Differential Signaling to Apoptotic and Necrotic Cell Death by Fas-associated Death Domain Protein FADD
Two general pathways for cell death have been defined, apoptosis and necrosis. Previous studies in Jurkat cells have demonstrated that the Fas-associated death domain (FADD) is required for Fas-mediated signaling to apoptosis and necrosis. Here we developed L929rTA cell lines that allow Tet-on inducible expression and FK506-binding protein (FKBP)-mediated dimerization of FADD, FADD-death effector domain (FADD-DED), or FADD-death domain (FADD-DD). We show that expression and dimerization of FADD leads to necrosis. However, pretreatment of the cells with the Hsp90 inhibitor geldanamycin, which leads to proteasome-mediated degradation of receptor interacting protein 1 (RIP1), reverts FKBP-FADD-induced necrosis to apoptosis. Expression and dimerization of FADD-DD mediates necrotic cell death. We found that FADD-DD is able to bind RIP1, another protein necessary for Fas-mediated necrosis. Expression and dimerization of FADD-DED initiates apoptosis. Remarkably, in the presence of caspase inhibitors, FADD-DED mediates necrotic cell death. Coimmunoprecipitation studies revealed that FADD-DED in the absence procaspase-8 C/A is also capable of recruiting RIP1. However, when procaspase-8 C/A and RIP1 are expressed simultaneously, FADD-DED preferentially recruits procaspase-8 C/A. Two general pathways for cell death have been defined, apoptosis and necrosis. Previous studies in Jurkat cells have demonstrated that the Fas-associated death domain (FADD) is required for Fas-mediated signaling to apoptosis and necrosis. Here we developed L929rTA cell lines that allow Tet-on inducible expression and FK506-binding protein (FKBP)-mediated dimerization of FADD, FADD-death effector domain (FADD-DED), or FADD-death domain (FADD-DD). We show that expression and dimerization of FADD leads to necrosis. However, pretreatment of the cells with the Hsp90 inhibitor geldanamycin, which leads to proteasome-mediated degradation of receptor interacting protein 1 (RIP1), reverts FKBP-FADD-induced necrosis to apoptosis. Expression and dimerization of FADD-DD mediates necrotic cell death. We found that FADD-DD is able to bind RIP1, another protein necessary for Fas-mediated necrosis. Expression and dimerization of FADD-DED initiates apoptosis. Remarkably, in the presence of caspase inhibitors, FADD-DED mediates necrotic cell death. Coimmunoprecipitation studies revealed that FADD-DED in the absence procaspase-8 C/A is also capable of recruiting RIP1. However, when procaspase-8 C/A and RIP1 are expressed simultaneously, FADD-DED preferentially recruits procaspase-8 C/A. Depending on the cell type, cellular context, or stimulus, a cell may die by apoptosis or necrosis (1Fiers W. Beyaert R. Declercq W. Vandenabeele P. Oncogene. 1999; 18: 7719-7730Crossref PubMed Scopus (770) Google Scholar). Apoptosis is characterized by membrane blebbing, shrinking and condensing of the cell and its organelles, and internucleosomal DNA degradation. Finally, the cell disintegrates, and apoptotic bodies are phagocytosed by professional phagocytes or neighboring cells, in most cases without any detrimental effect on the surrounding tissue (2Kerr J.F. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (13155) Google Scholar, 3Wyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6827) Google Scholar). Apoptosis is generally considered a process that depends on a family of cysteine proteases called caspases (4Lamkanfi M. Declercq W. Depuydt B. Kalai M. Saelens X. Vandenabeele P. Los M. Walczak H. Caspases: Their Role in Cell Death and Cell Survival. Landes Bioscience, Kluwer Academic Press, Georgetown, TX2003: 1-40Google Scholar). Necrosis is characterized by swelling of the cell and its organelles and results in disruption of the cell membrane and cell lysis, often accompanied by inflammation due to release of the cellular contents (1Fiers W. Beyaert R. Declercq W. Vandenabeele P. Oncogene. 