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Fas) Death-Inducing Signaling Complex
METHODS: A Companion to Methods in Enzymology 17, 287–291 (1999) Article ID meth.1999.0742, available online at http://www.idealibrary.com on
Isolation and Analysis of Components of CD95 (APO-1/Fas) Death-Inducing Signaling Complex Carsten Scaffidi, Peter H. Krammer, and Marcus E. Peter1 Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
CD95 (APO-1/Fas) is an apoptosis-inducing receptor belonging to the tumor necrosis factor receptor superfamily. Multimerization of CD95 leads to instant recruitment of the signaling molecules FADD and caspase-8 to the activated receptor forming the death-inducing signaling complex (DISC). DISC formation is the first essential step of CD95 signaling and results in activation of caspase-8 starting a signaling cascade that leads to apoptosis. Here we describe a method for analyzing the CD95 DISC. The method is based on coimmunoprecipitation of the signaling molecules with the activated CD95 receptor followed by Western blot detection of associated molecules. Therefore, this method can analyze the very first signaling events during CD95mediated apoptosis. © 1999 Academic Press
CD95 (APO-1/Fas) is a member of the tumor necrosis factor and nerve growth factor receptor superfamily which includes type I transmembrane cell surface receptors that possess characteristic cysteine-rich repeats in their extracellular domains. Within this superfamily, CD95 belongs to the subgroup of the apoptosis-inducing “death receptors” (1). These receptors are characterized by a death domain (DD) in their cytoplasmic region which is essential for triggering apoptosis. Signal transduction of CD95 has been shown to involve activation of caspases (2, 3), a family of cysteine proteases which are essential for many forms of apoptosis (4). To analyze the mechanism how CD95 triggering leads to caspase activation and subTo whom correspondence should be addressed. Fax: 1496221-411715. E-mail: [email protected]. 1
1046-2023/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
sequent cell death we focused on the identification of receptor-associated signaling molecules. Recently, several groups reported the identification of CD95associated molecules by using the yeast two-hybrid system (5). However, as we were interested in proteins that specifically associate with the activated CD95 receptor in mammalian cells, we have developed a biochemical approach to identify such molecules. Activation of CD95 requires cross-linking of the receptor either by agonistic antibodies such as IgG3 anti-APO-1 (6) or by the natural ligand CD95L, whereas mere receptor dimerization is not sufficient for its activation (7). Therefore, we concentrated on proteins that specifically coimmunoprecipitated only with the activated (cross-linked) CD95 receptor. With this approach we identified a set of signaling molecules, designated cytotoxicity-dependent APO1-associated proteins (CAP1– 4) that form the deathinducing signaling complex (DISC) with the activated CD95 receptor (8) (Fig. 1). Formation of the DISC is an instant process after CD95 triggering and can be detected within seconds after receptor cross-linking (8, 9). CAP1 and CAP2 represent the phosphorylated adapter molecule FADD (also called MORT-1) (10, 11) which binds through its C-terminal DD to the DD of CD95 (8). The N terminus of FADD was shown to be essential for the recruitment of CAP3 and CAP4 to the DISC through its death effector domain (DED) (12). The identification of CAP4 as a member of the caspase family established the link between receptor-associated molecules and caspase activation (13). At its N terminus CAP4 also contains two DEDs with the N-terminal one being essential for binding to FADD in the DISC (14). Therefore, CAP4 was named FLICE (for FADD-like 287
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ICE) (13). It was also called MACH, Mch5, or caspase-8 (15, 16). Recruitment of caspase-8 to the DISC results in its proteolytic activation as the first step in CD95 signaling (14). This leads to the release of active caspase-8 subunits p18 and p10 into the cytoplasm which results in activation of other caspases and cleavage of various cellular proteins resulting in death of the cell. Recently, a new class of viral DED-containing proteins has been described acting as an inhibitor of CD95-mediated apoptosis by interfering with the DISC (17). They were called v-FLIPs (for viral FLICE-inhibitory proteins). v-FLIPs are encoded by g-herpes viruses such as herpes virus saimiri, the Karposi sarcoma virus-associated human herpes virus 8, and other viruses (18). These v-FLIPs contain two DEDs and were shown to bind to the CD95 DISC, preventing recruitment of caspase-8 to the CD95–FADD complex and thereby inhibiting complete DISC formation. Recently, a cellular homolog of v-FLIP, c-FLIP, was identified, showing homology to caspase-8 (19). Like its viral counterpart c-FLIP is also able to bind to the activated CD95 DISC, inhibiting caspase-8 activation and thereby CD95mediated apoptosis (20). As FLIPs and maybe other death-inhibitory or -promoting molecules act directly at the receptor level, the investigation of the DISC is important to study the first steps in death receptor signaling. Here we describe the technical details for analyzing the CD95 DISC.
