Current models for Fas (CD95)-mediated apoptosis suggest that FLICE/caspase-8 is recruited and activated, which results in cell death. However, the role of additional molecules in Fas signaling and FLICE activation is not clear. A chimeric Fas/FLICE (F/F) receptor, containing the extracellular/transmembrane portion of Fas and the caspase region of FLICE, mediated anti-Fas apoptosis. FLICE protease subunits were generated from the F/F precursor. Killing induced by Fas, but not F/F, was blocked by a dominant negative FADD. Apoptosis triggered through Fas and F/F was inhibited by coexpression of CrmA and p35, but not Bcl-xL. F/F bypassed Fas resistance in COS-7 cells and blocking by the death effector domain (DED)-containing viral protein MC159. These results show that: 1) F/F induces cell death, indicating that FLICE activation is sufficient for apoptosis and does not require additional Fas- or FADD-binding proteins; and 2) F/F bypasses proximal defects in Fas signaling that prevent FLICE recruitment or activation.
Fas/APO-1/CD95 is a member of the TNF/NGF receptor family that plays a critical role in cell homeostasis in both normal and pathologic processes (reviewed in Refs. 1 and 2). Fas contains an intracytoplasmic motif called a death domain (DD),2 which recruits molecules such as FADD/MORT to the plasma membrane through homotypic DD/DD protein interactions. Current data suggest that FADD, through another protein interaction motif called a DED (death effector domain), serves as an adapter to recruit FLICE/MACH/caspase-8 to the receptor complex (2, 3, 4). Following its proteolytic activation, FLICE initiates the apoptotic cascade, presumably by cleaving relevant downstream substrates. This model is supported by the ability of caspase inhibitors and dominant negative variants of FADD and FLICE to block Fas-mediated cell death (4, 5). However, whether additional molecules in the activated Fas receptor complex participate in apoptotic signaling is unknown, and the mechanism by which FLICE is recruited and converted to the active caspase is not clear. Several noncaspase signaling pathways have been implicated in Fas-induced cytotoxicity including sphingomyelinases and protein kinases. In addition to FADD, a variety of molecules that interact with the cytoplasmic portion of Fas have been identified that are potential mediators or modulators of the apoptotic signal including RIP, FAP, Daxx, UBC9, and FAF. Yet to be identified Fas-interacting molecules or DD/DED-recruited proteins such as TRADD, TRAF, caspase-10, or Casper could also be important for Fas-mediated cell death. Because overexpression of a dominant negative FADD would be expected to disrupt or prevent assembly of the activated Fas receptor complex, the exact role of these proteins in apoptotic signaling has not been established. To explore these issues, chimeric Fas/FLICE receptors (F/F) were made that lacked the intracellular portion of Fas and contained no DD or DED motifs.
Materials and Methods
Cell lines, reagents, and Abs
COS-7 cells (American Type Culture Collection, Rockville, MD; CRL1651), L1210 cells (6), and the mouse T cell hybridoma 2B4.11 cells (2B4) (7) have been described. Hybridoma cells secreting the anti-Myc mAb 9E10 were obtained from Dr. Allan Weissman (National Institutes of Health, Bethesda, MD). Dexamethasone (Dex) was purchased from Sigma (Sigma Chemical Co., St. Louis, MO), and anti-human Fas Ab CH-11 was purchased from Kamiya Biomedical Co. (Thousand Oaks, CA).
Expression vectors for Fas (pCI-Fas), Bcl-xL (CMV-Bcl-xL), CrmA (CMV-CrmA), and β-galactosidase (CMV-β-gal) have been described (7). pCI-MC159 (8) was kindly provided by Dr. Jeffrey Cohen (National Institutes of Health, Bethesda, MD). An expression plasmid for p35 (pCI-p35) was made from pRC-p35 (9), generously provided by Dr. Lois Miller (University of Georgia, Athens, GA). A dominant negative version of FADD (amino acids 91–208) containing only the DD was made by PCR from full length FADD, kindly provided by Dr. Michael Lenardo (National Institutes of Health, Bethesda, MD): 5′ primer (TAT ATG GCG CCT GGG GAA GAA GAC CTG TGT) and 3′ primer (TAG ATC TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA ACC GGA ACC GGA CGC TTC GGA GGT AGA TGC GTC). The PCR product was cloned and screened in pCR II (Invitrogen, San Diego, CA) and then moved to pCI (Promega, Madison, WI) as an EcoRI fragment.
