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• W2064950130 abstract "Activation of caspase-12 from procaspase-12 is specifically induced by insult to the endoplasmic reticulum (ER) (Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98–103), yet the functional consequences of caspase-12 activation have been unclear. We have shown that recombinant caspase-12 specifically cleaves and activates procaspase-9 in cytosolic extracts. The activated caspase-9 catalyzes cleavage of procaspase-3, which is inhibitable by a caspase-9-specific inhibitor. Although cytochrome creleased from mitochondria has been believed to be required for caspase-9 activation during apoptosis (Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405–413, Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997)Cell 91, 479–489), caspase-9 as well as caspase-12 and -3 are activated in cytochrome c-free cytosols in murine myoblast cells under ER stress. These results suggest that caspase-12 can activate caspase-9 without involvement of cytochrome c. To examine the role of caspase-12 in the activation of downstream caspases, we used a caspase-12-binding protein, which we identified in a yeast two-hybrid screen, for regulation of caspase-12 activation. The binding protein protects procaspase-12 from processing in vitro. Stable expression of the binding protein renders procaspase-12 insensitive to ER stress, thereby suppressing apoptosis and the activation of caspase-9 and -3. These data suggest that procaspase-9 is a substrate of caspase-12 and that ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochromec-independent manner. Activation of caspase-12 from procaspase-12 is specifically induced by insult to the endoplasmic reticulum (ER) (Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98–103), yet the functional consequences of caspase-12 activation have been unclear. We have shown that recombinant caspase-12 specifically cleaves and activates procaspase-9 in cytosolic extracts. The activated caspase-9 catalyzes cleavage of procaspase-3, which is inhibitable by a caspase-9-specific inhibitor. Although cytochrome creleased from mitochondria has been believed to be required for caspase-9 activation during apoptosis (Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405–413, Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997)Cell 91, 479–489), caspase-9 as well as caspase-12 and -3 are activated in cytochrome c-free cytosols in murine myoblast cells under ER stress. These results suggest that caspase-12 can activate caspase-9 without involvement of cytochrome c. To examine the role of caspase-12 in the activation of downstream caspases, we used a caspase-12-binding protein, which we identified in a yeast two-hybrid screen, for regulation of caspase-12 activation. The binding protein protects procaspase-12 from processing in vitro. Stable expression of the binding protein renders procaspase-12 insensitive to ER stress, thereby suppressing apoptosis and the activation of caspase-9 and -3. These data suggest that procaspase-9 is a substrate of caspase-12 and that ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochromec-independent manner. endoplasmic reticulum glutathione S-transferase The caspase protease family plays a central role in the implementation of apoptosis in vertebrates (4Thornberry N.A. Lazebnik Y. Science (Wash. D. C.). 1998; 281: 1312-1316Crossref PubMed Scopus (6112) Google Scholar, 5Cryns V.L. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1157) Google Scholar). Caspases are constitutively expressed in healthy cells, where they are synthesized as precursor proteins (procaspases). Caspases are activated upon processing of procaspases into ∼20-kDa (p20) and 10-kDa (p10) mature fragments, in addition to the N-terminal prodomain. The caspase family is broadly divided into two groups: initiator caspases (caspase-8, -9, and -12) and effector caspases (caspase-3, -6, and -7). Initiator caspases undergo autoprocessing for activation in response to apoptotic stimuli. Active initiator caspases in turn process precursors of the effector caspases responsible for dismantling cellular structures. Recent studies have suggested the existence of a novel apoptotic pathway in which caspase-12 functions as the initiator caspase in response to a toxic insult to the ER,1 such as by treatment with tunicamycin (an inhibitor of glycosylation), thapsigargin (an inhibitor of the ER-specific calcium ATPase), or calcium ionophores (1Nakagawa T. Zhu H. Morishima N., Li, E., Xu, J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar). Caspase-12 is specifically activated in cells subjected to ER stress. Furthermore, caspase-12-deficient cells are resistant to inducers of ER stress, suggesting that caspase-12 is significant in ER stress-induced apoptosis (1Nakagawa T. Zhu H. Morishima N., Li, E., Xu, J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar). ER stress has received growing attention because it is considered a cause of pathologically relevant apoptosis, and it is particularly implicated in neurodegenerative disorders (6Aridor M. Balch W.E. Nat. Med. 1999; 5: 745-751Crossref PubMed Scopus (254) Google Scholar). However, the mechanism of caspase-12-mediated apoptosis has been unknown, mainly due to the lack of identification of caspase-12 substrates. In this study, we have examined the susceptibility of procaspases to active caspase-12 and have shown that procaspase-9 can specifically be cleaved by caspase-12 in vitro. Recent studies show that multiple death signals converge on the mitochondrion (7Wei M.C. Zong W.-X. Cheng E.H.-Y. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science (Wash. D. C.). 2001; 292: 727-730Crossref PubMed Scopus (3299) Google Scholar). Damaged mitochondria release cytochromec, which facilitates conformational changes in Apaf-1, the specific activator of procaspase-9 (2Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2716) Google Scholar, 3Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6157) Google Scholar). The cytochromec·Apaf-1 complex called an apoptosome (8Zhou Q., Li, Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1771) Google Scholar, 9Saleh A. Srinivasula S.M. Acharya S. Fishel R. Alnemri E.S. J. Biol. Chem. 1999; 274: 17941-17945Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar) is thought to recruit procaspase-9 through interaction between Apaf-1 and procaspase-9 and facilitate autoactivation of caspase-9. Active caspase-9 then activates caspase-3, the major effector caspase that is responsible for destruction of various substrates (4Thornberry N.A. Lazebnik Y. Science (Wash. D. C.). 1998; 281: 1312-1316Crossref PubMed Scopus (6112) Google Scholar, 5Cryns V.L. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1157) Google Scholar). Cytochromec release from mitochondria has also been observed in ER stress-induced apoptosis of several cell lines, including mouse embryonic fibroblast cells (10Ito Y. Mishra N. Kumar S. Narula N. Kharbanda S. Saxena S. Kufe D. Mol. Cell. Biol. 2001; 21: 6233-6242Crossref PubMed Scopus (116) Google Scholar, 11Ha¨cki J. Egger L. Monney L. Conus S., T., R. Fellay I. Borner C. Oncogene. 2000; 19: 2286-2295Crossref PubMed Scopus (279) Google Scholar). The in vitro cleavage of procaspase-9 by caspase-12 described above can be achieved in the absence of cytochrome c, suggesting the presence of the ER stress-specific caspase cascade, which comprises caspase-12, -9, and -3 in this order. For examination of the role of caspase-12 in activation of the caspase cascade in vivo, however, it would be desirable to use conditions in which cytochrome c is not released from mitochondria; otherwise, caspase-9 could be activated by the cytochrome c·Apaf-1 mechanism, independent of caspase-12. We thus used a murine myoblast cell line, C2C12, to study caspase-12, because our preliminary data showed that ER stress induces the activation of caspase-12 and apoptosis in the cell line without the release of cytochrome c from mitochondria. This result suggests that cytochrome c release is not essential for ER