Recently, specific monoclonal antibodies against the DISC components FADD, caspase-8, and c-FLIP became available (9) (and unpublished data), making it possible to detect receptor-associated molecules by Western blot analysis and establishing DISC analysis as a standard method. Here we describe the technical details for analyzing the CD95 DISC.
DESCRIPTION OF METHOD To analyze the CD95 receptor only when stimulated we made use of the following experimental system. The CD95 receptor is activated by the agonistic antibody anti-APO-1 (IgG3) (6) which leads to receptor crosslinking on the cell surface due to coaggregation of the IgG3 heavy chains. After being washed, the cells are lysed and only the receptors which have been stimulated by binding of the antiAPO-1 antibody are immunoprecipitated using protein A–Sepharose. By using the anti-APO-1 antibody for both stimulation and immunoprecipitation of CD95 only the activated receptor is immunoprecipitated whereas the receptor that was not crosslinked by anti-APO-1 remains unaffected (8). Therefore, the method described here can discriminate between activated and nonactivated CD95. The nonactivated CD95 receptor is immunoprecipitated to
FIG. 1. Analysis of the CD95 DISC by high-resolution 2D isoelectric focusing (IEF)–SDS–PAGE. 3 3 107 K50 cells (BL-60 cells stably expressing CD95 (8)) were metabolically labeled with 0.5 mCi [35S]methionine/cysteine (Amersham), lysed in lysis buffer, and subsequently supplemented with 3 mg of anti-APO-1 (B) or stimulated with 2 mg/ml anti-APO-1 for 5 min prior to lysis (C). Lysates were precleared with isotype-matched control antibody FII-23 covalently coupled to CNBr-activated Sepharose 4B beads and, subsequently, CD95 receptors were immunoprecipitated using protein A–Sepharose. Immunoprecipitates were analyzed by 2D IEF–SDS–PAGE and autoradiography. The stippled box indicates the migration position of CD95. Arrowheads indicate migration positions of CAP1-4.
CD95 DISC ANALYSIS
control which proteins associate in a stimulationdependent fashion. Specific activation-dependent signaling molecules are only detectable in the immunoprecipitate of the activated receptor, whereas nonspecific binding proteins will be present in both immunoprecipitates. Stimulation of CD95 Receptor and Cell Lysis DISC analysis is most suitable for cells with high levels of CD95 expression such as the B-lymphoblastoid cell line SKW6.4, the Burkitt lymphomas Raji or BJAB, or the leukemic T-cell line H9 or HuT78 (14). To analyze cell lines with lower CD95 expression some modifications may be applied as will be discussed below. For stimulation, cells (1 3 107 cells at a density of 2 3 106/ml) are incubated with 2 mg/ml anti-APO-1 at 37°C. Standard stimulation time is 5 min but kinetics may also be performed (9, 14). As a control, the same number of cells is left untreated. To remove unbound antibody and to terminate stimulation, cells are cooled to 4°C and washed once with ice-cold phosphate-buffered saline (PBS). The cell pellet is then resuspended in 1 ml of lysis buffer (30 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, small peptide inhibitors (8), 1 % Triton X-100 (Serva), and 10 % glycerol) and incubated on ice for 15 min and the lysate is cleared by centrifugation at 14,000g at 4°C. Immunoprecipitation of CD95 DISC As a control, nonactivated CD95 receptors are immunoprecipitated from nonstimulated cells. The lysate of untreated cells is supplemented with 1 mg of anti-APO-1 which binds CD95; however, crosslinking and DISC formation are no longer possible under these conditions. This amount of anti-APO-1 may be equivalent to the stimulated sample given the calculation that 10% of the antibody used for stimulation has bound to CD95 on the cell surface. In the following, both lysates are treated identically. To immunoprecipitate the DISC, lysates are supplemented with 30 ml of protein A–Sepharose 4B beads (Sigma) and incubated for more than 1 h rotating at 4°C. During that time the anti-APO-1 antibody together with stimulated or nonstimulated receptors binds to the protein A–Sepharose beads. The beads are then removed by centrifugation at 600g at 4°C for 1 min, supplemented with reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) sample buffer and boiled for 3 min at 95°C. Caspase-8 in vivo complexed to the DISC can also
