Activation of Initiator Caspases through a Stable Dimeric Intermediate
2002; Elsevier BV; Volume: 277; Issue: 52 Linguagem: Inglês
10.1074/jbc.m210356200
ISSN1083-351X
AutoresMin Chen, Aaron Orozco, David M. Spencer, Jin Wang,
Tópico(s)Autophagy in Disease and Therapy
ResumoStructural and biochemical studies have revealed that procaspases form dimers prior to proteolytic activation. How the two procaspases interact in the dimer is unclear. To study the mechanisms of dimer-dependent caspase activation we used a heterodimeric system so that two caspase molecules can be specifically brought together. Surprisingly, only one caspase partner in the dimer needs to be enzymatically active for caspase processing and activation to occur. Caspase activation is inefficient in the dimer in the absence of intramolecular processing, suggesting that caspase activation is initiated via intramolecular processing. Homodimerization of caspase-8 or caspase-9 leads to the formation of a stable dimeric complex. However, heterodimerization between caspase-8 and caspases-3, -9, or -10 failed to induce stable dimer formation or caspase activation. Our data suggest that the formation of a stable dimeric intermediate initiates caspase activation. Structural and biochemical studies have revealed that procaspases form dimers prior to proteolytic activation. How the two procaspases interact in the dimer is unclear. To study the mechanisms of dimer-dependent caspase activation we used a heterodimeric system so that two caspase molecules can be specifically brought together. Surprisingly, only one caspase partner in the dimer needs to be enzymatically active for caspase processing and activation to occur. Caspase activation is inefficient in the dimer in the absence of intramolecular processing, suggesting that caspase activation is initiated via intramolecular processing. Homodimerization of caspase-8 or caspase-9 leads to the formation of a stable dimeric complex. However, heterodimerization between caspase-8 and caspases-3, -9, or -10 failed to induce stable dimer formation or caspase activation. Our data suggest that the formation of a stable dimeric intermediate initiates caspase activation. death effector domain fas-associated death domain protein death-inducing signaling complex caspase-recruitment domain chemical inducer of dimerization FK506-binding protein green fluorescence protein hemagglutinin cysteine to serine mutation FKBP rapamycin-binding FKBP containing a F36V mutation "frozen" aspartate to alanine 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside FK506-binding protein 12 Caspases are a family of cysteine proteases that cleave target proteins at specific aspartate residues (1Alnemri E.S. J. Cell. Biochem. 1997; 64: 33-42Crossref PubMed Scopus (290) Google Scholar, 2Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1306) Google Scholar). 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Recent studies suggest that procaspases at high local concentrations form a globular dimeric structure (18Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1465) Google Scholar, 28Chai J., Wu, Q. Shiozaki E. Srinivasula S.M. Alnemri E.S. Shi Y. Cell. 2001; 107: 399-407Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 29Riedl S.J. Fuentes-Prior P. Renatus M. Kairies N. Krapp S. Huber R. Salvesen G.S. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14790-14795Crossref PubMed Scopus (196) Google Scholar, 30Acehan D. Jiang X. Morgan D.G. Heuser J.E. Wang X. Akey C.W. Mol. Cell. 2002; 9: 423-432Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 31Renatus M. Stennicke H.R. Scott F.L. Liddington R.C. Salvesen G.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14250-14255Crossref PubMed Scopus (372) Google Scholar). In contrast to the inactive monomeric procaspases, dimeric procaspases display protease activities (29Riedl S.J. Fuentes-Prior P. Renatus M. Kairies N. Krapp S. Huber R. Salvesen G.S. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14790-14795Crossref PubMed Scopus (196) Google Scholar, 31Renatus M. Stennicke H.R. Scott F.L. Liddington R.C. Salvesen G.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14250-14255Crossref PubMed Scopus (372) Google Scholar). Therefore, high local concentrations of caspase-8 and caspase-10 in the DISC and caspase-9 in the apoptosome may lead to caspase activation through dimer formation. Comparisons between the crystal structures of procaspases and active caspases suggest that conformational changes lead to exposure of the protease active sites in active caspases (18Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1465) Google Scholar, 28Chai J., Wu, Q. Shiozaki E. Srinivasula S.M. Alnemri E.S. Shi Y. Cell. 2001; 107: 399-407Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 29Riedl S.J. Fuentes-Prior P. Renatus M. Kairies N. Krapp S. Huber R. Salvesen G.S. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14790-14795Crossref PubMed Scopus (196) Google Scholar, 30Acehan D. Jiang X. Morgan D.G. Heuser J.E. Wang X. Akey C.W. Mol. Cell. 2002; 9: 423-432Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 31Renatus M. Stennicke H.R. Scott F.L. Liddington R.C. Salvesen G.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14250-14255Crossref PubMed Scopus (372) Google Scholar). Therefore, dimer formation may activate caspases by inducing conformational changes that facilitate proteolytic processing of caspase zymogens into active protease subunits. Although dimerization is sufficient to activate caspases, the exact mechanism of caspase activation in the dimer has not been resolved. It is possible that one procaspase cleaves an adjacent procaspase by an intermolecular processing mechanism. Alternatively, each procaspase in the oligomer may cleave itself by an intramolecular processing mechanism. In this study, we provide evidence that the formation of a stable dimeric intermediate induces caspase activation by an intramolecular processing mechanism. A synthetic rapamycin analog, AP20840 (Ariad Pharmaceutics), was used as the chemical inducer of dimerization (CID) to induce dimer formation between one FK506-binding protein containing a F36V mutation (FV) and one FKBP rapamycin-binding (FRB) domain of the FKBP rapamycin-associated protein. Monoclonal and polyclonal anti-HA1.1 antibodies were obtained from Convance (Berkeley, CA). Monoclonal anti-FLAG M2 was from Sigma. Protein G-Sepharose beads were from Amersham Biosciences. FKBP containing an FV has been described (32Clackson T. Yang W. Rozamus L.W. Hatada M. Amara J.F. Rollins C.T. Stevenson L.F. Magari S.R. Wood S.A. Courage N.L., Lu, X. Cerasoli F., Jr. Gilman M. Holt D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10437-10442Crossref PubMed Scopus (420) Google Scholar). The FRB domain used in this study contains FKBP rapamycin-associated protein amino acid residues 2025–2114 with a threonine to leucine mutation at amino acid 2098. A single copy of FV or FRB was used to construct fusion proteins to ensure that each fusion protein has only one binding site for the rapamycin analog, AP20840. Part of the prodomain and the entire protease domain of caspase-8 (amino acid residues 207–479), caspase-10 (amino acid residues 210–522), or caspase-9 (amino acid residues 139–415) were fused in frame to the C terminus of FV or FRB. The entire coding region of caspase-3, caspase-6, or caspase-7 was fused to the C terminus of FV or FRB. An HA or FLAG tag was fused to the N terminus of each Fv and FRB construct, respectively. The cysteine residues in the QACQG protease active site of caspase-8 (amino acid residue 360) and caspase-10 (amino acid residue 415) or the QACGG protease active site of caspase-9 (amino acid residue 315) were mutated to serine by site-directed mutagenesis (Stratagene). The frozen mutant of caspase-8 (caspase-8DD/AA) was generated by mutating amino acid residues 374 and 384 from aspartate into alanine by site-directed mutagenesis according to the protocol of the supplier (Stratagene). Plasmids were verified by DNA sequencing, and those free of errors were used for experiments. HeLa cells in 6-well plates were transfected with wild type and cysteine to serine mutation (C/S) mutant caspase constructs at the indicated amounts plus 1 μg of pcDNA3-lacZ by the FuGENE 6 method (Roche, Indianapolis, IN). 18 h after transfection the cells were fixed with phosphate-buffered saline containing 2% formaldehyde and 0.2% glutaraldehyde at room temperature for 5 min. The cells were then stained by incubation in phosphate-buffered saline containing 1 μg/ml X-gal, 5 mm ferricyanide, 5 mm ferrocyanide, 2 mm MgCl2, 0.02% Triton X-100, and 0.01% SDS at 37 °C. Jurkat T cells were transfected with 10 μg of both FV and FRB fusion constructs plus 3 μg of a GFP plasmid by electroporation (Bio-Rad). After an 8-h culture, live cells were purified by Ficoll gradient separation, and 200 μl cells (2.5 × 105/ml) were added to 96-well flat-bottom tissue culture plates. The cells were incubated with CID at the indicated concentrations. 24 h later the cells were harvested and stained with phosphatidylethanolamine-conjugated annexin V propidium iodide (Sigma). Propidium iodide−, annexin V−, and GFP+ cells were quantitated by flow cytometry. Percentage of cell loss (% apoptosis) was calculated as described previously (33Wang J. Lobito A.A. Shen F. Hornung F. Winoto A. Lenardo M.J. Eur. J. Immunol. 