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Fas-associated Factor 1, FAF1, Is a Member of Fas Death-inducing Signaling Complex

2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês

10.1074/jbc.m302200200

ISSN

1083-351X

Autores

Seung-Wook Ryu, Soo-Jin Lee, Min-Young Park, Joon-il Jun, Yong‐Keun Jung, Eunhee Kim,

Tópico(s)

NF-κB Signaling Pathways

Resumo

FAF1 has been introduced as a Fas-binding protein. However, the function of FAF1 in apoptotic execution is not established. Based on the fact that FAF1 is a Fas-binding protein, we asked if FAF1 interacted with other members of the Fas-death-inducing signaling complex (Fas-DISC) such as Fas-associated death domain protein (FADD) and caspase-8. FAF1 could interact with caspase-8 and FADD in vivo as well as in vitro. The death effector domains (DEDs) of caspase-8 and FADD interacted with the amino acid 181–381 region of FAF1, previously known to have apoptotic potential. Considering that FAF1 directly binds to Fas and caspase-8, FAF1 shows similar protein-interacting characteristics to that of FADD. In the coimmunoprecipitation with an anti-Fas antibody (APO-1) in Jurkat cells, endogenous FAF1 was associated with the precipitates in which caspase-8 was present. By confocal microscopic analysis, both Fas and FAF1 were detected in the cytoplasmic membrane before Fas activation, and in the cytoplasm after Fas activation. FADD and caspase-8 colocalized with Fas in Jurkat cells validating the presence of FAF1 in the authentic Fas-DISC. Overexpression of FAF1 in Jurkat cells caused significant apoptotic death. In addition, the FAF1 deletion mutant lacking the N terminus where Fas, FADD, and caspase-8 interact protected Jurkat cells from Fas-induced apoptosis demonstrating dominant-negative phenotype. Cell death by overexpression of FAF1 was suppressed significantly in both FADD- and caspase-8-deficient Jurkat cells when compared with that in their parental Jurkat cells. Collectively, our data show that FAF1 is a member of Fas-DISC acting upstream of caspase-8. FAF1 has been introduced as a Fas-binding protein. However, the function of FAF1 in apoptotic execution is not established. Based on the fact that FAF1 is a Fas-binding protein, we asked if FAF1 interacted with other members of the Fas-death-inducing signaling complex (Fas-DISC) such as Fas-associated death domain protein (FADD) and caspase-8. FAF1 could interact with caspase-8 and FADD in vivo as well as in vitro. The death effector domains (DEDs) of caspase-8 and FADD interacted with the amino acid 181–381 region of FAF1, previously known to have apoptotic potential. Considering that FAF1 directly binds to Fas and caspase-8, FAF1 shows similar protein-interacting characteristics to that of FADD. In the coimmunoprecipitation with an anti-Fas antibody (APO-1) in Jurkat cells, endogenous FAF1 was associated with the precipitates in which caspase-8 was present. By confocal microscopic analysis, both Fas and FAF1 were detected in the cytoplasmic membrane before Fas activation, and in the cytoplasm after Fas activation. FADD and caspase-8 colocalized with Fas in Jurkat cells validating the presence of FAF1 in the authentic Fas-DISC. Overexpression of FAF1 in Jurkat cells caused significant apoptotic death. In addition, the FAF1 deletion mutant lacking the N terminus where Fas, FADD, and caspase-8 interact protected Jurkat cells from Fas-induced apoptosis demonstrating dominant-negative phenotype. Cell death by overexpression of FAF1 was suppressed significantly in both FADD- and caspase-8-deficient Jurkat cells when compared with that in their parental Jurkat cells. Collectively, our data show that FAF1 is a member of Fas-DISC acting upstream of caspase-8. Apoptosis requires the transmission of apoptotic signals from the plasma membrane receptors to caspases. In receptor-mediated apoptosis, apoptotic initiation is due to the formation of a protein-signaling complex that involves the physical association of caspases followed by their activation (1Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Google Scholar, 2Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 6: 2794-2804Google Scholar). Fas, a member of tumor necrosis factor receptor superfamily, has an intracellular death domain (DD) 1The 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-1β-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. in its cytoplasmic region (3Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Google Scholar, 4Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Google Scholar, 5Krammer P.H. Nature. 