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be used to test putative caspase targets for being caspase-8 substrates as the immunoprecipitated DISC was shown to contain caspase-8 as the only proteolytic activity (14). To this end the number of cells used for immunoprecipitation of the DISC should be increased (e.g., 5 3 107 SWK6.4 cells). The immunoprecipitated DISC is then incubated with putative caspase-8 substrates (e.g., in vitro translated 35S-labeled or recombinant proteins) in cleavage buffer [100 mM NaCl, 50mM Hepes, pH 7.4, 0.1% Chaps, 10 mM dithiothreitol, 10% sucrose] for 24 h at 4°C. Cleavage is then monitored by autoradiography or Western blot analysis (14). Control Immunoprecipitations To control for specificity and to remove nonspecific binding proteins, lysates may be treated with a control antibody before DISC precipitation. Therefore, we used the isotype-matched control antibody FII-23 (8) covalently coupled to CNBr-activated Sepharose 4B (Pharmacia). During this control immunoprecipitation no protein A or G-Sepharose must be used as this would lead to binding of the anti-APO-1 antibody to the beads and, therefore, to immunoprecipitation of the DISC. After incubation of lysates with FII-23 beads at 4°C for 1 h, the beads are removed and washed as described above. In cases with limiting cell numbers, nonstimulated CD95 receptors can be immunoprecipitated from the stimulated lysate as for unknown reasons stimulation of cells with anti-APO-1 leads only to cross-linking of approximately 50% of CD95, whereas approximately 50% of the receptors remain monomeric. These monomeric receptors can selectively be immunoprecipitated from the stimulated lysate after DISC precipitation using anti-APO-1 antibody covalently coupled to CNBr-activated Sepharose 4B (8). This immunoprecipitation can then serve as an unstimulated control and cells from the untreated sample may be saved. Western Blot Analysis of Immunoprecipitates For Western blot detection of caspase-8 and FADD (9, 20), DISC immunoprecipitates are boiled in reducing sample buffer and separated by 12% SDS– PAGE. Proteins are transferred onto a nitrocellulose membrane (Hybond C, Amersham) using a semidry blotting system and transfer buffer [25 mM Tris, 190 mM glycine, 0.04% SDS, 20% methanol] at 0.8 mA/ cm2 for 1.5 h. Transfer efficiency may be controlled by using a colored protein molecular weight standard marker such as the Rainbow marker (Amer-
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sham). The membrane is then blocked with 2% bovine serum albumin in TPBS (0.05% Tween 20 in PBS) for 1 h at room temperature. After washing three times for 10 min in TPBS the same membrane may be used for detection of both caspase-8 and FADD. Therefore, the membrane is cut slightly above 30 kDa (as judged by the molecular weight marker) and the upper part is incubated with the anti-caspase-8 monoclonal antibody (mAb) (C15, 1:20 diluted in TPBS) and the lower part is incubated with an anti-FADD mAb (Transduction Laboratories) for 16 h at 4°C. After washing as described above the membranes are incubated with secondary antibody. A secondary antibody directed against mouse IgG would cross-react with the anti-APO-1 antibody used for immunoprecipitation resulting in strong signals at about 50 and 25 kDa which closely correspond to the migration positions of caspase-8 (55 kDa) and FADD (27 kDa), respectively. To avoid this, isotype-specific antibodies are used (anti-IgG2b for C15 and anti-IgG1 for anti-FADD). Secondary antibodies (Southern Biotechnologies) are diluted
1:20,000 in TPBS and incubated for 1 h at room temperature. Blots are washed and developed with enhanced chemiluminescence following the manufacturer’s instructions (Amersham). Results Figure 2 shows a typical result of a DISC analysis of SKW6.4 cells. Caspase-8 and FADD are only detectable in the immunoprecipitates of stimulated CD95 (Fig. 2, lane 4) but are absent in the immunoprecipitate of the nonstimulated receptors (Fig. 2, lane 3) or the monomeric receptors from stimulated cells (Fig. 2, lane 5). They are also not detectable in the immunoprecipitation using FII-23 control antibody (Fig. 2, lanes 1 and 2). This demonstrates that both FADD and caspase-8 only associate with CD95 after receptor cross-linking. Caspase-8 is detected as a double band due to two different isoforms, caspase8/a and -8/b, which are expressed and recruited to the DISC in various cell lines (9). Not only fulllength caspase-8 can be detected in the DISC, but also the cleavage intermediates p43 and p41 which are generated by cleaving the p10 active subunit from caspase-8/a and 8/b, respectively. Also the prodomains of the two isoforms, migrating at 26 and 24 kDa, respectively, can be detected in the CD95 DISC (9). Therefore, the method described here cannot only detect association of FADD and caspase-8 with activated receptors, but can also detect proteolytic activation of caspase-8 at the DISC as the first signaling step in CD95-mediated apoptosis (14).