2000; 30: 155-163Crossref PubMed Scopus (123) Google Scholar). To detect caspase processing, 293T cells were transfected with 1 μg of different fusion constructs by the FuGENE 6 method (Roche). The cells were cultured at 37 °C for 24 h and treated with 100 nm CID for different periods. The cells were then lysed in lysis buffer (50 mmHEPES, pH 7.0, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1:1 protease inhibitor mixture from Roche, and 20 μm benzyloxycarbonyl-Val-Ala-Asp) and used for Western blot analysis using anti-HA1.1 (Convance) or anti-FLAG antibody and developed by the chemiluminescent method (Pierce). Different constructs were transfected into 293T cells by the FuGENE 6 method (Roche). 18 h later the cells were incubated with or without 100 nm CID at 37 °C for 1 h. The cells were then lysed in lysis buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 1 mmEDTA, 10% glycerol, 10 μm benzyloxycarbonyl-Val-Ala-Asp, and 1:1 protease inhibitor mixture from Roche). The cell lysates were used for Western blot analysis with anti-HA antibodies or for immunoprecipitation with anti-FLAG. Protein G beads (AmershamBiosciences) coated with anti-FLAG M2 (Sigma) were incubated with cell lysates at 4 °C for 2 h. The beads were then washed three times with lysis buffer followed by SDS-PAGE and Western blot analysis with rabbit anti-HA1.1 (Convance). The blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (AmershamBiosciences) and developed by the chemiluminescent method (Pierce). Crystal structural and biochemical studies suggest that procaspases dimerize prior to proteolytic activation (18Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1465) Google Scholar, 28Chai J., Wu, Q. Shiozaki E. Srinivasula S.M. Alnemri E.S. Shi Y. Cell. 2001; 107: 399-407Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 29Riedl S.J. Fuentes-Prior P. Renatus M. Kairies N. Krapp S. Huber R. Salvesen G.S. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14790-14795Crossref PubMed Scopus (196) Google Scholar, 30Acehan D. Jiang X. Morgan D.G. Heuser J.E. Wang X. Akey C.W. Mol. Cell. 2002; 9: 423-432Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 31Renatus M. Stennicke H.R. Scott F.L. Liddington R.C. Salvesen G.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14250-14255Crossref PubMed Scopus (372) Google Scholar). A protease-deficient caspase could potentially form a dimer with a wild type caspase and thereby dominantly inhibit the activation of the wild type caspase. To examine the potential dominant negative effects of caspase mutants that contain a mutated protease active site, we examined their interactions with wild type caspases in HeLa cells. As expected, expression of wild type caspase-8, caspase-9, or caspase-10 induced significant apoptosis in HeLa cells (Fig. 1). Protease-deficient mutants with a C/S in the protease active site caused no detectable apoptosis (Fig. 1). We then co-transfected the wild type and C/S mutants of these caspases. However, the caspase-C/S mutants displayed no inhibitory effects on wild type caspases in inducing apoptosis (Fig. 1). We also observed no dominant inhibitory effects when 3-fold more mutant caspases than wild type caspases were used in the experiments (data not shown). By contrast, baculovirus p35 significantly inhibited apoptosis induction by these caspases (Fig. 1). Similar data were observed in MCF-7 cells (data not shown). Therefore, caspases containing mutated active sites are inefficient inhibitors of their wild type counterparts. The puzzling observations that C/S mutants are incapable of inhibiting the functions of wild type caspases prompted us to study the molecular mechanism of caspase activation. Because caspases can undergo dimer-dependent activation, we set out to examine how enzymatically inactive C/S caspases interact with wild type caspases after their dimerization. Chemically induced homodimerization has been successfully used to study caspase activation (23Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar, 24Yang X. Chang H.Y. Baltimore D. Mol. Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 25MacCorkle R.A. Freeman K.W. Spencer D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3655-3660Crossref PubMed Scopus (175) Google Scholar). In the present study, we adapted a system for chemically induced heterodimerization. Because rapamycin is a bivalent agent that binds to both FK506-binding protein 12 (FKBP12) and the FRB domain of FKBP rapamycin-associated protein simultaneously (34Choi J. Chen J. Schreiber S.L. Clardy J. Science. 