2000; 407: 789-795Google Scholar, 6Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Google Scholar). The DD is essential for the transduction of apoptotic signal. The Fas-associated death domain protein (FADD) binds to the DD of Fas through its DD in the C terminus (7Boldin M.P. Varfolomeev E.E. Pancer Z. Mett I.L. Camonis J.H. Wallach D. J. Biol. Chem. 1995; 270: 7795-7798Google Scholar, 8Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Google Scholar). In addition to DD, FADD has a DED at its N terminus, another protein interaction module. Therefore, FADD can recruit caspase-8 to the DISC by homotypic interactions between the DEDs of FADD and caspase-8 (2Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 6: 2794-2804Google Scholar, 9Muzio M. Chinnaiyan A.M. Kischkel 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: 817-827Google Scholar). The Fas-DISC formation is the first event that occurs during Fas-initiated cell death signaling. Following Fas-DISC formation, caspase-8 subsequently can be activated by autocleavage leading to the release of the active subunits p18 and p10 (2Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 6: 2794-2804Google Scholar, 10Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Google Scholar). In addition, the activated caspase-8 activates downstream effector caspases such as caspase-3, caspase-6, and caspase-7 (11Fernandes-Alnemri T. Takahashi A. Armstrong R. Krebs J. Fritz L. Tomaselli K.J. Wang L. Yu Z. Croce C.M. Salveson G. Cancer Res. 1995; 55: 6045-6052Google Scholar, 12Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Google Scholar, 13Hasegawa J. Kamada S. Kamiike W. Shimizu S. Imazu T. Matsuda H. Tsujimoto Y. Cancer Res. 1996; 56: 1713-1718Google Scholar, 14Schlegel J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Google Scholar, 15Kamada S. Washida M. Hasegawa J. Kusano H. Funahashi Y. Tsujimoto Y. Oncogene. 1997; 15: 285-290Google Scholar, 16Takahashi A. Hirata H. Yonehara S. Imai Y. Lee K.K. Moyer R.W. Turner P.C. Mesner P.W. Okazaki T. Sawai H. Kishi S. Yamamoto K. Okuma M. Sasada M. Oncogene. 1997; 14: 2741-2752Google Scholar). 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 (17Goltsev Y.V. Kovalenko A.V. Arnold E. Varfolomeev E.E. Brodianskii V.M. Wallach D. J. Biol. Chem. 1997; 272: 19641-19644Google Scholar, 18Han D.K. 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: 11333-11338Google Scholar, 19Hu S. Vincenz C. Ni J. Gentz R. Dixit V.M. J. Biol. Chem. 1997; 272: 17255-17257Google Scholar, 20Inohara N. Koseki T. Hu Y. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10717-10722Google Scholar, 21Irmler 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: 190-195Google Scholar, 22Shu H.B. Halpin D.R. Goeddel D.V. Immunity. 1997; 6: 751-763Google Scholar, 23Srinivasula S.M. Ahmad M. Ottilie S. Bullrich F. Banks S. Wang Y. Fernandes-Alnemri T. Croce C.M. litwack G. Tomaselli K.J. Armstrong R.C. Alnemri E.S. J. Biol. Chem. 1997; 272: 18542-18545Google Scholar, 24Rasper D.M. Vaillancourt J.P. Hadano S. Houtzager V.M. Seiden I. Keen S.L. Tawa P. Xanthoudakis S. Nasir J. Martindale D. Koop B.F. Peterson E.P. Thornberry N.A. Huang J. MacPherson D.P. Black S.C. Hornung F. Lenardo M.J. Hayden M.R. Roy S. Nicholson D.W. Cell Death Differ. 1998; 5: 271-288Google Scholar). 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 (25Imai Y. Kimura T. Murakami A. Yajima N. Sakamaki K. Yonehara S. Nature. 1999; 398: 777-785Google Scholar). 