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J. Biol. Chem., Vol. 278, Issue 26, 24003-24010, June 27, 2003
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¶
From the
Research Center for Biomedicinal
Resources and Division of Life Science, PaiChai University, Daejeon 302-735,
Korea, and the Department of Life Science,
Kwangju Institute of Science and Technology, Kwangju 500-712, Korea
Received for publication, March 3, 2003 , and in revised form, March 26, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In addition to FADD and caspase-8, another DED-containing protein related to DISC has been reported. Viral FLICE inhibitory protein (v-FLIP) is composed of two DEDs and binds to the Fas·FADD complex and inhibits the recruitment of caspase-8 to Fas-DISC. A human homolog of v-FLIP has many different names c-FLIP, FLAME, I-FLICE, Casper, CASH, usurpin, MRIT, and CLARP, respectively (17–24). FLICE-associated huge protein (FLASH) is another protein with binding activity to the DEDs of caspase-8 and FADD through its DED-like domain and is a component of the Fas-DISC (25). In addition, FLASH enhances the activation of caspase-8 in Fas-mediated apoptosis. Thus, DED-containing proteins seem to modulate the apoptotic process.
Two different cell types in Fas signaling pathways have been identified (26). Type I cells are characterized by recruitment of caspase-8 to the DISC following Fas activation, leading to direct activation of downstream caspases, including caspase-3 and caspase-7. In type I cells, the blocking of mitochondrial apoptotic function by overexpression of Bcl-2 has no effect on caspase activation. In type II cells, the amount of active caspase-8 generated in the DISC is low. In addition, DISC formation in type II cells is strongly reduced, and overexpression of Bcl-2 or Bcl-XL blocks caspase-8 and caspase-3 activation. Thus, Fas-mediated apoptosis in type II cells is dependent on mitochondrial activity.
FAF1 is a Fas-associating molecule, which enhances Fas-mediated apoptosis (27). In our previous work, mere intrinsic overexpression of FAF1 initiated apoptosis in the absence of extrinsic death signals in BOSC23 cells (28). This apoptotic potential required the region comprising amino acids 181–381 of FAF1. Mouse FAF1 (mFAF1), however, was able to enhance but unable to initiate apoptosis in L-cells (27). Thus, the apoptotic potential of FAF1 is not clear. The N terminus of FAF1 binds to the DD of Fas even though it does not contain the typical death domain (29). Although FAF1 contains domains found in the proteins of ubiquitination pathway, the function of FAF1 in relation to ubiquitin is largely unknown. FAF1 localizes in the nucleus, the perinuclear cytoplasm, and the nucleoli depending on the cell type (28, 30, 31). FAF1 is not only a binding partner of protein kinase CK2 but also a substrate target for serine residues 289 and 291 (31, 32). Also, the interaction of protein kinase CK2 and FAF1 is enhanced in vivo upon induction of apoptosis (33).
In this study, we demonstrate that FAF1 is a component of Fas-DISC, and DISC is formed by interaction of the DED-like region (amino acid 181–381 of FAF1) of FAF1 and the DEDs of caspase-8 and FADD. Therefore, this study provides a molecular explanation regarding the proapoptotic role of FAF1 in Fas-mediated signaling.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmids—Construction of the HA-tagged FADD, GST-fused FAF1,
GFP-tagged FAF1, pcDNA3/FAF1, FLAG-tagged FAF1, and FLAG-tagged
FAF1-(181–650), which where named to FAF1-
FID, was reported
previously (28,
29,
34). FLAG-tagged
FAF1-(366–650) was cloned by PCR using FLAG-tagged FAF1 as a template
and named to FAF1-FID·DEDID. pcDNA3/FAF1-DEDID was
generated by PCR using the pJG4–5/FAF1(s)
(29). FAF1(s) is an endogenous
isoform of FAF1 with an internal in-frame deletion that overlaps most of the
DEDID (FAF1-DEDID corresponds to amino acids 181–381 of FAF1, and amino
acids 188–339 of FAF1 is deleted in FAF1(s)). The construction of
His-tagged caspase-8 (pET21b/caspase-8) and His-tagged caspase-8C
(pET21b/caspase-8C) was reported previously
(35). To clone the FAF1
deletion mutants (FAF1-(1–305), FAF1-FID, and FAF1-DEDID) into
glutathione S-transferase (GST) fusion vector (Amersham Biosciences,
Uppsala, Sweden), PCRs with primers containing EcoRI and
XhoI linkers were performed using GST-FAF1 full-length cDNA as a
template. The pcDNA3/caspase-8 DED construct was generated by PCR using the
PET21b/caspase-8 plasmid as a template with primers containing
HindIII and BamHI linkers. To subclone the FADD and its
deletion mutants (FADD-DD and FADD-DED) into GST fusion vector, PCRs were
performed using pcDNA3/FADD plasmid as a template. pcDNA3.1/FADD-DED was
kindly provided by Dr. S. H. Kim (Seoul National University, Korea).