CONCLUDING REMARKS
FIG. 2. Analysis of the CD95 DISC by Western blot. 1 3 107 SKW6.4 cells were stimulated with 2 mg/ml anti-APO-1 for 5 min (1) or left untreated (2). After cell lysis the untreated sample was supplemented with 2 mg of anti-APO-1 and both lysates were precleared with an isotype-matched control antibody (FII-23) covalently coupled to CNBr-activated Sepharose 4B beads and subsequently CD95 receptors were immunoprecipitated using protein A–Sepharose. After that nonactivated receptors were immunoprecipitated from the stimulated lysate by using antiAPO-1 covalently coupled to CNBr-activated Sepharose 4B. Immunoprecipitates were analyzed by SDS–PAGE followed by Western blotting with anti-caspase-8 mAb (C15) or anti-FADD mAb as described in the text.
The method described here is a powerful tool to investigate DISC formation as the very first event after CD95 cross-linking. DISC formation is an essential step in CD95 signaling as cells that are defective in DISC formation, for example, by ectopic expression of a truncated CD95 receptor (8), a dominant-negative deletion mutant of FADD (12), or a FLIP molecule (17, 19) are resistant to CD95mediated apoptosis. To make DISC analysis suitable for different applications, some modifications of the above-described method will be discussed. To analyze cells with lower expression of CD95 the number of cells used per sample should be increased (up to 5–10 3 107). In addition, we recently described cell lines that form only very little DISC despite high
CD95 DISC ANALYSIS
CD95 cell surface expression (21). These cells, designated type II cells, activate only a little caspase-8 at the receptor level and depend, therefore, on mitochondrial activity during apoptosis as death can be blocked by Bcl-2 overexpression. The reason why type II cells, such as the T-cell lines Jurkat and CEM, form only little DISC is unknown at the moment. Analysis of the DISC in these cells is, therefore, only possible by increasing the number of cells or the concentration of anti-APO-1 used for stimulation. Recently, it became clear that the CD95 system is also involved in apoptosis induction by some cytotoxic drugs (22–24). Cells that were defective in CD95 signaling were, therefore, also found to be resistant to drug-induced apoptosis, at least in part (24 –26). As DISC analysis as described here is suitable for being established as a standard method for investigating the early CD95 signaling events it may be useful in determine the block in, e.g., drugresistant cancer cells.
ACKNOWLEDGMENTS This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium fu¨r Forschung und Technologie, and the Tumor Center Heidelberg/ Mannheim.
REFERENCES 1. Peter, M. E., Scaffidi, C., Medema, J. P., Kischkel, F., and Krammer, P. H. (1999) Results. Probl. Cell Differ. 23, 25– 63. 2. Los, M., Van de Craen, M., Penning, L. C., Schenk, H., Westendorp, M., Baeuerle, P. A., Droge, W., Krammer, P. H., Fiers, W., and Schulze-Osthoff, K. (1995) Nature 375, 81– 83. 3. Enari, M., Hug, H., and Nagata, S. (1995) Nature 375, 78 – 81. 4. Salvesen, G. S., and Dixit, V. M. (1997) Cell 91, 443– 446. 5. Peter, M. E., Kischkel, F. C., Hellbardt, S., Chinnaiyan, A. M., Krammer, P. H., and Dixit, V. M. (1996) Cell Death Differ. 3, 161–170. 6. Trauth, B. C., Klas, C., Peters, A. M., Matzku, S., Mo¨ller, P., Falk, W., Debatin, K.-M., and Krammer, P. H. (1989) Science 245, 301–305. 7. Dhein, J., Daniel, P. T., Trauth, B. C., Oehm, A., Mo¨ller, P., and Krammer, P. H. (1992) J. Immunol. 149, 3166 – 3173.