Construction of chimeric receptors
F/F chimeric constructs were generated by PCR using primers incorporating appropriate restriction sites and Myc epitope tags. cDNA for the extracellular and transmembrane portion of human Fas (amino acids 1–222) was made by PCR from pCI-Fas using a 5′ primer (ACA ACC ATG CTG GGC ATC TGG ACC) and a 3′ 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 fragment. Full length FLICE cDNA was cloned by PCR from Jurkat mRNA (MicroFastTrack kit, Invitrogen) using homologous 5′ (GGC GGT TCT AGA GGC GGT TCC ACC ATG GAC TTC AGC AGA AAT CTT TAT) and 3′ (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 5′ 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 3′ 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 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 which linked Fas to FLICE with a seven-amino acid spacer (GGSRGGS). Mutant F/F was made by changing the active site cysteine (FLICE, C360) to serine with primers that added a new XbaI restriction site (for screening) using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) (GTG TTT TTT ATT CAG GCT TCT AGA GGG GAT AAC TAC CAG, CTC GTA GTT ATC CCC TCT AGA AGC CTG AAT AAA AAA CAC). This changed the amino acid sequence of the active site region from QACQG to QASRG. F/F chimeric receptor cDNAs were confirmed by DNA sequencing.
Transient transfection death assay
This assay has been described (7). Briefly, cells were transfected by electroporation with expression vector DNA along with the CMV-β-gal reporter plasmid. The transfected cells were cultured in medium, Dex, or with anti-Fas Ab overnight and then analyzed for β-gal activity. As with other death receptors, overexpression of Fas and F/F receptors by themselves led to cytotoxicity. This effect was minimized by reducing the amount of plasmid DNA transfected.
Electrophoretic protein analysis
COS-7 cells were transfected by electroporation with 5 μg of F/F DNA, and 107 cells were cultured overnight at 37°C in 5 ml of medium (6-well plates). Cells were labeled in 1.5 ml of methionine-free medium containing 0.2 mCi/ml of[35S]methionine (Trans35S label; ICN Radiochemicals, Irvine, CA) for 3 h at 37°C. After labeling, half of the wells were stimulated with 200 ng/ml of CH-11 anti-Fas Ab for 90 min. Detergent lysis of cells, immunoprecipitation, and SDS-PAGE have all been described (10). Briefly, cells were lysed in 1 ml of lysis buffer, and postnuclear supernatants were immunoprecipitated with 9E10 anti-Myc prebound to protein A beads for 2 h at 4°C. Immunoprecipitates were washed, eluted in reducing sample buffer, and resolved on 12% SDS-PAGE gels. Gels were fixed, impregnated with Enlightning (Dupont NEN, Boston, MA), and imaged with a Storm 820 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Results and Discussion
Construction of chimeric receptors
Fas consists of extracellular, transmembrane, and DD-containing cytoplasmic regions (Fig. 1). FLICE/caspase-8 contains a prodomain with two DEDs and a C-terminal caspase precursor with large and small protease subunits. Functional and biochemical evidence suggests that Fas ligation induces trimerization of the receptor, which brings FADD to the membrane complex through DD/DD interactions (2, 3). FLICE is then recruited to the plasma membrane through DED motifs, where it becomes proteolytically activated by cleavage at aspartate residues to form the mature caspase subunits. To determine whether FLICE/caspase-8 activation is sufficient to induce apoptosis or whether additional Fas- or FADD-binding proteins are required, chimeric molecules were generated containing the extracellular/transmembrane portion of Fas and the C-terminal caspase region of FLICE (Fig. 1). This chimeric receptor contains only a small amount of intracellular Fas and lacks DD/DEDs from Fas, FADD, and FLICE. F/F links Fas to FLICE C-terminal to the DEDs but before the cleavage sites that separate the prodomain from the caspase precursor (11, 12). A mutant F/F was made in which the active site cysteine was changed to serine to inactivate the proteolytic activity of the chimeric receptor.
Functional activity of chimeric receptors
To test the cytotoxic activity of the chimeric receptors, wild-type human Fas and the fusion constructs were transiently expressed in the Fas-negative mouse tumor L1210 and the mouse T cell hybridoma 2B4. We have previously reported that transient expression of human Fas in 2B4 conferred sensitivity to killing by anti-human Fas Abs (7). Transfected cells were cultured in medium alone or with increasing doses of anti-human Fas Ab CH-11. Expression of normal human Fas in L1210 and 2B4 led to cell death induced by the anti-Fas Ab (Fig. 2). Similarly, F/F showed potent cytotoxic activity. In contrast, the mutant F/F receptor was inactive. Identical results were obtained when individual transfected cells were followed by coexpression of the green fluorescent protein (data not shown). Thus, the functional activity of the chimeric receptors was similar to normal human Fas and required the protease active site cysteine.