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 (26Scaffidi C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Google Scholar). 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 (27Chu K. Niu X. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11894-11898Google Scholar). In our previous work, mere intrinsic overexpression of FAF1 initiated apoptosis in the absence of extrinsic death signals in BOSC23 cells (28Ryu S.W. Kim E. Biochem. Biophys. Res. Commun. 2001; 286: 1027-1032Google Scholar). 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 (27Chu K. Niu X. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11894-11898Google Scholar). 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 (29Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar). 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 (28Ryu S.W. Kim E. Biochem. Biophys. Res. Commun. 2001; 286: 1027-1032Google Scholar, 30Fröhlich T. Risau W. Flamme I. J. Cell Sci. 1998; 111: 2353-2363Google Scholar, 31Jensen H.H. Hjerrild M. Guerra B. Larsen M.R. Hojrup P. Boldyreff B. Int. J. Biochem. Cell Biol. 2001; 33: 577-589Google Scholar). FAF1 is not only a binding partner of protein kinase CK2 but also a substrate target for serine residues 289 and 291 (31Jensen H.H. Hjerrild M. Guerra B. Larsen M.R. Hojrup P. Boldyreff B. Int. J. Biochem. Cell Biol. 2001; 33: 577-589Google Scholar, 32Kusk M. Ahmed R. Thomsen B. Bendixen C. Issinger O.G. Boldyreff B. Mol. Cell. Biochem. 1999; 191: 51-58Google Scholar). Also, the interaction of protein kinase CK2 and FAF1 is enhanced in vivo upon induction of apoptosis (33Guerra B. Boldyreff B. Issinger O.G. Int. J. Oncol. 2001; 19: 1117-1126Google Scholar). 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. Materials and Reagents—For confocal microscopy, antibodies against FAF1 (M-20), Fas (C-20), FADD (N-18), caspase-8 (N-19), and PARP (F-2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against FITC-conjugated goat anti-rabbit and rabbit anti-goat, and TRITC-conjugated rabbit anti-goat were purchased from Sigma (St. Louis, MO). For Western analysis, antibodies against FADD (A66–2) and caspase-8 (IC12) were purchased from Transduction Laboratories (Lexington, KY) and Cell Signaling Technology (Beverly, MA), respectively. FAF1 monoclonal antibody was a gift from Dr. J. S. Lim (Korea Research Institute of Bioscience and Biotechnology, Korea). For stimulation of Fas, antibody against Fas (CH-11) was purchased from Upstate Biotechnology (Lake Placid, NY). For immunoprecipitation of DISC, antibody against Fas (APO-1) kindly provided by Dr. P. H. Krammer (German Cancer Research Center, Heidelberg, Germany) was used. Horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma. The glutathione beads and Ni2+-nitrilotriacetic acid beads were purchased from Peptron (Daejeon, Korea) and Qiagen (Heidelberg, Germany), respectively. The protein A/G-agarose beads were purchased from Santa Cruz Biotechnology. All restriction enzymes were purchased from TaKaRa Shuzo Co. (Shiga, Japan). 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-ΔFID, was reported previously (28Ryu S.W. Kim E. Biochem. Biophys. Res. Commun. 2001; 286: 1027-1032Google Scholar, 29Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar, 34Ryu S.W. Chae S.K. Kim E. Biochem. Biophys. Res. Commun. 2000; 279: 6-10Google Scholar). FLAG-tagged FAF1-(366–650) was cloned by PCR using FLAG-tagged FAF1 as a template and named to FAF1-ΔFID·DEDID. pcDNA3/FAF1-ΔDEDID was generated by PCR using the pJG4–5/FAF1(s) (29Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar). 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 (35Kim I.K. Chung C.W. Woo H.N. Hong G.S. Nagata S. Jung Y.K. Biochem. Biophys. Res. Commun. 