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8. Kischkel, F. C., Hellbardt, S., Behrmann, I., Germer, M., Pawlita, M., Krammer, P. H., and Peter, M. E. (1995) EMBO J. 14, 5579 –5588. 9. Scaffidi, C., Medema, J. P., Krammer, P. H., and Peter, M. E. (1997) J. Biol. Chem. 272, 26953–26958. 10. Chinnaiyan, A. M., O’Rourke, K., Tewari, M., and Dixit, V. M. (1995) Cell 81, 505–512. 11. Boldin, M. P., Varfolomeev, E. E., Pancer, Z., Mett, I. L., Camonis, J. H., and Wallach, D. (1995) J. Biol. Chem. 270, 7795–7798. 12. Chinnaiyan, A. M., Tepper, C. G., Seldin, M. F., O’Rourke, K., Kischkel, F. C., Hellbardt, S., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) J. Biol. Chem. 271, 4961– 4965. 13. Muzio, 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., and Dixit, V. M. (1996) Cell 85, 817– 827. 14. Medema, J. P., Scaffidi, C., Kischkel, F. C., Shevchenko, A., Mann, M., Krammer, P. H., and Peter, M. E. (1997) EMBO J. 16, 2794 –2804. 15. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803– 815. 16. Fernandes-Alnemri, T., Armstrong, R. C., Krebs, J., Srinivasula, S. M., Wang, L., Bullrich, F., Fritz, L. C., Trapani, J. A., Tomaselli, K. J., Litwack, G., and Alnemri, E. S. (1996) Proc. Natl. Acad. Sci. USA 93, 7464 –7469. 17. Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E., Neipel, F., Mattmann, C., Burns, K., Bodmer, J. L., Schroter, M., Scaffidi, C., Krammer, P. H., Peter, M. E., and Tschopp, J. (1997) Nature 386, 517–521. 18. Tschopp, J., Thome, M., Hoffmann, K., and Meinl, E. (1998) Curr. Opin. Genet. Dev. 8, 82– 87. 19. Irmler, 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., and Tschopp, J. (1997) Nature 388, 190 –195. 20. Scaffidi, C., Schmitz, I., Krammer, P. H., and Peter, M. E. (1999) J. Biol. Chem. 274, 1541–1548. 21. Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K. J., Debatin, K. M., Krammer, P. H., and Peter, M. E. (1998) EMBO J. 17, 1675–1687. 22. Mu¨ller, M., Strand, S., Hug, H., Heinemann, E. M., Walczak, H., Hofmann, W. J., Stremmel, W., Krammer, P. H., and Galle, P. R. (1997) J. Clin. Invest. 99, 403– 413. 23. Fulda, S., Sieverts, H., Friesen, C., Herr, I., and Debatin, K. M. (1997) Cancer Res. 57, 3823–3829. 24. Friesen, C., Herr, I., Krammer, P. H., and Debatin, K. M. (1996) Nature Med. 2, 574 –577. 25. Los, M., Herr, I., Friesen, C., Fulda, S., Schulze-Osthoff, K., and Debatin, K. M. (1997) Blood 90, 3118 –3129. 26. Friesen, C., Fulda, S., and Debatin, K. M. (1997) Leukemia 11, 1833–1841.
Isolation and Analysis of Components of CD95 (APO-1/Fas) Death-Inducing Signaling Complex Carsten Scaffidi, Peter H. Krammer, and Marcus E. Peter1 Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
CD95 (APO-1/Fas) is an apoptosis-inducing receptor belonging to the tumor necrosis factor receptor superfamily. Multimerization of CD95 leads to instant recruitment of the signaling molecules FADD and caspase-8 to the activated receptor forming the death-inducing signaling complex (DISC). DISC formation is the first essential step of CD95 signaling and results in activation of caspase-8 starting a signaling cascade that leads to apoptosis. Here we describe a method for analyzing the CD95 DISC. The method is based on coimmunoprecipitation of the signaling molecules with the activated CD95 receptor followed by Western blot detection of associated molecules. Therefore, this method can analyze the very first signaling events during CD95mediated apoptosis. © 1999 Academic Press
CD95 (APO-1/Fas) is a member of the tumor necrosis factor and nerve growth factor receptor superfamily which includes type I transmembrane cell surface receptors that possess characteristic cysteine-rich repeats in their extracellular domains. Within this superfamily, CD95 belongs to the subgroup of the apoptosis-inducing “death receptors” (1). These receptors are characterized by a death domain (DD) in their cytoplasmic region which is essential for triggering apoptosis. Signal transduction of CD95 has been shown to involve activation of caspases (2, 3), a family of cysteine proteases which are essential for many forms of apoptosis (4). To analyze the mechanism how CD95 triggering leads to caspase activation and subTo whom correspondence should be addressed. Fax: 1496221-411715. E-mail: [email protected]. 1
1046-2023/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