stress-induced apoptosis. We took advantage of the fact that cytochromec is not released to examine the mechanism of caspase cascade activation in the absence of mitochondrial damage, focusing on events that occur downstream of caspase-12 activation. C2C12 cells (RIKEN Cell Bank, Tsukuba, Japan) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Invitrogen), 50 units/ml penicillin, and 50 μg/ml streptomycin (Invitrogen) at 37 °C with 5% CO2. Apoptosis was induced in cultured cells by adding the following reagents in culture medium unless otherwise stated: 2 μg/ml tunicamycin, 1 μm thapsigargin, and 10 μg/ml etoposide. Apoptosis was induced in C2C12 cells, and then the cells were stained with the MitoSensor reagent (CLONTECH) according to the manufacturer's protocol. The C2C12 cell 100,000 × g supernatant was prepared according to the method described in Liu and Wang (12Liu X. Wang X. Methods Enzymol. 2000; 322: 177-182Crossref PubMed Google Scholar). Briefly, cells were disrupted in buffer containing 250 mm sucrose by a Dounce homogenizer. The supernatant was centrifuged in a microcentrifuge for 10 min, and subsequently at 100,000 × g for 30 min in a tabletop ultracentrifuge (Beckman Coulter, Inc.). Anti-MAGE-3 (melanoma-associated antigen-3) polyclonal antibody was generated by immunization of rabbit with a synthetic peptide (CHISYPPLHEWVLREGEE) as described previously (13Nakanishi K. Maruyama M. Shibata T. Morishima N. J. Biol. Chem. 2001; 276: 41237-41244Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Primary antibodies for Western blot analysis were used at the following dilutions: anti-MAGE-3 polyclonal antibody, 1:400; anti-FLAG monoclonal antibody (Sigma-Aldrich), 1:1,000; anti-hexahistidine tag monoclonal antibody (CLONTECH), 1:5,000; anti-caspase-12 rat monoclonal antibody (1Nakagawa T. Zhu H. Morishima N., Li, E., Xu, J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar), 1:100; anti-MAGE-3 monoclonal antibody (14Kocher T. Schultz-Thater E. Gudat F. Schaefer C. Casorati G. Juretic A. Willimann T. Harder F. Heberer M. Spagnoli G.C. Cancer Res. 1995; 55: 2236-2239PubMed Google Scholar), 1:2; anti-caspase-9 monoclonal antibody (Medical and Biological Laboratories (MBL), Nagoya, Japan), 1:1,000; anti-caspase-3 (cleaved form) antibody (Cell Signaling TechnologyInc.), 1:1,000; anti-caspase-7 monoclonal antibody (BD Biosciences), 1:250; anti-cytochrome c monoclonal antibody (BD Biosciences), 1:500; anti-α-tubulin monoclonal antibody (Oncogene Science), 1:1,000; anti-BiP monoclonal antibody (BD Biosciences), 1:500. Primary antibodies on Western blots were detected as described previously (15Morishima N. Genes Cells. 1999; 4: 401-414Crossref PubMed Scopus (98) Google Scholar). The split LexA protein system was used for two-hybrid screening according to the method of Brent as described by Gyuris et al. (16Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1316) Google Scholar). The caspase-12 p10 fragment (Thr319-Asn419) was used as the bait for the screening of a HeLa cell cDNA library. From ∼2 × 107 transformants, we obtained 15 positive clones, all of which contained sequences derived from the MAGE-3 mRNA. The 5′ ends of the cDNAs were located between codons 81 and 94. We cloned the full-length coding region of MAGE-3 (314 amino acids) for further analysis by polymerase chain reaction amplification of a human testis cDNA library (CLONTECH). Immunoprecipitation was performed after COS-1 cells (1.8 × 105 seed cells) were transfected with 5 μg of DNA. At 2 days post-transfection, cells were lysed at 0 °C in phosphate-buffered saline containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium lauryl sulfate, and a COMPLETE protease inhibitor mixture (Roche Molecular Biochemicals). Cell lysates were incubated with 30 μl of anti-FLAG (M2) affinity resin (Sigma-Aldrich) for 4–5 h at 0 °C. Proteins that bound the affinity resin were analyzed by Western blotting. Caspase-12- or MAGE-3 cDNA was cloned into the vector pRSET (Invitrogen) and used for transformation of BL21(DE3) pLysS