1996; 273: 239-242Crossref PubMed Scopus (728) Google Scholar), FKBP12 and FRB domains can be fused to distinct proteins for rapamycin-dependent heterodimer formation. For improved specificity, we used a FKBP12 variant, FV (32Clackson T. Yang W. Rozamus L.W. Hatada M. Amara J.F. Rollins C.T. Stevenson L.F. Magari S.R. Wood S.A. Courage N.L., Lu, X. Cerasoli F., Jr. Gilman M. Holt D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10437-10442Crossref PubMed Scopus (420) Google Scholar), containing a phenylalanine to valine mutation at amino acid residue 36 and the FRB domain of FKBP rapamycin-associated protein (amino acid residues 2025–2114) containing a threonine to leucine mutation of amino acid 2098. A synthetic rapamycin analog, AP20840, was used throughout this study as a CID. Only one FV or one FRB domain was used in each fusion protein so that each fusion protein has only one CID interacting domain. We replaced the prodomain of caspase-8 with either FV or FRB. To test whether the system is suitable for studying caspase activation, we measured apoptosis induction after dimer formation between FV-caspase-8 and FRB-caspase-8 in Jurkat T cells. CID did not induce apoptosis in cells expressing FV or FRB only (Fig. 2 A). CID also failed to cause apoptosis in cells expressing FV and FRB-caspase-8 or FV-caspase-8 and FRB (Fig. 2 A). In contrast, significant apoptosis was induced by dimer formation between FV-caspase-8 and FRB-caspase-8. Therefore, two caspase-8 molecules are required for caspase-8 activation and apoptosis induction. We also observed similar data with caspase-9 (Fig. 2 B) and caspase-10 (Fig. 2 C). We also tested caspase-8 containing a cysteine to serine mutation in the conserved proteolytic active site (caspase-8C/S) that abolishes the protease activity. As expected, CID-induced dimer formation between FV-caspase-8C/S and FRB-caspase-8C/S caused no apoptosis (Fig. 2 A). Likewise, homodimerization of caspase-9C/S or caspase-10C/S also induced no apoptosis (Fig. 2,B and C). To investigate why protease-deficient caspases are poor inhibitors of wild type caspases, we measured apoptosis induction by dimerization between wild type caspase-8 and caspase-8C/S. Surprisingly, CID-induced dimer formation between FV-caspase-8 and FRB-caspase-8C/S caused significant levels of apoptosis (Fig. 2 A). Dimerization between FV-caspase-8C/S and FRB-caspase-8 also induced significant apoptosis (Fig. 2 A). However, the levels of apoptosis induced by one wild type and one C/S mutant of caspase-8 were slightly lower than the levels induced by two wild type caspases-8. We also observed a similar phenomenon between wild type and C/S mutant of caspase-9 (Fig. 2 B) or caspase-10 (Fig. 2 C). Apoptosis induced by dimerization between a wild type caspase and a C/S mutant may explain the difficulties in using protease-deficient caspases as dominant negative molecules to inhibit wild type caspases (Fig. 1). Although dimerization is sufficient to activate caspases, the exact model of caspase activation has not been resolved. It is still unclear whether each caspase in the dimer cleaves itself by intramolecular processing or cleaves the other caspase by intermolecular processing. If intermolecular processing were responsible for caspase activation, dimerization between wild type caspase-8 and caspase-8C/S would lead to processing of the mutant by wild type caspase-8 (Fig. 3 A). On the other hand the intramolecular processing model would predict that dimerization between wild type caspase-8 and caspase-8C/S results in autoprocessing of wild type caspase-8 but not caspase-8C/S (Fig. 3 A). To distinguish between these models, we examined caspase processing after dimerization between caspase-8 and its C/S mutant. As expected, dimerization between FV-caspase-8 and FRB-caspase-8 resulted in the processing of both caspase-8 molecules (Fig. 3 B, panels a and e). Also, CID-induced dimer formation of FV with FRB-caspase-8 or FV-caspase-8 with FRB caused no caspase processing (data not shown). Therefore, two caspase-8 molecules are required for caspase activation to occur. Interestingly, FV-caspase-8 was processed after dimerization with FRB-caspase-8C/S (Fig 3 B, panel b). Therefore, caspase-8C/S contributes to the activation of wild type caspase-8 in a dimer. Similarly, FRB-caspase-8 was processed after dimerization with FV-caspase-8C/S (Fig. 3 B, panel g). Because caspase-8C/S does not have a functional protease active site, it cannot cleave itself or the other caspase in the dimer (Fig. 3 B, panels d and h). Therefore, it is likely that the wild type caspase-8 had cleaved itself by intramolecular processing (Fig. 3 A). Because caspase-8C/S has no functional proteolytic