sequent cell death we focused on the identification of receptor-associated signaling molecules. Recently, several groups reported the identification of CD95associated molecules by using the yeast two-hybrid system (5). However, as we were interested in proteins that specifically associate with the activated CD95 receptor in mammalian cells, we have developed a biochemical approach to identify such molecules. Activation of CD95 requires cross-linking of the receptor either by agonistic antibodies such as IgG3 anti-APO-1 (6) or by the natural ligand CD95L, whereas mere receptor dimerization is not sufficient for its activation (7). Therefore, we concentrated on proteins that specifically coimmunoprecipitated only with the activated (cross-linked) CD95 receptor. With this approach we identified a set of signaling molecules, designated cytotoxicity-dependent APO1-associated proteins (CAP1– 4) that form the deathinducing signaling complex (DISC) with the activated CD95 receptor (8) (Fig. 1). Formation of the DISC is an instant process after CD95 triggering and can be detected within seconds after receptor cross-linking (8, 9). CAP1 and CAP2 represent the phosphorylated adapter molecule FADD (also called MORT-1) (10, 11) which binds through its C-terminal DD to the DD of CD95 (8). The N terminus of FADD was shown to be essential for the recruitment of CAP3 and CAP4 to the DISC through its death effector domain (DED) (12). The identification of CAP4 as a member of the caspase family established the link between receptor-associated molecules and caspase activation (13). At its N terminus CAP4 also contains two DEDs with the N-terminal one being essential for binding to FADD in the DISC (14). Therefore, CAP4 was named FLICE (for FADD-like 287
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ICE) (13). It was also called MACH, Mch5, or caspase-8 (15, 16). Recruitment of caspase-8 to the DISC results in its proteolytic activation as the first step in CD95 signaling (14). This leads to the release of active caspase-8 subunits p18 and p10 into the cytoplasm which results in activation of other caspases and cleavage of various cellular proteins resulting in death of the cell. Recently, a new class of viral DED-containing proteins has been described acting as an inhibitor of CD95-mediated apoptosis by interfering with the DISC (17). They were called v-FLIPs (for viral FLICE-inhibitory proteins). v-FLIPs are encoded by g-herpes viruses such as herpes virus saimiri, the Karposi sarcoma virus-associated human herpes virus 8, and other viruses (18). These v-FLIPs contain two DEDs and were shown to bind to the CD95 DISC, preventing recruitment of caspase-8 to the CD95–FADD complex and thereby inhibiting complete DISC formation. Recently, a cellular homolog of v-FLIP, c-FLIP, was identified, showing homology to caspase-8 (19). Like its viral counterpart c-FLIP is also able to bind to the activated CD95 DISC, inhibiting caspase-8 activation and thereby CD95mediated apoptosis (20). As FLIPs and maybe other death-inhibitory or -promoting molecules act directly at the receptor level, the investigation of the DISC is important to study the first steps in death receptor signaling. Here we describe the technical details for analyzing the CD95 DISC.
Recently, specific monoclonal antibodies against the DISC components FADD, caspase-8, and c-FLIP became available (9) (and unpublished data), making it possible to detect receptor-associated molecules by Western blot analysis and establishing DISC analysis as a standard method. Here we describe the technical details for analyzing the CD95 DISC.
DESCRIPTION OF METHOD To analyze the CD95 receptor only when stimulated we made use of the following experimental system. The CD95 receptor is activated by the agonistic antibody anti-APO-1 (IgG3) (6) which leads to receptor crosslinking on the cell surface due to coaggregation of the IgG3 heavy chains. After being washed, the cells are lysed and only the receptors which have been stimulated by binding of the antiAPO-1 antibody are immunoprecipitated using protein A–Sepharose. By using the anti-APO-1 antibody for both stimulation and immunoprecipitation of CD95 only the activated receptor is immunoprecipitated whereas the receptor that was not crosslinked by anti-APO-1 remains unaffected (8). Therefore, the method described here can discriminate between activated and nonactivated CD95. The nonactivated CD95 receptor is immunoprecipitated to
FIG. 1. Analysis of the CD95 DISC by high-resolution 2D isoelectric focusing (IEF)–SDS–PAGE. 3 3 107 K50 cells (BL-60 cells stably expressing CD95 (8)) were metabolically labeled with 0.5 mCi [35S]methionine/cysteine (Amersham), lysed in lysis buffer, and subsequently supplemented with 3 mg of anti-APO-1 (B) or stimulated with 2 mg/ml anti-APO-1 for 5 min prior to lysis (C). Lysates were precleared with isotype-matched control antibody FII-23 covalently coupled to CNBr-activated Sepharose 4B beads and, subsequently, CD95 receptors were immunoprecipitated using protein A–Sepharose. Immunoprecipitates were analyzed by 2D IEF–SDS–PAGE and autoradiography. The stippled box indicates the migration position of CD95. Arrowheads indicate migration positions of CAP1-4.