cells. The histidine-tagged proteins synthesized inEscherichia coli were purified on a Probond Ni-column resin (Invitrogen). The glutathione S-transferase (GST)-MAGE-3 fusion was generated by inserting MAGE-3 cDNA into a pGEX-4T-3 vector (Amersham Biosciences). GST-MAGE-3 was purified from E. coli lysates by batch-chromatography with glutathione Sepharose-4B beads (Amersham Biosciences). GST-MAGE-3 protein (1 μg) and histidine-tagged caspase-12 (0.1 μg) were incubated with 10 μl of glutathione Sepharose-4B beads (Amersham Biosciences) for 1 h at room temperature in 150 μl of 20 mm phosphate buffer, pH 7.0, containing 200 mm NaCl and 0.02% Triton X-100. Anti-hexahistidine monoclonal antibody (CLONTECH) was used for the detection of caspase-12 p20. Proteins that bound glutathione resin were analyzed by Western blotting. In vitro synthesis of 35S-labeled proteins and their detection by autoradiography were achieved as described previously (15Morishima N. Genes Cells. 1999; 4: 401-414Crossref PubMed Scopus (98) Google Scholar). For mutant analysis, mutations at specific aspartic acid residues in procaspases were introduced by the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Introduction of mutation was confirmed by DNA sequencing. 35S-labeled procaspase (0.2 μl of the labeled protein solution) was incubated for a cleavage assay at 37 °C with caspase-12 (0.28 μg) for 4 h. Resistance of procaspase-12 to cleavage by active caspase-12 was examined by addition of recombinant MAGE-3 to the procaspase-12 cleavage assay solution. Procaspase-12 whose active site Cys residue had been replaced with Ser was synthesized in vitro in the presence of [35S]methionine. 35S-Labeled mutant procaspase-12 (0.2 μl of the labeled protein solution) was incubated with active caspase-12 (0.28 μg) for 45 min in the presence or absence of MAGE-3. Cytochrome c-free cytosol from C2C12 cells (10 μg of proteins) was treated with recombinant caspase-12 p30 (0.8 μg) at 37 °C for 4 h. Activation of caspase-9 and -3 was examined by Western blot analysis. Five micrograms of proteins were loaded on each lane. As a positive control for caspase-9 activation, the cytochrome c-free cytosol was incubated with 10 μm bovine cytochrome c (Sigma-Aldrich) and 1 mm dATP for 60 min at 37 °C. For inhibition of caspase-9 activity, LEHD-fluoromethylketone (BioVision, Palo Alto, CA) was added to the cytosol before the addition of caspase-12. MAGE-3 stable cell lines of C2C12 were generated as follows. MAGE-3 cDNA was cloned into pcDNA3.1(−) vector (Invitrogen). The plasmid DNA was linearized by ScaI digestion before transfection. Transfection was performed with a Superfect transfection reagent (QIAGEN) according to the manufacturer’s protocol. MAGE-3 cDNA cloned into the pcDNA3.1(−) vector (Invitrogen) was used for stable transfection. Stable transfectants were grown in medium containing 600 μg/ml G418 (Invitrogen) for 2 weeks before cloning. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were incubated with either anti-caspase-12 monoclonal (1Nakagawa T. Zhu H. Morishima N., Li, E., Xu, J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar) or anti-MAGE-3 polyclonal antibody (this study). Primary antibodies were detected with either Alexa594-coujugated anti-rabbit IgG antibody (1:500) or a combination of biotin-labeled anti-rat IgG antibodies (1:500) and Alexa488-coujugated avidin (1:1,000). These reagents were obtained from Molecular Probes. Images were captured with a cooled charge-coupled device camera mounted on an Olympus IX70 microscope. We examined how caspase-12 processing is linked to the activation of other caspases. For an in vitro cleavage assay, we produced recombinant caspase-12 (p30) whose N-terminal prodomain had been removed and replaced with a hexahistidine tag. The p30 protein undergoes efficient autoprocessing into p20 and p10 peptides when overexpressed in E. coli (p30* in Fig. 1A). A mutant p30 (p30C/S), whose active site Cys is substituted with Ser, is not processed in E. coli (Fig. 1A). The mature