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

Abstract

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. 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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. 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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 M. M.R. P. J. Biochem. Cell Biol. 2001; Scholar). FAF1 is not a binding of protein but a for and M. M.R. P. J. Biochem. Cell Biol. 2001; Scholar, M. R. C. Cell. Biochem. 1999; Scholar). the interaction of protein and FAF1 is in vivo of apoptosis J. 2001; Scholar). In we that FAF1 is a component of Fas-DISC, and DISC is by interaction of the DED-like region acid 181–381 of of FAF1 and the DEDs of caspase-8 and FADD. Therefore, a the of FAF1 in Fas-mediated signaling. and confocal FAF1 Fas FADD caspase-8 and were from and and were from analysis, FADD and caspase-8 were from and Cell FAF1 antibody was a from J. S. of and of Fas, antibody Fas was from of antibody Fas (APO-1) by P. H. Krammer Cancer was were from The glutathione and acid were from and The protein were from were from of the FADD, FAF1, FAF1, FAF1, and which where to was previously (28Ryu S.W. Kim E. Biochem. Biophys. Res. Commun. 2001; 286: 1027-1032Google Scholar, S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar, S.W. Chae S.K. Kim E. Biochem. Biophys. Res. Commun. 2000; Scholar). was by FAF1 as a and to was generated by the (29Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar). is an endogenous of FAF1 with an deletion that of the to amino acids 181–381 of FAF1, and amino acids of FAF1 is in The of caspase-8 and was previously Nagata S. Biochem. Biophys. Res. Commun. 2000; Scholar). the FAF1 deletion and glutathione with and were as a The DED was generated by the as a with and the FADD and its deletion and were as a was by S. H. Kim In proteins were in with cells were in The proteins were with for at and for protein The and DED were in with of was with of in the for at In were with proteins in the binding and for at in the were with The were an and by a Cell cells were in and and in antibody and Jurkat cells were and with for were at after anti-Fas antibody with and The and protein were to the and The were with and to or were by and a membrane an were for in and at with or were in and were with of were and with DISC Jurkat cells were for at in and the was with and for cells were at different after anti-Fas antibody with and in a anti-Fas antibody was to from the cells. protein was by and to The anti-Fas (APO-1) and protein were to the and for at The were with and to or Cell Death cells were or by an the Jurkat cells were in and in of of was to a and with an cells were with anti-Fas antibody for were in for and on with and by cells of different types of Jurkat cells FADD and caspase-8 P. Kuo C. P. Blenis J. Cell Differ. 1999; were with FAF1 were and on the as The of apoptotic cells was on the of cells a fluorescence and for were and of caspase-8, and FADD was by of Fas and FAF1, Jurkat cells with or of Fas with were on a were and with were with anti-Fas or antibody for with PBS, and with antibody with with and for were with and on of FAF1 and Fas-DISC, Jurkat cells with or of Fas with were on a were and with were with anti-Fas for with PBS, and with with for The cells were with were with and for with PBS, and with with for were on were a confocal FADD and with FAF1 in 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, S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar). Therefore, we if FAF1 was a component of the signaling complex with Fas first the binding of FAF1 with Fas-DISC In binding were by FAF1, in FADD, and caspase-8. in in of FADD and caspase-8 to and and proteins were in E. as and In FADD and caspase-8 were on to which but not Thus, interactions between FAF1 and Fas-DISC members were in the in binding FAF1 with FADD and in Jurkat show that FAF1 with FADD and caspase-8 in cells, endogenous FAF1 and FADD were by the antibody and the The were by caspase-8 and FADD, in Fas-DISC member was detected in the it was not detected in the Thus, the interaction of Fas-DISC and FAF1 was in and in and show direct association of FAF1 with DISC The of FAF1 to the DED of FADD and Based on the fact that Fas-DISC complex is by interactions between their protein we which FAF1 domains were required for the binding to the Fas-DISC the binding domains to Fas-DISC an in binding was deletion and in and to the previously known apoptotic effector domain (28Ryu S.W. Kim E. Biochem. Biophys. Res. Commun. 2001; 286: 1027-1032Google was to interact with in FADD The in FAF1 was to to but not to In binding between FAF1 and caspase-8, was as the caspase-8 region deletion of caspase-8, the DED of caspase-8 was to to FAF1 but not to the C terminus of caspase-8 where the activity The N terminus acids of FAF1 was previously as the binding 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 and domains in FADD. The DED-interacting of the region of amino acids 181–381 of FAF1 was as the domain with the DED domains of FADD and caspase-8 and Thus, we the region of amino acids 181–381 of FAF1 has to the DED domains of FADD and caspase-8. of FAF1 that the amino acid of the DED domains to was not significant In addition, the amino acid found in the DEDs M. D. C. Bodmer J.L. A. Tschopp J. were not in Thus, is as The of with protein data that have The of DED domains to be in the interaction M. D. C. Bodmer J.L. A. Tschopp J. Scholar, Vincenz C. Biochem. Sci. 2001; Scholar). FAF1 with the Fas-DISC before Fas or including FADD, caspase-8, and FLASH, the Fas-DISC through homotypic domain interactions FAF1 with Fas-DISC members and has a DEDID, we if FAF1 was a member of Fas-DISC. FAF1 is in the Fas-DISC, were with an human antibody Fas in Jurkat cells before and after of Fas with the anti-Fas in endogenous FAF1 was in the Fas-DISC before Fas activation but not caspase-8. However, caspase-8 was to the Fas-DISC after Fas activation. In