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Or FADD-Binding Proteins Fas/FLICE Receptor Cutting Edge: Apoptosis Induced by a Chimeric Fas/FLICE Receptor: Lack of Requirement for Fas- or FADD-Binding Proteins This information is current as Sarfraz A. Memon, Jing-zhou Hou, M. Belen Moreno and Charles of September 28, 2021. M. Zacharchuk J Immunol 1998; 160:2046-2049; ; http://www.jimmunol.org/content/160/5/2046 Downloaded from References This article cites 15 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/160/5/2046.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. c Cutting Edge: Apoptosis Induced by a Chimeric Fas/FLICE Receptor: Lack of Requirement for Fas- or FADD-Binding Proteins Sarfraz A. Memon, Jing-zhou Hou, M. Belen Moreno, and Charles M. Zacharchuk1 stream substrates. This model is supported by the ability of caspase Current models for Fas (CD95)-mediated apoptosis suggest inhibitors and dominant negative variants of FADD and FLICE to that FLICE/caspase-8 is recruited and activated, which results block Fas-mediated cell death (4, 5). However, whether additional Downloaded from in cell death. However, the role of additional molecules in Fas molecules in the activated Fas receptor complex participate in ap- signaling and FLICE activation is not clear. A chimeric Fas/ optotic signaling is unknown, and the mechanism by which FLICE FLICE (F/F) receptor, containing the extracellular/transmem- is recruited and converted to the active caspase is not clear. Several brane portion of Fas and the caspase region of FLICE, medi- noncaspase signaling pathways have been implicated in Fas-in- ated anti-Fas apoptosis. FLICE protease subunits were duced cytotoxicity including sphingomyelinases and protein ki- generated from the F/F precursor. Killing induced by Fas, but nases. In addition to FADD, a variety of molecules that interact http://www.jimmunol.org/ not F/F, was blocked by a dominant negative FADD. Apoptosis with the cytoplasmic portion of Fas have been identified that are triggered through Fas and F/F was inhibited by coexpression potential mediators or modulators of the apoptotic signal including of CrmA and p35, but not Bcl-xL. F/F bypassed Fas resistance RIP, FAP, Daxx, UBC9, and FAF. Yet to be identified Fas-inter- in COS-7 cells and blocking by the death effector domain acting molecules or DD/DED-recruited proteins such as TRADD, (DED)-containing viral protein MC159. These results show TRAF, caspase-10, or Casper could also be important for Fas- that: 1) F/F induces cell death, indicating that FLICE activa- mediated cell death. Because overexpression of a dominant nega- tion is sufficient for apoptosis and does not require additional tive FADD would be expected to disrupt or prevent assembly of Fas- or FADD-binding proteins; and 2) F/F bypasses proximal the activated Fas receptor complex, the exact role of these proteins by guest on September 28, 2021 defects in Fas signaling that prevent FLICE recruitment or in apoptotic signaling has not been established. To explore these activation. The Journal of Immunology, 1998, 160: issues, chimeric Fas/FLICE receptors (F/F) were made that lacked 2046–2049. the intracellular portion of Fas and contained no DD or DED motifs. as/APO-1/CD95 is a member of the TNF/NGF receptor family that plays a critical role in cell homeostasis in both Materials and Methods F normal and pathologic processes (reviewed in Refs. 1 and Cell lines, reagents, and Abs 2). Fas contains an intracytoplasmic motif called a death domain (DD),2 which recruits molecules such as FADD/MORT to the COS-7 cells (American Type Culture Collection, Rockville, MD; CRL1651), L1210 cells (6), and the mouse T cell hybridoma 2B4.11 cells plasma membrane through homotypic DD/DD protein interac- (2B4) (7) have been described. Hybridoma cells secreting the anti-Myc tions. Current data suggest that FADD, through another protein mAb 9E10 were obtained from Dr. Allan Weissman (National Institutes of interaction motif called a DED (death effector domain), serves as Health, Bethesda, MD). Dexamethasone (Dex) was purchased from Sigma an adapter to recruit FLICE/MACH/caspase-8 to the receptor com- (Sigma Chemical Co., St. Louis, MO), and anti-human Fas Ab CH-11 was purchased from Kamiya Biomedical Co. (Thousand Oaks, CA). plex (2–4). Following its proteolytic