CD95 DISC ANALYSIS
control which proteins associate in a stimulationdependent fashion. Specific activation-dependent signaling molecules are only detectable in the immunoprecipitate of the activated receptor, whereas nonspecific binding proteins will be present in both immunoprecipitates. Stimulation of CD95 Receptor and Cell Lysis DISC analysis is most suitable for cells with high levels of CD95 expression such as the B-lymphoblastoid cell line SKW6.4, the Burkitt lymphomas Raji or BJAB, or the leukemic T-cell line H9 or HuT78 (14). To analyze cell lines with lower CD95 expression some modifications may be applied as will be discussed below. For stimulation, cells (1 3 107 cells at a density of 2 3 106/ml) are incubated with 2 mg/ml anti-APO-1 at 37°C. Standard stimulation time is 5 min but kinetics may also be performed (9, 14). As a control, the same number of cells is left untreated. To remove unbound antibody and to terminate stimulation, cells are cooled to 4°C and washed once with ice-cold phosphate-buffered saline (PBS). The cell pellet is then resuspended in 1 ml of lysis buffer (30 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, small peptide inhibitors (8), 1 % Triton X-100 (Serva), and 10 % glycerol) and incubated on ice for 15 min and the lysate is cleared by centrifugation at 14,000g at 4°C. Immunoprecipitation of CD95 DISC As a control, nonactivated CD95 receptors are immunoprecipitated from nonstimulated cells. The lysate of untreated cells is supplemented with 1 mg of anti-APO-1 which binds CD95; however, crosslinking and DISC formation are no longer possible under these conditions. This amount of anti-APO-1 may be equivalent to the stimulated sample given the calculation that 10% of the antibody used for stimulation has bound to CD95 on the cell surface. In the following, both lysates are treated identically. To immunoprecipitate the DISC, lysates are supplemented with 30 ml of protein A–Sepharose 4B beads (Sigma) and incubated for more than 1 h rotating at 4°C. During that time the anti-APO-1 antibody together with stimulated or nonstimulated receptors binds to the protein A–Sepharose beads. The beads are then removed by centrifugation at 600g at 4°C for 1 min, supplemented with reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) sample buffer and boiled for 3 min at 95°C. Caspase-8 in vivo complexed to the DISC can also
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be used to test putative caspase targets for being caspase-8 substrates as the immunoprecipitated DISC was shown to contain caspase-8 as the only proteolytic activity (14). To this end the number of cells used for immunoprecipitation of the DISC should be increased (e.g., 5 3 107 SWK6.4 cells). The immunoprecipitated DISC is then incubated with putative caspase-8 substrates (e.g., in vitro translated 35S-labeled or recombinant proteins) in cleavage buffer [100 mM NaCl, 50mM Hepes, pH 7.4, 0.1% Chaps, 10 mM dithiothreitol, 10% sucrose] for 24 h at 4°C. Cleavage is then monitored by autoradiography or Western blot analysis (14). Control Immunoprecipitations To control for specificity and to remove nonspecific binding proteins, lysates may be treated with a control antibody before DISC precipitation. Therefore, we used the isotype-matched control antibody FII-23 (8) covalently coupled to CNBr-activated Sepharose 4B (Pharmacia). During this control immunoprecipitation no protein A or G-Sepharose must be used as this would lead to binding of the anti-APO-1 antibody to the beads and, therefore, to immunoprecipitation of the DISC. After incubation of lysates with FII-23 beads at 4°C for 1 h, the beads are removed and washed as described above. In cases with limiting cell numbers, nonstimulated CD95 receptors can be immunoprecipitated from the stimulated lysate as for unknown reasons stimulation of cells with anti-APO-1 leads only to cross-linking of approximately 50% of CD95, whereas approximately 50% of the receptors remain monomeric. These monomeric receptors can selectively be immunoprecipitated from the stimulated lysate after DISC precipitation using anti-APO-1 antibody covalently coupled to CNBr-activated Sepharose 4B (8). This immunoprecipitation can then serve as an unstimulated control and cells from the untreated sample may be saved. Western Blot Analysis of Immunoprecipitates For Western blot detection of caspase-8 and FADD (9, 20), DISC immunoprecipitates are boiled in reducing sample buffer and separated by 12% SDS– PAGE. Proteins are transferred onto a nitrocellulose membrane (Hybond C, Amersham) using a semidry blotting system and transfer buffer [25 mM Tris, 190 mM glycine, 0.04% SDS, 20% methanol] at 0.8 mA/ cm2 for 1.5 h. Transfer efficiency may be controlled by using a colored protein molecular weight standard marker such as the Rainbow marker (Amer-