In Vitro Protein Binding—GST fusion proteins were expressed
in Escherichia coli BL21(DE3) with
isopropyl-
-D-thiogalactopyranoside induction. Subsequently,
cells were sonicated in ice-cold lysis buffer (200 mM Tris-Cl, pH
8.0, 0.5 M NaCl, 100 µM EDTA, 0.1% Triton X-100, 0.4
mM phenylmethylsulfonyl fluoride). The GST fusion proteins were
incubated with glutathione-coated beads for 4 h at 4 °C and normalized for
protein concentration. The pcDNA3/FAF1, pcDNA3/FADD, pET21b/caspase-8,
pET21b/caspase-8C, and pcDNA3/caspase-8 DED were translated in vitro
with TNT® Quick Coupled Transcription/Translation Systems
(Promega, Madison, WI). Briefly, 2 µg of DNA was incubated with 20 µCi
of [35S]methionine in the TNT® Quick Master mix for
90 min at 30 °C. In vitro translated products were mixed with
GST-fused proteins bound onto glutathione-coated beads in the binding buffer
(50 mM Hepes, pH 7.6, 50 mM NaCl, 5 mM EDTA,
0.1% Nonidet P-40, 10% glycerol) and then incubated for 4 h at 4 °C. After
washing three times in the lysis buffer, samples were treated with SDS-loading
buffer containing 5% -mercaptoethanol. The samples were loaded onto an
SDS-PAGE gel and visualized by using a BAS analyzer (Fuji Photo Film Co.,
Tokyo, Japan).
Cell Culture—Jurkat cells were all maintained in RPMI 1640 (Jeil Biotechservices Inc., Daegu, Korea), antibiotic-antimycotic (100 units/ml penicillin G sodium, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B) (Invitrogen, Grand Island, NY) and 10% fetal calf serum (Invitrogen) in 5% CO2. For immunoprecipitation assay using anti-FADD antibody (N-18), anti-FAF1 monoclonal antibody, and control antibody, IgG1, (BD Biosciences, San Jose, CA), 1 x 107 Jurkat cells were cultured and serum-starved with RPMI 1640 medium without fetal bovine serum for 12 h. Cells were harvested at 1 h after 50 ng/ml anti-Fas (CH-11) antibody treatment together with 5 µg/ml cycloheximide (CHX) and lysed. The antibodies and protein A/G-Sepharose beads were added to the samples and incubated for2hat4 °C. The beads were washed four times with lysis buffer and subjected to either SDS-PAGE or immunoblotting.
Western Blotting—Samples were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) using an electrotransporter (OWL, Portsmouth, PA). Membranes were blocked for 1 h in Tris-buffered saline containing 0.05% Tween 20, 2 mM CaCl2 dihydrate, 0.01% antifoam A, and 5% milk powder. After incubation at 4 °C with anti-FADD (A66–2), anti-FAF1 monoclonal, or anti-caspase-8 (IC12) antibodies, membranes were washed in Tris-buffered saline containing 0.05% Tween 20, 2 mM CaCl2 dihydrate, 0.01% antifoam A, and 5% milk powder. Membranes were incubated with 1:2000 dilutions of horseradish peroxidase-conjugated secondary antibodies. Membrane were washed and developed with ECL plus Western blotting detection reagents (Amersham Biosciences, Uppsala, Sweden).