addition, the association of FAF1 with the Fas-DISC was at after of Fas antibody and to the cell as apoptosis association of FAF1 with the Fas-DISC in cells as well not FAF1 with Fas-DISC to the has been previously that Fas-DISC is in the cytoplasmic membrane homotypic FAF1 has been to in 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 M. M.R. P. J. Biochem. Cell Biol. 2001; Scholar). found that FAF1 to the cytoplasm in and cells known to be to apoptosis not By confocal microscopic the endogenous FAF1 and Fas, both FAF1 and Fas in the cytoplasmic membrane in cells that were not with anti-Fas antibody A and the of Fas and FAF1 was from to after Fas in of Fas, which with a previous A. L. Peter M.E. Cell. Biol. and FAF1 was detected after of Fas with anti-Fas cells the of Fas and FAF1 in the cytoplasm A and Fas-DISC and FAF1 in cells before and after of Fas with the anti-Fas of was by confocal endogenous FAF1, Fas, FADD, and caspase-8 in Jurkat cells. in and endogenous FAF1, FADD, and caspase-8 colocalized with Fas in cells and not with anti-Fas was detected in the as for the of Fas-induced if in the Fas-induced apoptosis, the deletion were and by in Jurkat cells. in mere overexpression of both and to apoptosis, of FAF1 and apoptosis. In addition, of in which Fas region is Fas-mediated apoptosis significantly as of and Fas-mediated apoptosis significantly as data that deletion as in the Fas-mediated apoptotic Thus, it has been that the is essential in Fas-induced apoptosis. of FAF1 in FADD- and Jurkat and caspase-8 are for Fas receptor-mediated cell death. the of caspase-8 and FADD for cell death. FAF1 was by in parental Jurkat cells Jurkat cells and caspase-8-deficient Jurkat cells in of the FAF1 in parental Jurkat cells with apoptosis, when compared with that in parental Jurkat cells However, as compared with in parental Jurkat cells, cell death by FAF1 in or caspase-8-deficient cells was significantly In apoptosis The of FAF1 to apoptosis required FADD and caspase-8. The fact that overexpression of FAF1 a significant of apoptosis in FADD- and caspase-8-deficient Jurkat cells that FAF1 have another of cell death the apoptosis. This FAF1 as a member of Fas-DISC with FADD and caspase-8. FAF1 with the DED domains of FADD and caspase-8 through a DED-like DEDID, of FAF1 previously known to have an apoptotic potential. In addition, FAF1 lacking Fas-induced apoptosis in a dominant-negative the of in Fas-induced apoptosis. By confocal microscopic analysis, we that FAF1 as well as FADD and caspase-8 colocalized with Fas in Jurkat cells. show that caspase-8 downstream of FAF1, significant of apoptotic by FAF1 in caspase-8-deficient Jurkat cells. FAF1 with caspase-8 and FADD. The interaction occurs the of FAF1 and the DEDs of caspase-8 and of FADD. homotypic interaction domains such as death death effector caspase recruitment and domain have been K. P. Tschopp J. Biochem. Sci. 1997; Scholar). of the proteins interact their DED 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, K. P. Tschopp J. Biochem. Sci. 1997; Scholar, J. Armstrong R.C. Ottilie S. Wang Y. Banks S. Wang Alnemri E.S. Lenardo M.J. Tomaselli K.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: Scholar, A. Scaffidi C. C. Hofmann I. Krammer P.H. Peter M.E. EMBO J. 1998; 17: Scholar). However, death effector factor and FLASH interact with DED proteins a DED (25Imai Y. Kimura T. Murakami A. Yajima N. Sakamaki K. Yonehara S. Nature. 1999; 398: 777-785Google Scholar, L. Peter M.E. Lenardo M.J. J. Biol. Chem. 2001; Scholar). FAF1 is as another of a protein with the DEDs of caspase-8 and FADD a typical The DED has a composed of if FAF1 does not show significant with DED by amino acid analysis, the of FAF1 has potential when by the protein data Thus, of of FAF1 be able to its to DED or DED-like FLASH has been 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 domain that with the DED domains of caspase-8 and FADD, and FLASH is in association of FADD and caspase-8 Fas However, FLASH does not interact with Fas or its death domain. FLASH, FAF1 directly with as well as FADD- and FAF1 is FADD even though have similar protein-interacting Thus, it is that FAF1 a as a protein that signaling of the interaction of FAF1 with other Fas-DISC members such as FLASH of the of FAF1 as a Two different 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 Fas activation and by Fas activation. In type II cells, the DISC is Fas activation. Thus, Fas-mediated apoptosis in type I cells (25Imai Y. Kimura T. Murakami A. Yajima N. Sakamaki K. Yonehara S. Nature. 1999; 398: 777-785Google Scholar, C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Google Scholar). FLASH is to Fas 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 we that FAF1 was with Fas before Fas activation in both type I not and type II cells. In addition, the association of FAF1 with Fas was during cell death signaling Fas activation. Thus, that FAF1 as an for the formation of of FADD and caspase-8 has been to through 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 lacking amino acids as a dominant-negative of and tumor necrosis apoptosis. In lacking caspase-8 binding domain The dominant-negative effect of on Fas-induced cell death to the of in Fas-induced cell death. to the an endogenous FAF1 in which of is (29Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google Scholar). The of have been found both in the human and in the cell even though the protein has not been detected by Thus, if be able to function as an endogenous FAF1 in Fas-induced apoptosis. The of FAF1 with Fas (29Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Google and the of FAF1 with the DED of caspase-8. In FAF1 is similar to the protein FADD. both FAF1 and FADD have their in is that FAF1 in Fas-DISC to the of caspase-8 a FADD-like a between FADD and FAF1 in the formation of Fas-DISC, of apoptosis by FAF1 in Jurkat cells. P. H. Krammer for antibody and J. Blenis for and Jurkat cells. are to R. J. of for a of the