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sham). The membrane is then blocked with 2% bovine serum albumin in TPBS (0.05% Tween 20 in PBS) for 1 h at room temperature. After washing three times for 10 min in TPBS the same membrane may be used for detection of both caspase-8 and FADD. Therefore, the membrane is cut slightly above 30 kDa (as judged by the molecular weight marker) and the upper part is incubated with the anti-caspase-8 monoclonal antibody (mAb) (C15, 1:20 diluted in TPBS) and the lower part is incubated with an anti-FADD mAb (Transduction Laboratories) for 16 h at 4°C. After washing as described above the membranes are incubated with secondary antibody. A secondary antibody directed against mouse IgG would cross-react with the anti-APO-1 antibody used for immunoprecipitation resulting in strong signals at about 50 and 25 kDa which closely correspond to the migration positions of caspase-8 (55 kDa) and FADD (27 kDa), respectively. To avoid this, isotype-specific antibodies are used (anti-IgG2b for C15 and anti-IgG1 for anti-FADD). Secondary antibodies (Southern Biotechnologies) are diluted
1:20,000 in TPBS and incubated for 1 h at room temperature. Blots are washed and developed with enhanced chemiluminescence following the manufacturer’s instructions (Amersham). Results Figure 2 shows a typical result of a DISC analysis of SKW6.4 cells. Caspase-8 and FADD are only detectable in the immunoprecipitates of stimulated CD95 (Fig. 2, lane 4) but are absent in the immunoprecipitate of the nonstimulated receptors (Fig. 2, lane 3) or the monomeric receptors from stimulated cells (Fig. 2, lane 5). They are also not detectable in the immunoprecipitation using FII-23 control antibody (Fig. 2, lanes 1 and 2). This demonstrates that both FADD and caspase-8 only associate with CD95 after receptor cross-linking. Caspase-8 is detected as a double band due to two different isoforms, caspase8/a and -8/b, which are expressed and recruited to the DISC in various cell lines (9). Not only fulllength caspase-8 can be detected in the DISC, but also the cleavage intermediates p43 and p41 which are generated by cleaving the p10 active subunit from caspase-8/a and 8/b, respectively. Also the prodomains of the two isoforms, migrating at 26 and 24 kDa, respectively, can be detected in the CD95 DISC (9). Therefore, the method described here cannot only detect association of FADD and caspase-8 with activated receptors, but can also detect proteolytic activation of caspase-8 at the DISC as the first signaling step in CD95-mediated apoptosis (14).
CONCLUDING REMARKS
FIG. 2. Analysis of the CD95 DISC by Western blot. 1 3 107 SKW6.4 cells were stimulated with 2 mg/ml anti-APO-1 for 5 min (1) or left untreated (2). After cell lysis the untreated sample was supplemented with 2 mg of anti-APO-1 and both lysates were precleared with an isotype-matched control antibody (FII-23) covalently coupled to CNBr-activated Sepharose 4B beads and subsequently CD95 receptors were immunoprecipitated using protein A–Sepharose. After that nonactivated receptors were immunoprecipitated from the stimulated lysate by using antiAPO-1 covalently coupled to CNBr-activated Sepharose 4B. Immunoprecipitates were analyzed by SDS–PAGE followed by Western blotting with anti-caspase-8 mAb (C15) or anti-FADD mAb as described in the text.
The method described here is a powerful tool to investigate DISC formation as the very first event after CD95 cross-linking. DISC formation is an essential step in CD95 signaling as cells that are defective in DISC formation, for example, by ectopic expression of a truncated CD95 receptor (8), a dominant-negative deletion mutant of FADD (12), or a FLIP molecule (17, 19) are resistant to CD95mediated apoptosis. To make DISC analysis suitable for different applications, some modifications of the above-described method will be discussed. To analyze cells with lower expression of CD95 the number of cells used per sample should be increased (up to 5–10 3 107). In addition, we recently described cell lines that form only very little DISC despite high
CD95 DISC ANALYSIS
CD95 cell surface expression (21). These cells, designated type II cells, activate only a little caspase-8 at the receptor level and depend, therefore, on mitochondrial activity during apoptosis as death can be blocked by Bcl-2 overexpression. The reason why type II cells, such as the T-cell lines Jurkat and CEM, form only little DISC is unknown at the moment. Analysis of the DISC in these cells is, therefore, only possible by increasing the number of cells or the concentration of anti-APO-1 used for stimulation. Recently, it became clear that the CD95 system is also involved in apoptosis induction by some cytotoxic drugs (22–24). Cells that were defective in CD95 signaling were, therefore, also found to be resistant to drug-induced apoptosis, at least in part (24 –26). As DISC analysis as described here is suitable for being established as a standard method for investigating the early CD95 signaling events it may be useful in determine the block in, e.g., drugresistant cancer cells.
ACKNOWLEDGMENTS This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium fu¨r Forschung und Technologie, and the Tumor Center Heidelberg/ Mannheim.
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