 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 CD8
(15, 16),
the CD8-C chimera formed dimers with itself and other CD8
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. 3B). We found
that either CD8-empty or an inactive CD8-C chimera blocked the
lethality of CD8-C in a dose-response fashion (Fig. 3B).
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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. 4A). 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 4C). 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. 4B). This striking effect was also observed with the corresponding mutations in the Tac-C chimera (Fig. 4C 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. 4D). 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.
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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 (1, 2). 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 (8-11). 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 (25, 26). Since our chimeras constrain the N termini of the precursors in a parallel relationship, the initial active pseudoconformation is likely to differ significantly from the final mature structure. Interestingly, despite the trimeric symmetry of tumor necrosis factor receptor and CD95 (27), CD8 dimers appear to be sufficient for caspase-8 activation. A dimer could allow p18 and p11 subunits from two chains to form an enzyme tetramer. 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 (28, 29). 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 (30, 31).
Although proteolytic separation of caspase-8 from its prodomain in the DISC complex has been observed after prolonged treatment with anti-CD95 antibody (11), the necessity of this event was unknown. We found that substitution of alanines for the aspartates at the junction between the prodomain and the caspase domain (D210A/D216A) completely blocked apoptosis. We show that the D210A/D216A double mutant also gave no evidence of processing, although we could not evaluate cleavage between p18 and p10, because the antibody used was directed against the p18 subunit. 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 (12). Unlike other membrane signaling pathways such as phosphorylation or inositol phosphate generation, caspase-8 processing is an irreversible signal that commits the cell's fate to apoptosis. These downstream biochemical events involving caspase-8 may be targets for genetic alterations in autoimmune/lymphoproliferative syndrome patients who do not have mutations in the CD95 protein itself (32).
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Rm. 11N311, Bldg. 10, Laboratory of Immunology, National Institutes of Health, 10 Center Dr.,
MSC1892, Bethesda, MD 20892-1892. Tel.: 301-496-6754; Fax:
301-402-8530; E-mail: lenardo{at}nih.gov.
1 The 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.
2 D. A. Martin and M. J. Lenardo, unpublished observations.
3 D. Eicher and T. A. Waldmann, personal communication.
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REFERENCES |
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