DISC Immunoprecipitation—1 x 107 Jurkat cells were cultured for 24 h at 37 °C in RPMI 1640 medium containing 10% fetal bovine serum and antibiotics. After 24 h, the culture medium was exchanged with serum-free RPMI 1640 medium containing antibiotics and cultured for 12 h. After 12 h, cells were harvested at different times after anti-Fas antibody (50 ng/ml) treatment together with 5 µg/ml CHX and lysed in Triton X-100 lysis buffer. As a control, anti-Fas antibody was added to lysates prepared from the unstimulated cells. Lysate protein content was determined by Bradford assay and equalized prior to immunoprecipitation. The anti-Fas (APO-1) and protein A/G-Sepharose beads were added to the samples and incubated for 2 h at 4 °C. The beads were washed four times with lysis buffer and subjected to either SDS-PAGE or immunoblotting.
Cell Death Assay—Jurkat cells were transfected using either LipofectAMINE reagent (Invitrogen, Scotland, UK) or NucleofactorTM solution provided by using an Amaxa apparatus (Amaxa, Cologne, Germany). For using the Amaxa system, 5 x 106 Jurkat cells were harvested, washed once in PBS buffer, and resuspended in 100 µl of specified electroporation buffer. Then, 1 µg of plasmid DNA was mixed, transferred to a cuvette, and nucleofected with an Amaxa NucleofectorTM apparatus. After 30 h, cells were treated with 50 ng/ml anti-Fas antibody for 1 h. Cells were fixed in 3.7% formaldehyde for 15 min and mounted on slides using xylene substitute mountant/histomount solution, stained with 4',6-diamidino-2-phenylindole, dihydrochloride (Molecular Probes, Eugene, OR), and analyzed by microscopy. For using LipofectAMINE reagent, 5 x 106 cells of three different types of Jurkat cells (A3; parental, I2.1; FADD (–/–), and I9–2; caspase-8 (–/–)) (36) were transiently transfected with GFP-tagged FAF1 using LipofectAMINE reagent. Cells were fixed and mounted on the slides as above. The percentage of apoptotic cells was analyzed based on the morphological change of cells under a fluorescence microscope. Excitation and emission wavelengths for GFP were 488 and 505 nm, respectively. Existence of GFP-FAF1, caspase-8, and FADD was assessed by immunoblot analysis.
Indirect Immunofluorescence—For localization analysis of Fas and FAF1, Jurkat cells with or without stimulation of Fas together with CHX were plated on a poly(A)-lysine-coated coverglass. They were fixed and rinsed with 1x PBS containing 10% normal serum (Sigma). They were incubated with anti-Fas (C-20) or anti-FAF1 (M-20) antibody for 1 h, washed twice with 1x PBS, and incubated with secondary antibody conjugated with FITC (Sigma) together with RNase and propidium iodide for 30 min. They were washed three times with 1x PBS and mounted on slides using xylene substitute mountant/histomount solution.
For colocalization analysis of FAF1 and Fas-DISC, Jurkat cells with or without stimulation of Fas together with CHX were plated on a poly(A)-lysine-coated coverglass. They were fixed and rinsed with 1x PBS containing 10% normal serum. Cells were incubated with anti-Fas (C-20) antibodies for 1 h, washed twice with 1x PBS, and incubated with secondary anti-rabbit IgG antibodies conjugated with FITC for 1 h. The cells were washed three times with 1x PBS. Cells were incubated with anti-FAF1 (M-20), anti-FADD (N-18), anti-caspase-8 (N-19), and anti-PARP (F-2) antibodies for 1 h, respectively, washed twice with 1x PBS, and incubated with each secondary anti-IgG antibodies conjugated with TRITC (Sigma) for 1 h. Cells were mounted on slides using xylene substitute mountant/histomount solution. Dual color images were acquired using a Radiance 2000 confocal microscope (Bio-Rad, Richmond, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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FAF1 Interacts with FADD and Caspase-8 in Jurkat Cells—To show that FAF1 interacts with FADD and caspase-8 in mammalian cells, endogenous FAF1 and FADD were immunoprecipitated by the anti-FAF1 monoclonal antibody and the anti-FADD (N-18) antibody, respectively. The immunoprecipitates were analyzed by Western blot using antibodies against caspase-8 and FADD, respectively. As shown in Fig. 1B, each Fas-DISC member was detected in the immunoprecipitates, whereas it was not detected in the isotype control, IgG immunoprecipitates. Thus, the interaction of Fas-DISC components and FAF1 was demonstrated in vitro and in vivo, and these results show direct association of FAF1 with DISC members.