1999; 18: 7719-7730Crossref PubMed Scopus (770) Google Scholar, 5Denecker G. Vercammen D. Declercq W. Vandenabeele P. Cell. Mol. Life Sci. 2001; 58: 356-370Crossref PubMed Scopus (229) Google Scholar). Although apoptosis and necrosis have clearly distinguished morphological and biochemical features, they can be initiated by tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; TNFR1, 55-kDa TNF receptor; CrmA, cytokine response modifier A; DD, death domain; DED, death effector domain; FADD, Fas-associated death domain; FKBP, FK506-binding protein; PI, propidium iodide; RIP1, receptor-interacting protein 1; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone; FS, forward scatter; SS, side scatter; HA, hemagglutinin. or by Fas, depending on the cellular context. In L929sAhFas cells, TNF signals to necrotic cell death, whereas anti-Fas treatment of the same cells leads to apoptosis (6Vercammen D. Brouckaert G. Denecker G. Van de Craen M. Declercq W. Fiers W. Vandenabeele P. J. Exp. Med. 1998; 188: 919-930Crossref PubMed Scopus (484) Google Scholar). Although pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) inhibits anti-Fas-induced apoptosis, the cells nevertheless die necrotically (6Vercammen D. Brouckaert G. Denecker G. Van de Craen M. Declercq W. Fiers W. Vandenabeele P. J. Exp. Med. 1998; 188: 919-930Crossref PubMed Scopus (484) Google Scholar). Recently we showed that TNFR1-induced necrosis is reverted to apoptosis by pretreatment with the Hsp90 inhibitor geldanamycin, which leads to the proteasome-dependent degradation of Hsp90 client proteins such as receptor-interacting protein 1 (RIP1) (7Vanden Berghe T. Kalai M. van Loo G. Declercq W. Vandenabeele P. J. Biol. Chem. 2003; 278: 5622-5629Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Thus, both death domain-containing receptors, TNFR1 and Fas, can initiate apoptotic and necrotic cell death depending on the cellular context. The Fas-associated death domain protein (FADD), which is recruited to the receptor through its carboxy-terminal “death domain” (DD), is a key adaptor protein in death receptor-induced apoptosis (8Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 9Boldin M.P. Varfolomeev E.E. Pancer Z. Mett I.L. Camonis J.H. Wallach D. J. Biol. Chem. 1995; 270: 7795-7798Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar). The amino-terminal “death effector domain” (DED) of FADD interacts with a homologous DED within the prodomain of caspase-8 (10Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2122) Google Scholar, 11Muzio 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: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2757) Google Scholar), providing a platform for caspase-8 activation and consecutive downstream caspase activation either directly or through the cleavage of Bid and mitochondrial cytochrome c release (12Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3834) Google Scholar, 13Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3107) Google Scholar). The role of FADD in TNFR1- and Fas-induced necrosis is still not defined. In FADD-deficient Jurkat cells, TNFR1-induced apoptosis is blocked, and the cells die via a necrotic pathway. However, both cell death pathways are blocked when these cells are exposed to Fas ligand (14Holler N. Zaru R. Micheau O. Thome M. Attinger A. Valitutti S. Bodmer J.L. Schneider P. Seed B. Tschopp J. Nat. Immunol. 2000; 1: 489-495Crossref PubMed Scopus (1501) Google Scholar, 15Harper N. Hughes M. MacFarlane M. Cohen G.M. J. Biol. Chem. 2003; 278: 25534-25541Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). This suggests that TNFR1-induced necrosis can occur independently of FADD, whereas Fas-induced necrosis cannot. In a previous study we showed by transient transfection experiments that overexpression of FADD-DN (amino acids 79-208) lacking the intact DED (8Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2187) Google Scholar) is cytotoxic to L929sA cells in a CrmA-insensitive way, whereas overexpression of FADD-DED (amino acids 1-111) containing the DED is toxic in a CrmA inhibitory way (16Boone E. Vanden Berghe T. Van Loo G. De Wilde G. De Wael N. Vercammen