Cleavage of chimeric receptors
Activated caspases are generated from an inactive precursor by cleavage between the large and small enzyme subunits and from the prodomain. To analyze the processing of F/F chimeric molecules, the C-terminal Myc epitope-tagged F/F constructs were transiently expressed in COS-7 cells. Based on studies with FLICE, F/F would be expected to be clipped before and after the large caspase subunit producing fragments of approximately 35, 18, and 11 kDa (11). Because F/F was metabolically labeled, anti-Myc immunoprecipitation should show the conversion of the intact molecule into both large and small subunits of the activated FLICE heterotetramer. In the absence of stimulation, the major Myc-precipitated band was seen at 65 to 70 kDa, corresponding to full length F/F (Fig. 3). Loss of the F/F precursor was seen following anti-Fas treatment along with the appearance of subunit bands of approximately 12 and 18 kDa. No caspase subunits or precursor loss was detected with mutant F/F. An enhanced gel image is shown that clearly identifies the breakdown products (indicated with arrows).
Effect of dominant negative FADD, Bcl-xL, CrmA, and p35 on chimeric receptor cytotoxic activity
Overexpression of a DD-containing dominant negative variant of FADD has been shown to block TNF- and Fas-mediated apoptosis (4, 5). Since the chimeric receptors lack DDs, they should be unaffected by a dominant negative FADD. Fas and F/F were transiently expressed in 2B4 cells with increasing amounts of dominant negative C-FADD plasmid DNA (amino acids 91–208). As expected, expression of C-FADD led to a dose-dependent inhibition of anti-Fas-induced apoptosis (Fig. 4,A, filled squares). However, killing by F/F was not influenced by C-FADD expression (open squares). To demonstrate the specificity of C-FADD for Fas signaling, Dex-induced apoptosis was also tested and was not affected by the cotransfection of C-FADD. We have previously shown that CrmA and Bcl-2 family members differentially inhibit Fas- and Dex-mediated cytotoxicity in T cells supporting distinct apoptotic signaling pathways (7, 13). To see whether the chimeric receptors showed the same behavior, F/F and Fas were cotransfected with Bcl-xL, CrmA, and p35. In agreement with previous results, both viral caspase inhibitors blocked killing induced through Fas, while Bcl-xL had no effect (Fig. 4 B). The inhibitory profile was exactly the same with F/F, suggesting that apoptotic signaling must be very similar to normal Fas. Additional controls showed that Bcl-xL (and p35) had functional activity, since Dex killing was blocked. In combination, these results suggest that direct FLICE activation through F/F receptor aggregation is sufficient for anti-Fas-triggered apoptosis and does not require additional Fas-, DD-, or DED-binding proteins. Other molecules in the active Fas receptor complex could, however, play an important regulatory role or trigger additional biochemical pathways.
Chimeric receptors induce apoptosis in Fas-resistant cells
Resistance to Fas-mediated apoptosis has been reported in both normal and malignant cells despite adequate Fas expression. Although absence of FADD or FLICE might explain this phenotype, the molecular basis for Fas resistance is generally unknown. Recently, viral and cellular DED-containing proteins have been described that interfere with apoptosis induced by death receptors (8, 14). Because of their unique structure and lack of DEDs, F/F chimeric receptors can be used to investigate the basis of Fas-resistance. Proximal defects in the Fas receptor complex that prevent FLICE recruitment or activation will be bypassed by expression of the F/F molecule. In contrast, resistance to F/F would suggest direct interference with the FLICE protease or a defect in downstream targets. To demonstrate that F/F expression can bypass proximal blocking molecules, the DED-containing MC159 protein was tested. This poxvirus gene product contains two DEDs that bind to FADD and potently inhibit Fas- and TNFR1-induced apoptosis (8). As seen with the dominant negative FADD, cotransfection of MC159 clearly blocked Fas- but not F/F-mediated cytotoxicity (Fig. 5,A). Based on these results, F/F receptors would be expected to bypass defects in Fas signaling due to the expression of any DD- or DED-containing viral or cellular inhibitor. COS-7 cells have been shown to be resistant to overexpression of caspase-1 (ICE) and caspase-2 (Ich-1) (15). Interestingly, when human Fas was transiently expressed in COS-7 cells, no cell death was induced (Fig. 5 B). However, expression of F/F, but not mutant F/F, led to clear anti-Fas induced cytotoxicity. Transient expression of FADD or FLICE, alone or in combination with Fas, did not result in cell death (data not shown). Although the exact molecular defect in COS-7 cells is not known, the results are consistent with a proximal DED-blocking protein and illustrate the use of F/F chimeric receptors to investigate Fas resistance.
We are grateful to Drs. Jeffrey Cohen and Michael Lenardo for providing pCI-MC159 and pCI-FADD, respectively. We thank Drs. Jon Ashwell and Allan Weissman for reviewing the manuscript.
Abbreviations used in this paper: DD, death domain; F/F, Fas/FLICE; DED, death effector domain; 2B4, T cell hybridoma 2B4.11; Dex, dexamethasone; β-gal, β-galactosidase.