caspase-12 (p30*) exhibits proteolytic activity and cleaves procaspase-12 into 35- and 12-kDa fragments (Fig. 1B,lane 16). The cleavage site was located at Asp318, because a procaspase-12 mutant in which Asp318 was replaced with Ser was resistant to caspase-12 digestion (data not shown). Asp318 is also the cleavage site for autoprocessing in E. coli (Fig.1A), as revealed by amino acid sequencing of p10 by the Edman degradation method (data not shown). p30* cleaves caspase-9 (Fig.1, B and C) but not other caspase precursors (murine caspase-1 and -2, and human caspase-3, -6, and -8) under the experimental conditions. Note that processing site sequences between p20 and p10 are highly conserved between murine and human caspase-3, -6, -7, and -8. Mutation analysis of caspase-9 (Fig. 1C) indicates that caspase-12 cleaves at specific Asp residues in the linker region between p20 and p10 in the procaspase-9 polypeptide (LDSD349 and SEPD353 in the murine caspase-9, PEPD315 in the human caspase-9). Asp353 of the murine caspase-9 and Asp315 of human caspase-9 have been reported to be the cleavage sites for the activation of procaspase-9 (17Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (958) Google Scholar, 18Fujita E. Egashira J. Urase K. Kuida K. Momoi T. Cell Death Differ. 2001; 8: 335-344Crossref PubMed Scopus (94) Google Scholar). The caspase-9 cleavage observed in vitro thus suggests the possibility that caspase-9 can be activated by caspase-12 during ER stress-induced apoptosis. Under the experimental conditions used, murine caspase-9 contains another cleavage site(s) for caspase-12in vitro, cleavage at which generates 27- and 20-kDa fragments. We did not further analyze these additional cleavage site(s) because we could detect neither 27- nor 20-kDa caspase-9 fragments in apoptotic cells (described below; data not shown). Procaspase-7 seems to be only slightly processed by active caspase-12 (Fig. 1B,lane 10). The in vitro cleavage of procaspase-7 was not studied further because processing of procaspase-7 is undetectable in C2C12 cells subjected to ER stress (Fig.1D). Several reports have demonstrated that ER stress causes mitochondrial damage, which results in cytochrome crelease from mitochondria (e.g., Refs. 10Ito Y. Mishra N. Kumar S. Narula N. Kharbanda S. Saxena S. Kufe D. Mol. Cell. Biol. 2001; 21: 6233-6242Crossref PubMed Scopus (116) Google Scholar, 11Ha¨cki J. Egger L. Monney L. Conus S., T., R. Fellay I. Borner C. Oncogene. 2000; 19: 2286-2295Crossref PubMed Scopus (279) Google Scholar). Cytochromec in cytosol and Apaf-1 can induce activation of caspase-9 (2Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2716) Google Scholar, 3Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6157) Google Scholar). To examine whether there is an ER stress-specific caspase cascade that is initiated by caspase-12, we used a murine myoblast cell line, C2C12, because this cell line undergoes ER stress-induced apoptosis without cytochrome c release from mitochondria. Cytosolic extracts (S-100) of tunicamycin- or thapsigargin-treated C2C12 cells contain cytochrome c at the same level as that detected in S-100 fractions prepared from untreated cells (Fig.2A). Nevertheless, more than 50% of the cells undergo apoptosis (see below). Cytochromec release per se, however, is functional in C2C12 cells, because treatment of C2C12 cells with etoposide or serum deprivation induces apoptosis at a similar level of lethality and with a significant release of cytochrome c. After apoptosis was induced by ER stress inducers, the mitochondrial transmembrane potential was maintained in apoptotic cells (small cells with condensed nuclei), as in the case of untreated cells, which was exhibited by mitochondrial accumulation of fluorochromes and their conversion to emit the orange color (Fig. 2B). Etoposide-treatment of C2C12 cells resulted in decrease in mitochondrial transmembrane potential, which was monitored by the green color of the fluorochromes in the cytosol (Fig. 2B). These results suggest that mitochondria