D. Fiers W. Haegeman G. Vandenabeele P. J. Biol. Chem. 2000; 275: 37596-37603Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In another study using a caspase-8-deficient Jurkat cell line, it was shown that FADD is involved in signaling to necrotic cell death (17Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (275) Google Scholar, 18Matsumura H. Shimizu Y. Ohsawa Y. Kawahara A. Uchiyama Y. Nagata S. J. Cell Biol. 2000; 151: 1247-1256Crossref PubMed Scopus (208) Google Scholar). Here, we used stably transfected L929rTA inducible cell lines expressing chimeric FKBP-FADD, FKBP-FADD-DED, and FKBP-FADD-DD constructs to examine the type of cell death initiated after doxycycline induction and enforced FKBP-dependent dimerization. Cell Culture and Reagents—The mouse fibrosarcoma cell line L929rTA and its derivatives were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (0.1 mg/ml), and l-glutamine (0.03%). Recombinant human TNF was produced in-house (7Vanden Berghe T. Kalai M. van Loo G. Declercq W. Vandenabeele P. J. Biol. Chem. 2003; 278: 5622-5629Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Propidium iodide (PI) and geldanamycin were purchased from Sigma and used at 30 and 1 μm, respectively. Doxycycline (Duchefa Biochemicals, The Netherlands) was used at 1 μg/ml. AP1510, a dimerizer of FKBP (19Amara J.F. Clackson T. Rivera V.M. Guo T. Keenan T. Natesan S. Pollock R. Yang W. Courage N.L. Holt D.A. Gilman M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10618-10623Crossref PubMed Scopus (178) Google Scholar), kindly provided by ARIAD Pharmaceuticals Inc. (Cambridge, MA), was used at 1 μm. The pan-caspase inhibitor zVAD-fmk was from Enzyme Systems Products (Dublin, CA) and was used at 25 μm. The caspase-3-like substrate acetyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-aminomethylcoumarin was from the Peptide Institute (Osaka, Japan) and was used at 50 μm. Monoclonal antibodies to hemagglutinin (HA) epitope, poly(ADP-ribose) polymerase, and cytochrome c were from Berkeley Antibody Co. (Richmond, CA), Biomol Research Labs Inc. (Plymouth, PA), and Pharmingen (clone 7H8.2C12), respectively. Rabbit polyclonal antibodies against recombinant caspase-3 were prepared at the Centre d'Economie Rurale (Laboratoire d'Hormonologie Animale, Marloie, Belgium). Rabbit polyclonal antibodies against caspase-9 and Bid were purchased from Cell Signaling and Systems Inc. respectively. was from antibodies were from Monoclonal antibodies against were from Berkeley Antibody Co. The antibodies against and were from and respectively. of to of The the for a from the of of a the human FADD and the The of was from Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) by and the expression M. H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. S. G. G. W. H. 1995; PubMed Scopus Google Scholar) provided by to The DNA for the DED of FADD acids and for FADD lacking the DED acids (8Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2187) Google were by from human FADD (8Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 9Boldin M.P. Varfolomeev E.E. Pancer Z. Mett I.L. Camonis J.H. Wallach D. J. Biol. Chem. 1995; 270: 7795-7798Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar). DNA for a was and L929rTA cells P. W. Haegeman G. Mol. Biol. 1999; Scholar) were stably with of the for of CA), a S. C. H. J. Med. 1999; 1: PubMed Scopus Google Scholar), and of the expression transfected cells were for in medium containing were for and expression by using cell showed expression of transfected FADD constructs after induction with doxycycline for at Cell Death and were at in and at in a The cells were with AP1510, and as cells were with doxycycline for in with μm, after doxycycline zVAD-fmk was whereas geldanamycin was were on a of cell membrane was using the at and at as (7Vanden Berghe T. Kalai M. van Loo G. Declercq W. Vandenabeele P. J. Biol. Chem. 2003; 278: 5622-5629Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The of cells containing DNA was by of cells after to cells, as D. Vandenabeele P. Beyaert R. Declercq