The Apoptotic Effector Domain (Amino Acids 181–381) of FAF1 Binds to the DED Domains of FADD and Caspase-8 — Based on the fact that Fas-DISC complex is formed by interactions between their protein domains, we investigated which FAF1 domains were required for the binding to the Fas-DISC members. To map the binding domains to Fas-DISC proteins, an in vitro binding assay was performed using various deletion constructs (Figs. 1, C–F, and 2A). As shown in Fig. 1 (C and D), GST-FAF1-(181–381), corresponding to the previously known apoptotic effector domain (28), was sufficient to interact with in vitro translated FADD (Fig. 1C). The in vitro translated FAF1 was sufficient to bind to GST-FADD-DED but not to GST-FADD-DD (Fig. 1D). In binding analysis between FAF1 and caspase-8, GST-FAF1-(181–381) was also mapped as the caspase-8 interacting region (Fig. 1E). Among deletion constructs of caspase-8, the DED of caspase-8 was sufficient to bind to FAF1 but not to the C terminus of caspase-8 where the catalytic activity resides (Fig. 1F). The N terminus (amino acids 1–201) of FAF1 was previously mapped as the binding site of Fas (29). Thus, our data show that FAF1 has Fas- and caspase-8-interacting domains in tandem like FADD.
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The DED-interacting Domain of FAF1-(181–381) Contains Several Helices—As shown above, the region of amino acids 181–381 of FAF1 was mapped as the interacting domain with the DED domains of FADD and caspase-8 (Figs. 1, C–F, and 2A). Thus, we investigated whether the region of amino acids 181–381 of FAF1 has sequence similarities to the DED domains of FADD and caspase-8. Structure-based alignment analysis of FAF1 revealed that the amino acid similarity of the conventional DED domains to FAF1-(181–381) was not significant (Fig. 2B). In addition, the consensus amino acid residues (RXDL) found in the DEDs (37) were not conserved in FAF1-(181–381). Thus, FAF1-(181–381) is named as DEDID (DED-interacting domain) henceforth. The secondary structure analysis of FAF1-(181–381) with protein data base (www2.protein.Osaka-u.ac.jp) revealed that FAF1-(181–381) might have several helical structures (Fig. 2B). The helical structures of DED domains seemed to be important in the protein-protein interaction (37, 38).
FAF1 Is Preassociated with the Fas-DISC before Fas Activation—The DED- or DED-like-domain-containing proteins, including FADD, caspase-8, and FLASH, formed the Fas-DISC through homotypic domain interactions via DEDs. Because FAF1 interacts with Fas-DISC members and has a DEDID, we questioned if FAF1 was a member of Fas-DISC. To determine whether FAF1 is present in the Fas-DISC, Fas-DISCs were immunoprecipitated with an agonistic human antibody against Fas (anti-APO-1 antibody) in Jurkat cells before and after stimulation of Fas with the agonistic anti-Fas monoclonal antibody. As shown in Fig. 3, endogenous FAF1 was already present in the Fas-DISC before Fas activation but not caspase-8. However, caspase-8 was recruited to the Fas-DISC after Fas activation. In addition, the association of FAF1 with the Fas-DISC was enhanced at 1 h after treatment of Fas antibody and gradually weakened to the resting cell level as apoptosis proceeded. We also observed association of FAF1 with the Fas-DISC in H9 cells (Type 1 cells) as well (data not shown).