2000; 277: 311-316Google Scholar). To clone the FAF1 deletion mutants (FAF1-(1–305), FAF1-ΔFID, 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-β-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% β-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 × 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 × 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 Nucleofactor™ solution provided by using an Amaxa apparatus (Amaxa, Cologne, Germany). For using the Amaxa system, 5 × 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 Nucleofector™ 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 × 106 cells of three different types of Jurkat cells (A3; parental, I2.1; FADD (–/–), and I9–2; caspase-8 (–/–)) (36Juo P. Woo M.S.A. Kuo C. Signorelli P. Biemann H.P. Hannun Y.A. Blenis J. Cell Growth Differ. 1999; 10: 797-804Google Scholar) 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 1× 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 1× 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 1× 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 1× PBS containing 10% normal serum. Cells were incubated with anti-Fas (C-20) antibodies for 1 h, washed twice with 1× PBS, and incubated with secondary anti-rabbit IgG antibodies conjugated with FITC for 1 h. The cells were washed three times with 1× 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 1× 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). FADD and Caspase-8 Can Interact with FAF1 in Vitro— FAF1 has been introduced as a Fas-binding protein (27Chu K. Niu X. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11894-11898Google Scholar, 29Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar). Therefore, we questioned if FAF1 was a component of the death-inducing signaling complex assembled with Fas (Fas-DISC). We first analyzed the binding pattern of FAF1 with Fas-DISC components. In vitro protein-protein binding assays were carried out by GST-pull-down using GST-fused FAF1, in vitro translated FADD, and caspase-8. As shown in Fig. 1A, in vitro translations of FADD and caspase-8 gave rise to 27- and 55-kDa 35S-labeled proteins, respectively. Also, recombinant GST-FAF1 and GST proteins were produced in E. coli BL21(DE3) strain as 100- and 26-kDa proteins, respectively. In vitro translated FADD and caspase-8 were retained on matrices to which GST-FAF1 had bound but not GST only (Fig. 1A). Thus, interactions between FAF1 and Fas-DISC members were demonstrated in the in vitro binding assay. 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 (28Ryu S.W. Kim E. Biochem. Biophys. Res. Commun. 2001; 286: 1027-1032Google Scholar), 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 (29Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar). Thus, our data show that FAF1 has Fas- and caspase-8-interacting domains in tandem like FADD. 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 (37Kaufmann M. Bozic D. Briand C. Bodmer J.L. Zerbe O. Kohl A. Tschopp J. Grűutter M.G. FEBS Lett. 2002; 527: 250-254Google Scholar) 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 (37Kaufmann M. Bozic D. Briand C. Bodmer J.L. Zerbe O. Kohl A. Tschopp J. Grűutter M.G. FEBS Lett. 2002; 527: 250-254Google Scholar, 38Weber C.H. Vincenz C. Trends Biochem. Sci. 2001; 26: 475-481Google Scholar). 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). 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 (28Ryu S.W. Kim E. Biochem. Biophys. Res. Commun. 2001; 286: 1027-1032Google Scholar, 30Fröhlich T. Risau W. Flamme I. J. Cell Sci. 1998; 111: 2353-2363Google Scholar, 31Jensen H.H. Hjerrild M. Guerra B. Larsen M.R. Hojrup P. Boldyreff B. Int. J. Biochem. Cell Biol. 2001; 33: 577-589Google Scholar). 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 (39Algeciras-Schimnich A. Shen L. Barnhart B.C. Murmann A.E. Burkhardt J.K. Peter M.E. Mol. Cell. Biol. 2002; 22: 207-220Google Scholar), 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). 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 Nucleofector™ Solution V provided by Amaxa in Jurkat cells. As shown in Fig. 5, mere overexpression of both FAF1-ΔDEDID and FAF1-ΔFID·DEDID mutants failed to induce apoptosis, whereas those of FAF1 and FAF1-ΔFID induced apoptosis. In addition, transfection of FAF1-ΔFID in which Fas interacting region is deleted inhibited Fas-mediated apoptosis significantly as expected. Moreover, transfection of FAF1-ΔDEDID and FAF1-ΔFID·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. 