W. Fiers W. 1997; PubMed Scopus Google Scholar). were at in The doxycycline and were to the cells as were with and in caspase 1 and 1 cytochrome c cells were with and in in containing and and on for 1 This treatment of the cell membrane without the were by at for and at were by and to a membrane were with the Life after of the with and for was by 25 of cell with 50 substrate acetyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-aminomethylcoumarin Japan) in of 1 The release of was for 1 at at in a at of and of are expressed as the in as a of Coimmunoprecipitation cells were in at 1 transfection with the expression FADD, and FADD-DD in mouse procaspase-8 in and RIP1 in from we used a of procaspase-8 called procaspase-8 C/A. after transfection cells were for with the dimerizer AP1510, with and in a containing and 10% for 30 at were for 1 at with 1 of and exposed to protein with containing at were by in 10% and by using and of to FADD study the role of FADD in apoptotic and necrotic cell death we stably transfected L929rTA cell L929rTA cells expressing a protein M. S. G. G. W. H. 1995; PubMed Scopus Google Scholar, P. W. Haegeman G. Mol. Biol. 1999; Scholar) were transfected with for human FADD, FADD-DED (amino acids or FADD-DD (amino acids 79-208) the of a and the The of a results in the of the to the activation of the FADD In the constructs used the of the FADD were to of FKBP to the used by Kawahara (17Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (275) Google Scholar), using a dimerizer (19Amara J.F. Clackson T. Rivera V.M. Guo T. Keenan T. Natesan S. Pollock R. Yang W. Courage N.L. Holt D.A. Gilman M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10618-10623Crossref PubMed Scopus (178) Google Scholar) of the FADD we a the FKBP and the FADD T. E. 1999; PubMed Scopus Google Scholar). The of doxycycline and the expression and of the to cell death TNF treatment demonstrated that the cell lines a to TNFR1-induced necrosis Apoptosis Necrosis in examine which type of cell death is through the of FADD and FADD the L929rTA cells were with doxycycline and AP1510, and was by FADD and FADD-DD the swelling of whereas FADD-DED a apoptotic characterized by membrane and the of apoptotic bodies we the and of with doxycycline and AP1510, caspase-3 was in L929rTA cells expressing not in cells expressing or the FKBP-FADD-DD of the and of the of the caspase were in cells not in and FKBP-FADD-DD cells cell death in of these cell lines This activation of caspase-3 in cells expressing was accompanied by the cleavage of the protein Bid to its and the cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase, as demonstrated by the of the protein of a for apoptotic cell death G. Vercammen D. M. Vanden Berghe T. Brouckaert G. Van Loo G. B. Fiers W. J. Declercq W. Vandenabeele P. Cell Death 2001; PubMed Scopus Google Scholar), was after induction and results that induction and of a apoptosis, whereas induction and of and FKBP-FADD-DD in a necrotic cell death. of by zVAD-fmk Apoptosis to in cellular and of cells were also by Necrosis with a in the and in the of the with Apoptosis is with a in both (7Vanden Berghe T. Kalai M. van Loo G. Declercq W. Vandenabeele P. J. Biol. Chem. 2003; 278: 5622-5629Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, M. Van Loo G. Vanden Berghe T. A. W. Saelens X. Vandenabeele P. Cell Death PubMed Scopus Google Scholar). The of and FKBP-FADD-DD cells after doxycycline and treatment was of necrotic cell death, whereas induction and of in and SS, for apoptosis are in with the of the L929sAhFas cells die by necrosis when Fas-induced apoptotic death was blocked using caspase (6Vercammen D. Brouckaert G. Denecker G. Van de Craen M. Declercq W. Fiers W. Vandenabeele P. J. Exp. Med. 1998; 188: 919-930Crossref PubMed Scopus (484) Google Scholar). induction and of in the presence of the caspase inhibitor zVAD-fmk also in a from apoptosis to as demonstrated by the and of the and by the absence of a DNA Thus, FADD-DED cells via apoptotic caspases are blocked the cells by a necrotic pathway. Necrosis to