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FAF1 Colocalizes with Fas-DISC to the Cytoplasmic Membrane—It has been previously shown that Fas-DISC is formed in the cytoplasmic membrane via homotypic interactions. Previously, FAF1 has been reported to localize in diverse subcellular positions such as the nucleus, perinuclear cytoplasm, and nucleoli depending on the cell type (28, 30, 31). We found that FAF1 localized to the cytoplasm in NIH3T3, BOSC23, and HeLa cells known to be less sensitive to Fas-DISC-mediated apoptosis (data not shown). By confocal microscopic analysis using the antibodies against endogenous FAF1 and Fas, respectively, both FAF1 and Fas localized in the cytoplasmic membrane in cells that were not treated with anti-Fas antibody (Fig. 4, A and B). Then, the localization of Fas and FAF1 was monitored from 1 to 4 h after Fas treatment. As shown in Fig. 4A, clustering of Fas, which agrees with a previous report (39), and FAF1 was detected after 1 h of Fas stimulation with anti-Fas antibody. After 2 h, almost all cells showed the diffuse pattern of Fas and FAF1 in the cytoplasm (Fig. 4, A and C).
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To test whether Fas-DISC and FAF1 colocalize in cells before and after stimulation of Fas with the agonistic anti-Fas monoclonal antibody, analysis of colocalization was performed by confocal microscopy using antibodies against endogenous FAF1, Fas, FADD, and caspase-8 in Jurkat cells. As shown in Fig. 4 (B and C), endogenous FAF1, FADD, and caspase-8 completely colocalized with Fas in cells treated and not treated with anti-Fas antibody. PARP was detected in the nucleus as expected.
FAF1-DEDID Is Essential for the Mediation of Fas-induced
Apoptosis—To know if DEDID performs critical functions in the
Fas-induced apoptosis, the DEDID deletion mutants were prepared and
transiently transfected using NucleofectorTM Solution V provided by Amaxa
in Jurkat cells. As shown in Fig.
5, mere overexpression of both FAF1-DEDID and
FAF1-FID·DEDID mutants failed to induce apoptosis, whereas those
of FAF1 and FAF1-FID induced apoptosis. In addition, transfection of
FAF1-FID in which Fas interacting region is deleted inhibited
Fas-mediated apoptosis significantly as expected. Moreover, transfection of
FAF1-DEDID and FAF1-FID·DEDID, respectively, inhibited
Fas-mediated apoptosis significantly as well. These data suggest that DEDID
deletion mutants act as dominant negatives in the Fas-mediated apoptotic
pathway. Thus, it has been demonstrated that the DEDID is essential in
mediating Fas-induced apoptosis.
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Effect of FAF1 in FADD- and Caspase-8-deficient Jurkat Cells—FADD and caspase-8 are necessary for Fas receptor-mediated cell death. We therefore evaluated the requirement of caspase-8 and FADD for FAF1-induced cell death. To this end, GFP-tagged FAF1 was transiently transfected by LipofectAMINE reagent in parental Jurkat cells (A3), FADD-deficient Jurkat cells (I2.1), and caspase-8-deficient Jurkat cells (I9–2). As shown in Fig. 6A, transient transfection of the GFP-tagged FAF1 in parental Jurkat cells (52 ± 6%) efficiently induced morphological changes, consistent with apoptosis, when compared with that in vector-transfected parental Jurkat cells (5 ± 0.8%). However, as compared with results in FAF1-transfected parental Jurkat cells, cell death by FAF1 in either FADD-deficient (19 ± 3%) or caspase-8-deficient (24 ± 4%) cells was significantly suppressed. In all experiments, GFP vector itself induced only slight apoptosis (<5%). The ability of FAF1 to induce full-blown apoptosis required FADD and caspase-8. The fact that overexpression of FAF1 still induced a significant extent of apoptosis (19–24%) in FADD- and caspase-8-deficient Jurkat cells suggests that FAF1 might have another mechanism of cell death besides the Fas-DISC-mediated apoptosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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FAF1 interacts with caspase-8 and FADD. The interaction occurs among the
DEDID of FAF1 and the DEDs of caspase-8 and of FADD. Apoptotic signal
transmissions via homotypic interaction domains such as death domain, death
effector domain, caspase recruitment domain, and DED-recruiting domain have
been reported (40). Most of
the caspase-8-interacting proteins interact via their DED motifs
(21,
40–42).
However, death effector domain-associated factor and FLASH interact with DED
proteins without having a DED
(25,
43). Likewise, FAF1 here is
presented as another example of a protein interacting with the DEDs of
caspase-8 and FADD without having a typical DED. The DED has a structure
composed of six highly conserved 
-helices. Even if FAF1 does not show
significant homology with DED by amino acid sequence analysis, the DEDID of
FAF1 has several potential -helices when analyzed by the protein data
base
(www2.protein.Osakau.ac.jp)
like DEDs. Thus, crystal structural analysis of DEDID of FAF1 would be able to
provide information regarding its relevance to DED or DED-like domains.