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. This study enrolls FAF1 as a member of Fas-DISC together with FADD and caspase-8. FAF1 interacts with the DED domains of FADD and caspase-8 through a potentially helix-rich DED-like domain, DEDID, of FAF1 previously known to have an apoptotic potential. In addition, FAF1 mutants lacking DEDID inhibit Fas-induced apoptosis in a dominant-negative fashion emphasizing the essentiality of DEDID in Fas-induced apoptosis. By confocal microscopic analysis, we demonstrated that FAF1 as well as FADD and caspase-8 completely colocalized with Fas in Jurkat cells. We also show that caspase-8 functions downstream of FAF1, because significant inhibition of apoptotic induction by FAF1 occurred in caspase-8-deficient Jurkat cells. 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 (40Hofmann K. Bucher P. Tschopp J. Trends Biochem. Sci. 1997; 22: 155-156Google Scholar). Most of the caspase-8-interacting proteins interact via their DED motifs (21Irmler 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: 190-195Google Scholar, 40Hofmann K. Bucher P. Tschopp J. Trends Biochem. Sci. 1997; 22: 155-156Google Scholar, 41Bertin 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.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1172-1176Google Scholar, 42Stegh A.H. Schickling O. Ehret A. Scaffidi C. Peterhansel C. Hofmann T.G. Grummt I. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 5974-5986Google Scholar). However, death effector domain-associated factor and FLASH interact with DED proteins without having a DED (25Imai Y. Kimura T. Murakami A. Yajima N. Sakamaki K. Yonehara S. Nature. 1999; 398: 777-785Google Scholar, 43Zheng L. Schickling O. Peter M.E. Lenardo M.J. J. Biol. Chem. 2001; 276: 31945-31952Google Scholar). 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 α-helices. Even if FAF1 does not show significant homology with DED by amino acid sequence analysis, the DEDID of FAF1 has several potential α-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 (25Imai Y. Kimura T. Murakami A. Yajima N. Sakamaki K. Yonehara S. Nature. 1999; 398: 777-785Google Scholar). 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 (26Scaffidi C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Google Scholar). 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 (25Imai Y. Kimura T. Murakami A. Yajima N. Sakamaki K. Yonehara S. Nature. 1999; 398: 777-785Google Scholar, 26Scaffidi C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Google Scholar). FLASH is only recruited to Fas upon Fas activation in both type I and II cells (25Imai Y. Kimura T. Murakami A. Yajima N. Sakamaki K. Yonehara S. Nature. 1999; 398: 777-785Google Scholar). 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 (2Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 6: 2794-2804Google Scholar, 9Muzio M. Chinnaiyan A.M. Kischkel 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: 817-827Google Scholar). 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-ΔDEDID lacking caspase-8 binding domain functioned similarly. The dominant-negative effect of FAF1-ΔDEDID on Fas-induced cell death testifies to the essentiality of DEDID in mediating Fas-induced cell death. FAF1-ΔDEDID corresponds to the FAF1(s), an endogenous short isoform FAF1 in which most of DEDID is deleted (29Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar). 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 (29Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar), 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. We thank Dr. P. H. Krammer for kindly donating anti-APO-1 antibody and Dr. J. Blenis (Harvard Medical School) for kindly providing caspase-8- and FADD-deficient Jurkat cells. We are grateful to Dr. R. J. Youle (National Institutes of Health) for a critical reading of the manuscript.

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