Apoptosis by Hsp90 we showed that pretreatment with the Hsp90 inhibitor geldanamycin leads to degradation of Hsp90 client proteins such as RIP1 and reverts TNFR1-induced necrosis to apoptosis (7Vanden Berghe T. Kalai M. van Loo G. Declercq W. Vandenabeele P. J. Biol. Chem. 2003; 278: 5622-5629Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). was after induction and of in the presence of geldanamycin, by the of a DNA Necrosis by FKBP-FADD-DD was by geldanamycin, to apoptosis was results clearly that depending on the cellular context, FADD can necrotic or apoptotic cell death, whereas FADD-DD signals to necrosis. FADD-DED whereas FADD-DD the recruited to the and FKBP-FADD-DD we these from cells transfected with the in cells the of procaspase-8 and FKBP-FADD-DED, dimerization of to of procaspase-8 C/A whereas FKBP-FADD-DD was of recruiting procaspase-8 C/A we of RIP1 by FKBP-FADD-DD in cells RIP1 and FKBP-FADD-DD Remarkably, was also capable of RIP1 However, of procaspase-8 C/A the of of RIP1 to In the of RIP1 to FKBP-FADD-DD was not by of procaspase-8 C/A results that can both procaspase-8 C/A and RIP1, to procaspase-8 C/A is In FKBP-FADD-DD can RIP1 to bind to procaspase-8 C/A. The role of FADD in TNFR1- and Fas-induced necrosis is In FADD-deficient Jurkat cells, TNFR1-induced apoptosis is blocked, whereas necrosis still In the same cells, both Fas-induced apoptosis and necrosis are (14Holler N. Zaru R. Micheau O. Thome M. Attinger A. Valitutti S. Bodmer J.L. Schneider P. Seed B. Tschopp J. Nat. Immunol. 2000; 1: 489-495Crossref PubMed Scopus (1501) Google Scholar, 15Harper N. Hughes M. MacFarlane M. Cohen G.M. J. Biol. Chem. 2003; 278: 25534-25541Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). This suggests that TNFR1-induced necrosis can occur independently of FADD, whereas Fas-induced necrosis In Fas-induced FADD to as a platform to initiate both apoptosis and necrosis. In of a role for FADD in necrotic signaling is the that inducible and FADD-DED initiate apoptosis or depending on the presence or absence of H. Shimizu Y. Ohsawa Y. Kawahara A. Uchiyama Y. Nagata S. J. Cell Biol. 2000; 151: 1247-1256Crossref PubMed Scopus (208) Google Scholar). In the same enforced dimerization of FADD-DD was not However, we found that transient overexpression of FADD-DD was able to initiate necrotic cell death (16Boone E. Vanden Berghe T. Van Loo G. De Wilde G. De Wael N. Vercammen D. Fiers W. Haegeman G. Vandenabeele P. J. Biol. Chem. 2000; 275: 37596-37603Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). we used stably transfected cell lines expressing chimeric FKBP-FADD, FKBP-FADD-DED, and FKBP-FADD-DD constructs and the type of cell death initiated after doxycycline induction and enforced FKBP-dependent dimerization. The of cell death by FADD, and FADD-DD are in a and dimerization of to a necrotic cell death that overexpression and dimerization of FADD to apoptosis, FADD is to the apoptotic from Fas to the enforced dimerization of apoptotic cell death in cells not enforced dimerization of FADD in Jurkat cells leads to apoptotic cell death. However, in caspase-8-deficient Jurkat cells enforced dimerization of FADD to a necrotic cell death (17Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (275) Google Scholar). In cells overexpression of TNFR1, death domain or FADD leads to that these cells have a cellular (16Boone E. Vanden Berghe T. Van Loo G. De Wilde G. De Wael N. Vercammen D. Fiers W. Haegeman G. Vandenabeele P. J. Biol. Chem. 2000; 275: 37596-37603Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, Haegeman G. Fiers W. J. Cell Biol. 1997; PubMed Scopus Google Scholar, P. Van Loo G. I. Vandenabeele P. Beyaert R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). FKBP-FADD-induced necrosis can to apoptosis by the cells with geldanamycin, a Hsp90 inhibitor (7Vanden Berghe T. Kalai M. van Loo G. Declercq W. Vandenabeele P. J. Biol. Chem. 2003; 278: 5622-5629Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). This suggests that depending on the cellular context, for the presence or absence of client proteins such as RIP1, enforced dimerization of FADD can necrotic or apoptotic cell death. of Fas in cells by FADD clearly apoptotic cell death. the that of FADD to Fas a or in FADD, which is for the apoptotic 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: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2757) Google Scholar, S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. D. A. M. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). and dimerization of FKBP-FADD-DD also to a necrotic cell This is in with that overexpression of a FADD lacking the DED of recruiting necrosis in and cells A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). However, as Matsumura H. Shimizu Y. Ohsawa Y. Kawahara A. Uchiyama Y. Nagata S. J. Cell Biol. 2000; 151: 1247-1256Crossref PubMed Scopus (208) Google Scholar) in Jurkat cells after enforced dimerization of This be due to the that they used in FADD-DD The of FADD at S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), whereas the protein used by Matsumura at in the absence of the is that of the FADD-DD is required for recruiting RIP1 and necrosis S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). a of the demonstrated that on side of the domain is of in and G. T. T. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the FKBP-FADD-DD cells with geldanamycin not the cell death response from necrosis to apoptosis, procaspase-8 not be This also suggests that the FADD-DD not FADD, which initiate apoptotic response in the presence of the receptor-interacting protein RIP1 was as a in the to necrotic cell death (14Holler N. Zaru R. Micheau O. Thome M. Attinger A. Valitutti S. Bodmer J.L. Schneider P. Seed B. Tschopp J. Nat. Immunol. 2000; 1: 489-495Crossref PubMed Scopus (1501) Google Scholar). its role in the activation of through of the to the TNF it was shown that in Jurkat cells the of RIP1 is required for necrotic cell death, that a RIP1 substrate may the necrotic cell death (14Holler N. Zaru R. Micheau O. Thome M. Attinger A. Valitutti S. Bodmer J.L. Schneider P. Seed B. Tschopp J. Nat. Immunol. 2000; 1: 489-495Crossref PubMed Scopus (1501) Google Scholar). using FADD as demonstrated that RIP1 is able to bind FADD E.E. M.P. Goncharov T.M. Wallach D. J. Exp. Med. 1996; PubMed Scopus Google Scholar). van The of RIP1 by FADD-DD in studies suggests that necrotic signaling in L929sA cells through RIP1. showed the morphological and biochemical of apoptotic cell death caspase activation was using the caspase inhibitor zVAD-fmk, apoptotic cell death by of was blocked nevertheless cells by necrosis. This suggests that the DED can also initiate necrotic cell death in the absence of in caspase-8-deficient Jurkat cells transfected with a chimeric was shown to a necrotic cell death (17Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (275) Google Scholar, 18Matsumura H. Shimizu Y. Ohsawa Y. Kawahara A. Uchiyama Y. Nagata S. J. Cell Biol. 2000; 151: 1247-1256Crossref PubMed Scopus (208) Google Scholar). The that was also capable of RIP1 the role of FADD-DED in the presence of caspase However, expression of both and RIP1 the of of RIP1 to not the of of RIP1. results that can both and RIP1, the to to be in cellular of caspase-8 and caspase-8 is able to and RIP1 signaling activation Oncogene. 2000; PubMed Scopus Google Scholar, Y. A. Y. 1999; PubMed Scopus Google Scholar, N. C. Tschopp J. 2000; PubMed Scopus Google Scholar). results may FADD-DED mediates apoptosis by recruiting and procaspase-8 and by the signaling by RIP1. However, in the absence of as in caspase-8-deficient Jurkat cells (17Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (275) Google Scholar), or caspase is blocked by zVAD-fmk RIP1 is not and necrotic cell death. In the results show that apoptotic and necrotic cell death pathways can initiate at the of the FADD-DD recruits RIP1, in necrotic whereas FADD-DED preferentially recruits procaspase-8 and initiates apoptosis. However, in the presence of caspase FADD-DED necrotic cell death by its to RIP1. We ARIAD Pharmaceuticals Inc. (Cambridge, for providing