in C2C12 cells do not suffer severe damages from ER stress, thus releasing little cytochrome c into cytosol. Treatment of C2C12 cells with ER stress inducers, either tunicamycin or thapsigargin, results in the processing of procaspase-12 (48 kDa, Fig.2C) and apoptosis. A 35-kDa fragment was detected by antibodies specific to the p20 region (1Nakagawa T. Zhu H. Morishima N., Li, E., Xu, J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar). Caspase-9 and caspase-3 are also activated in C2C12 cells treated with ER stress inducers (Fig.2C). The activation of caspase-3, one of the most downstream caspases, suggests that the ER stress-specific caspase cascade comprises caspase-12, -9, and -3. It has been suggested that calpain is involved in activation of caspases in cultured glial cells after deprivation of oxygen and glucose (19Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1029) Google Scholar). In the apoptotic C2C12 cells, however, cleavage of a calpain substrate, Bcl-XL, was not detected (Fig. 2C), suggesting that caspase activation in C2C12 cells treated with ER stress inducers is independent of calpain. We then examined whether caspase-9 activation occurs by the cleavage of procaspase-9 by active caspase-12 without the release of cytochrome c in cell extracts. Incubation of the S-100 fraction of untreated C2C12 cells with active caspase-12 results in a pattern of cleavage of procaspase-9 that is similar to that observed in S-100 of apoptotic C2C12 cells, the cleavage products being a doublet of 35-kDa fragments (Fig. 3A, lanes 2and 4). A control experiment showed that addition of cytochrome c and dATP to S-100 of untreated cells also caused processing of procaspase-9 into 35-kDa fragments that appeared as a doublet on the blot (Fig. 3B, lane 3). The lower band was less intense than the upper band in the case of caspase-12-induced processing (Fig. 3B, lane 2) and the apoptotic S-100 fractions (lane 4). The ratio of these 35-kDa fragments was different from that observed in the cytochrome c-treated S-100 (Fig. 3B, lane 3). It remains to be revealed whether the difference in the ratio of these fragments reflects a difference in mechanism of processing. The cleavage of procaspase-9 is not suppressed in the presence of a caspase-9-specific inhibitor LEHD-fluoromethylketone (20Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1834) Google Scholar), indicating that procaspase-9 is cleaved by active caspase-12, independent of the inherent autoprocessing activity of procaspase-9 (Fig. 3B, upper panel, lane 4). We then examined caspase-9 activation through detection of specific cleavage at Asp175 within the procaspase-3 polypeptide, the downstream target of caspase-9 (3Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6157) Google Scholar). Incubation of the S-100 fraction with caspase-12 causes processing of caspase-3 at Asp175 of ITED175 (Fig.3B, lower panel, lane 3), suggesting that the activated caspase-9 in the S-100 fraction cleaved procaspase-3. The specific cleavage of procaspase-3, but not procaspase-9, can be inhibited by LEHD-fluoromethylketone, (Fig.3B, lane 4). This result indicates that cleavage of procaspase-3 is dependent on caspase-9, as already observed for apoptosis induced by various stimuli other than ER stress (3Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6157) Google Scholar,21Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO (Eur. Mol. Biol. Organ.) J. 1998; 17: 37-49Crossref PubMed Scopus (1104) Google Scholar, 22Kuida K. Haydar T.F. Kuan C.Y., Gu, Y. Taya C. Karasuyama H., Su, M.S. Rakic P. Flavell R.A. Cell. 1998; 94: 325-337Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar, 23Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. Elia A. de la Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe S.W. Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1150) Google Scholar). We have recently isolated by yeast two-hybrid screening from a HeLa cell cDNA library a human cancer antigen, MAGE-3, as a protein that specifically binds the caspase-12 p10 fragment (see “Experimental Procedures”). Because MAGE-3 can suppress the activity of procaspase-12, as described below, we used the protein to examine the significance of caspase-12 activation in ER stress-induced apoptosis in C2C12 cells. MAGE-3 is a member of theMAGE gene family and is expressed in various types of tumor but not in normal tissues except for the testis (24Van Pel A. van der Bruggen P. Coulie P.G. Brichard V.G. Lethe B. van den Eynde B. Uyttenhove C. Renauld J.C. Boon T. Immunol. Rev. 1995; 145: 229-250Crossref PubMed Scopus (217) Google Scholar). Although the specific interaction between caspase-12 and MAGE-3 is intriguing, it remains unclear whether MAGE-3 plays any role in caspase regulation in human cells (see “Discussion”). MAGE-3 does not bind to other caspases, such as caspase-9 (of either murine or human origin), as tested by the two-hybrid assay (results of murine caspase-1, -9, and -11 and human caspase-3, -6, and -7 are shown in Fig.4A). The MAGE-3 protein can also bind both the caspase-12 p10 fragment and procaspase-12 in mammalian cells. When MAGE-3 is expressed in COS-1 cells by transient transfection it can be co-precipitated with FLAG-tagged p10 (Fig. 4B, lane 3) or FLAG-tagged procaspase-12 (lane 7) using an anti-FLAG antibody. MAGE-3 was not co-precipitated with FLAG-tagged p10 fragments of murine caspase-2 and human caspase-8, whose binding ability could not be examined by the two-hybrid assay because of significant background activity (data not shown). Fig. 4C shows that p30C/S (unprocessed p30) co-precipitates with GST-tagged MAGE-3 (lanes 3 and 4). Under the same conditions, however, p30* (processed) is not efficiently co-precipitated by GST-MAGE-3 (Fig.4C, lanes 1and 2), suggesting that MAGE-3 does not efficiently bind the p10 fragment in active caspase-12. It is possible that the p10 fragment within mature caspase-12 is not fully accessible to MAGE-3 because of steric hindrance by the p20 portion. X-ray crystallographic analyses of caspase-1 and caspase-3 have suggested that they undergo a conformational change upon maturation (25Wilson K.P. Black J.A. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Nature (Lond.). 1994; 370: 270-275Crossref PubMed Scopus (751) Google Scholar, 26Walker N.P. Talanian R.V. Brady K.D. Dang L.C. Bump N.J. Ferenz C.R. Franklin S. Ghayur T. Hackett M.C. Hammill L.D. Cell. 1994; 78: 343-352Abstract Full Text PDF PubMed Scopus (525) Google Scholar, 27Rotonda J. Nicholson D.W. Fazil K.M. Gallant M. Gareau Y. Labelle M. Peterson E.P. Rasper D.M. Ruel R. Vaillancourt J.P. Thornberry N.A. Becker J.W. Nat. Struct. Biol. 1996; 3: 619-625Crossref PubMed Scopus (400) Google Scholar). This conformational change may occur in caspase-12 and result in the p10 fragment being less exposed for binding to MAGE-3. Consistent with the binding of MAGE-3 to unprocessed caspase-12, MAGE-3 protects procaspase-12 from cleavage by active p30* in a dose-dependent manner (Fig. 4D, lanes 2–7). Substitution of MAGE-3 with bovine serum albumin fails to inhibit cleavage (Fig. 4D, lane 9). It is less likely that MAGE-3 blocks active caspase-12 by acting as a competitive inhibitor. In Fig. 4D, lane 7, small amounts of the 35- and 12-kDa fragments can be detected, indicating the presence of caspase-12 activity. Under such conditions, excessive levels of active caspase-12 are expected to be protected from inhibition by MAGE-3. However, an enhancement of cleavage was not detected in the presence of 4-fold higher levels of caspase-12 (lane 8). In contrast, when twice as much substrate is added to the reaction mixture in the presence of MAGE-3, both p35 and p12 cleavage products are produced at the same levels as in the absence of MAGE-3 (Fig.4E, lane 2). It is more likely that MAGE-3 protects procaspase-12 from processing by specifically binding the p10 portion of the precursor. This result is consistent with our observation that the affinity of MAGE-3 for p30C/S is muc" @default.
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