activation, FLICE initiates the apoptotic cascade, presumably by cleaving relevant down- Plasmids Expression vectors for Fas (pCI-Fas), Bcl-xL (CMV-Bcl-xL), CrmA Laboratory of Immune Cell Biology, Division of Basic Sciences, National Cancer (CMV-CrmA), and b-galactosidase (CMV-b-gal) have been described (7). Institute, National Institutes of Health, Bethesda, MD 20892 pCI-MC159 (8) was kindly provided by Dr. Jeffrey Cohen (National In- Received for publication November 20, 1997. Accepted for publication January stitutes of Health, Bethesda, MD). An expression plasmid for p35 (pCI- 5, 1998. p35) was made from pRC-p35 (9), generously provided by Dr. Lois Miller The costs of publication of this article were defrayed in part by the payment of page (University of Georgia, Athens, GA). A dominant negative version of charges. This article must therefore be hereby marked advertisement in accordance FADD (amino acids 91–208) containing only the DD was made by PCR with 18 U.S.C. Section 1734 solely to indicate this fact. from full length FADD, kindly provided by Dr. Michael Lenardo (National 1 Address correspondence and reprint requests to Dr. Charles M. Zacharchuk, Na- Institutes of Health, Bethesda, MD): 59 primer (TAT ATG GCG CCT tional Institutes of Health, Building 10, Room 1B40, 9000 Rockville Pike, Bethesda, GGG GAA GAA GAC CTG TGT) and 39 primer (TAG ATC TCA AGC MD 20892-1152. E-mail address: [email protected] GTA GTC TGG GAC GTC GTA TGG GTA ACC GGA ACC GGA CGC 2 Abbreviations used in this paper: DD, death domain; F/F, Fas/FLICE; DED, death TTC GGA GGT AGA TGC GTC). The PCR product was cloned and effector domain; 2B4, T cell hybridoma 2B4.11; Dex, dexamethasone; b-gal, screened in pCR II (Invitrogen, San Diego, CA) and then moved to pCI b-galactosidase. (Promega, Madison, WI) as an EcoRI fragment. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 c The Journal of Immunology 2047 Construction of chimeric receptors F/F chimeric constructs were generated by PCR using primers incorporat- ing appropriate restriction sites and Myc epitope tags. cDNA for the ex- tracellular and transmembrane portion of human Fas (amino acids 1–222) was made by PCR from pCI-Fas using a 59 primer (ACA ACC ATG CTG GGC ATC TGG ACC) and a 39 primer containing an XbaI linker tail (GGA ACC GCC TCT AGA ACC GCC TAT TGC CAC TGT TTC AGG ATT TAA GGT) so that caspase cDNAs could be cloned and ligated in frame to the truncated Fas cDNA. The PCR product was cloned and screened in pCR II and then moved into the pCI vector as an EcoRI frag- ment. Full length FLICE cDNA was cloned by PCR from Jurkat mRNA (MicroFastTrack kit, Invitrogen) using homologous 59 (GGC GGT TCT AGA GGC GGT TCC ACC ATG GAC TTC AGC AGA AAT CTT TAT) and 39 (TAG ATC TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA ACC GGA ACC ATC AGA AGG GAA GAC AAG TTT TTT TCT) primers. After screening in pCR II, the FLICE cDNA was used to generate the C-terminal Myc-tagged caspase portion of FLICE (amino acids 181– 479) with a 59 primer containing the XbaI restriction site linker for ligation to Fas (GGC GGT TCT AGA GGC GGT TCC ACC ATG TTC AGC AAA GAG AGA AGC AGC AGC) and a 39 primer adding the Myc epitope and a stop codon (ATA TAG ATC TCA GTT CAG GTC CTC CTC GGA AAT CAG CTT CTG CTC ACC GGA ACC ATC AGA AGG GAA GAC Downloaded from AAG TTT TTT). The F/F PCR product was cloned and screened in pCR II and then moved into XbaI digested pCI-Fas as an XbaI/SpeI fragment FIGURE 1. Structure of Fas, FLICE, and the chimeric F/F receptors. which linked Fas to FLICE with a seven-amino acid spacer (GGSRGGS). The extracellular and transmembrane portion of Fas (amino acids 1–222) Mutant F/F was made by changing the active site cysteine (FLICE, C360) was joined by a peptide linker (see Materials and Methods) to the caspase to serine with primers that added a new XbaI restriction site (for screening) portion of FLICE (amino acids 181–479) to make the chimeric F/F recep- using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, tors (ligation sites shown as horizontal lines). DD, DED, and caspase sub- CA) (GTG TTT TTT ATT CAG GCT TCT AGA GGG GAT AAC TAC units are labeled. F/F and mutant F/F proteins are shown with the wild-type http://www.jimmunol.org/ CAG, CTC GTA GTT ATC CCC TCT AGA AGC CTG AAT AAA AAA CAC).
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