Recently, FLASH has been reported as a member of Fas-DISC (25). FLASH contains a DED-recruitment domain that interacts with the DED domains of caspase-8 and FADD, and FLASH is involved in association of FADD and caspase-8 upon Fas stimulation. However, FLASH does not interact with either full-length Fas or its death domain. Unlike FLASH, FAF1 directly interacts with Fas-DD as well as FADD- and caspase-8-DEDs. FAF1 is much bigger than FADD even though they have similar protein-interacting characteristics. Thus, it is conceivable that FAF1 might play a role as a scaffolding protein that tethers Fas/FADD/caspase-8 signaling modules. Further studies of the interaction profile of FAF1 with other Fas-DISC members such as FLASH will provide us information of the putative role of FAF1 as a scaffold.
Two different mechanisms exist regarding formation of Fas-DISC (26). In type I cells, Fas-DISC is preformed without Fas activation and enhanced by Fas activation. In type II cells, the DISC is formed upon Fas activation. Thus, Fas-mediated apoptosis rapidly progresses in type I cells (25, 26). FLASH is only recruited to Fas upon Fas activation in both type I and II cells (25). In this study, we showed that FAF1 was already interacting with Fas before Fas activation in both type I (data not shown) and type II cells. In addition, the association of FAF1 with Fas was enhanced during cell death signaling upon Fas activation. Thus, these results suggest that FAF1 might serve as an early seed for the fast formation of DISC.
Interaction of FADD and caspase-8 has been reported to occur through
homophilic interaction between the DEDs of FADD and caspase-8
(2,
9). The FADD mutant, lacking
N-terminal amino acids 1–79 (DED), functions as a dominant-negative
inhibitor of Fas- and tumor necrosis factor-mediated apoptosis. In this study,
FAF1-DEDID lacking caspase-8 binding domain functioned similarly. The
dominant-negative effect of FAF1-DEDID on Fas-induced cell death
testifies to the essentiality of DEDID in mediating Fas-induced cell death.
FAF1-DEDID corresponds to the FAF1(s), an endogenous short isoform FAF1
in which most of DEDID is deleted
(29). The cDNAs of FAF1(s)
have been found both in the human liver and in the HeLa cell cDNA libraries
even though the protein has not yet been detected by Western analysis. Thus,
FAF1(s), if translated, would be able to function as an endogenous inhibitor
against FAF1 in Fas-induced apoptosis.
The FID of FAF1 interacts with Fas (29), and the DEDID of FAF1 interacts with the DED of caspase-8. In this respect, FAF1 is similar to the adapter protein FADD. Moreover, both FAF1 and FADD have their interacting modules in tandem. It is conceivable that FAF1 exists in Fas-DISC to amplify the aggregation of caspase-8 via a FADD-like mechanism. We expect a cooperative relationship between FADD and FAF1 in the formation of Fas-DISC, because diminution of apoptosis by FAF1 occurred in FADD-deficient Jurkat cells.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Division of Life Science, PaiChai University, 439-6 Doma-2-dong, Seo-gu, Daejeon, 302-735, Korea. Tel./Fax: 82-42-520-5463; E-mail: ehkim{at}mail.pcu.ac.kr.
1 The abbreviations used are: DD, death domain; FADD, Fas-associated death
domain protein; DED, death effector domain; Fas-DISC, Fas-death-inducing
signaling complex; FLICE, FADD-like interleukin-1-converting enzyme;
FLASH, FLICE-associated huge protein; FITC, fluorescein isothiocyanate; PARP,
poly(ADP-ribose) polymerase; TRITC, tetramethylrhodamine isothiocyanate; GST,
glutathione S-transferase; GFP, green fluorescence protein; CHX,
cycloheximide; PBS, phosphate-buffered saline; DEDID, DED-interacting domain;
FID, Fas-interacting domain.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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utter, M. G. (2002)
FEBS Lett. 527,
250–254[CrossRef][Medline]
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