site, its homodimerization caused no caspase processing (Fig. 3 B,panels d and h). However, caspase-8C/S was cleaved following dimer formation with enzymatically active caspase-8 (Fig. 3 B, panels c and f). Wild type caspase-8 is likely to be responsible for the processing of caspase-8C/S. Wild type caspase-8 may cleave caspase-8C/S before its intramolecular processing. Alternatively, wild type caspase-8 may undergo intramolecular processing first before acquiring the ability to process other caspases in trans. To distinguish between these two possibilities, we mutated the two aspartate residues that are required for processing between the large (p20) and the small (p10) subunits of the caspase-8 protease domain. Unlike wild type caspase-8, this "frozen" aspartate to alanine (DD/AA) cleavage mutant failed to undergo intramolecular processing between p20 and p10 after dimerization with caspase-8C/S (Fig. 4,panel f versus panel h). We then tested whether the DD/AA mutant retains the ability to confer intermolecular processing after dimerization with another caspase-8. Caspase-8DD/AA can support the processing of a wild type caspase-8 after its dimerization (Fig. 4, panel c). However, caspase-8DD/AA failed to induce the processing of caspase-8C/S after its dimerization (Fig. 4, panel d). Therefore, the caspase-8DD/AA that lacks the potential for intramolecular processing cannot cleave caspase-8C/S following dimerization. This suggests that intramolecular processing is the first step in the cleavage of initiator caspase after dimer formation. In the absence of intramolecular processing, intrans processing does not take place efficiently. Caspase-8 and caspase-10 can both be recruited into the DISC (15Wang J. Chun H.J. Wong W. Spencer D.M. Lenardo M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13884-13888Crossref PubMed Scopus (312) Google Scholar, 16Sprick M.R. Rieser E. Stahl H. Grosse-Wilde A. Weigand M.A. Walczak H. EMBO J. 2002; 21: 4520-4530Crossref PubMed Scopus (297) Google Scholar, 35Kischkel F.C. Lawrence D.A. Tinel A. LeBlanc H. Virmani A. Schow P. Gazdar A. Blenis J. Arnott D. Ashkenazi A. J. Biol. Chem. 2001; 276: 46639-46646Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar), raising the possibility that these two caspases could activate each other in the DISC. However, we found that heterodimerization between caspase-8 and caspase-10 did not induce apoptosis (Fig. 5 A). We also failed to observe caspase processing between caspase-8 and caspase-10 after dimerization (Fig. 5 B, lane 6). This suggests that caspase-8 and caspase-10 induce apoptosis independently, although they may come into close proximity in the DISC and may activate similar downstream pathways. We next tested whether apoptosis and caspase processing occur after dimerization between caspase-8 and caspase-9 or with effector caspases, including caspases-3, -6, and -7 (Fig. 5 B). Interestingly, we detected no caspase activation or apoptosis in each case when two different caspases were dimerized (Fig. 5, A andB, lanes 3–12). Therefore, dimer-mediated caspase activation specifically takes place between the same procaspases. One possible explanation for the stringent requirement for homotypic processing is that two different caspases may not be able to achieve a stable dimeric structure for caspase activation. Therefore, we performed co-immunoprecipitation analysis of the caspase dimers. We immunoprecipitated FRB-caspase-8 and analyzed the associated FV-caspase-8 by Western blot. As expected, FV-caspase-8 was co-precipitated with FRB-caspase-8 (Fig. 6 A, upper panel,lane 2). Moreover, the majority of the associated FV-caspase-8 in the immunoprecipitate was processed (Fig. 6 A, upper panel, lane 2). The association between FV-caspase-8 and FRB-caspase-8 is most likely through caspase-8 protease domains because FV and FRB cannot stably associate in the immunoprecipitate (data not shown). In contrast, we observed no stable dimer formation between FRB-caspase-8 and FV-caspase-9, FV-caspase-10, or FV-caspase-3 (Fig. 6 A, upper panel, lanes 4, 6, and 8). Thus, caspase-8 cannot form stable dimers with the protease domains of other caspases. This may explain the failure of these different caspases to activate each other. We also tested the interactions of caspase-9 with other caspases by co-immunoprecipitation. Because dimerization between two wild type caspases-9 leads to rapid cleavage and disappearance of caspase-9 (data not shown), we used the C/S mutant of caspase-9 in this immunoprecipitation study. We found that FRB-caspase-9C/S can co-precipitate FV-caspase-9C/S but not FV-
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