- Email: [email protected]
Pyrin N-Terminal Homology Domain- and Caspase Recruitment Domain-Dependent Oligomerization of ASC
Biochemical and Biophysical Research Communications 280, 652– 655 (2001) doi:10.1006/bbrc.2000.4190, available online at http://www.idealibrary.com on
Pyrin N-Terminal Homology Domain- and Caspase Recruitment Domain-Dependent Oligomerization of ASC Junya Masumoto, Shun’ichiro Taniguchi, and Junji Sagara 1 Department of Molecular Oncology and Angiology, Research Center on Aging and Adaptation, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Nagano, Japan
Received December 3, 2000
ASC was first identified as a caspase recruitment domain (CARD)-containing proapoptotic molecule that forms insoluble aggregates during apoptosis. Here, we report both the pyrin N-terminal homology domain (PYD) and CARD domains are involved in the aggregation of ASC. Preliminary experiments indicated that overexpression of ASC formed filament-like aggregates in COS-7 cells. Expression experiments using green fluorescent protein (GFP) constructs showed that not only the GFP-ASC-CARD but also the GFP-ASC-PYD formed filament-like aggregates in COS-7 cells. We confirmed these filament-like aggregates of both the ASC-PYD and the ASC-CARD due to homophilic interaction by immunoprecipitation method. We also demonstrated that the ASC-PYD associated with the ASC-CARD by heterophilic interaction. These observations suggest that the dimerization of the PYD as well as the CARD plays an important role in the oligomerization of ASC as an adaptor molecule. © 2001 Academic Press Key Words: ASC; PYD; CARD; adaptor; homophilic interaction; heterophilic interaction; aggregation; oligomerization; filament formation.
Apoptosis plays important roles in regulating the growth and development of organisms, and also mediates normal and neoplastic tissue growth by removing This work was supported by a Grant-in-aid 12670109 from the Ministry of Education, Science and Culture, Japan. Abbreviations used: ASC, apoptosis-associated speck-like protein containing a CARD; CARD, caspase recruitment domain; DD, death domain; DED, death effector domain; PYD, pyrin N-terminal homology domain; bcl, B-cell lymphoma/leukemia; RAIDD, RIP-associated ICH-1/Ced-3-homologous protein with a death domain; CRADD, caspase and RIP adaptor with death domain; PCR, polymerase chain reaction; GFP, green fluorescent protein; FADD, Fas-associated protein with death domain; DISC, death-inducing signaling complex. 1 To whom correspondence should be addressed. Fax: 81-263-372724. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
excess cells (1, 2). Apoptosis is implemented by death machinery linked to signaling pathways in which death adaptor domains act together in the effectorproximal part of apoptotic signaling components (3, 4). The caspase recruitment domain (CARD) was proposed as the third example of a heterodimerization domain involved in apoptosis signaling pathways in addition to the death domain (DD) and the death effector domain (DED) (5–7). ASC is a CARD-containing molecule composed of an N-terminal pyrin N-terminal homology domain (PYD) and a C-terminal CARD. Under physiological conditions, ASC is a cytosolic soluble protein that aggregates during apoptosis (8). While the aggregation of ASC is thought to reflect the nature of the CARD of ASC as an oligomerization domain, the function of the PYD of ASC remains unknown. In this study, we show that not only the CARD but also the PYD is involved in the aggregation of ASC. Finally, by immunoprecipitation analysis, we demonstrate the direct homophilic and heterophilic interactions of the ASC-PYD and ASC-CARD domains, suggesting that the PYD domain is a novel homophilic interaction motif. MATERIALS AND METHODS Construction of expression plasmids. The entire open reading frame of ASC was inserted into the EcoRI and SalI sites of pEGFP-C2 (Clontech) to produce pEGFP-ASC-WT. Deletion mutants of pEGFP-ASC-1-100(CARD) and pEGFP-ASC-⌬101195(PYD) were constructed by polymerase chain reaction (PCR) using the entire open reading frame of ASC-including gt11 clone (8) as a template following insertion into the EcoRI and SalI sites of pEGFP-C2. Deletion mutants of pFLAG-CMV-4-ASC-⌬1-100(CARD) and pFLAG-CMV-4-ASC-⌬101-195(PYD) were constructed as described above by insertion into the EcoRI and KpnI sites of pFLAGCMV-4 (Sigma). pcDNA3-ASC and anti-ASC monoclonal antibody were described previously (8). Schematic structures of these expression products are presented in Fig. 1. Immunofluorescence analysis. After COS-7 cells were transfected with pcDNA3-ASC, cells were fixed with 70% ethanol at ⫺20°C 30
652
Vol. 280, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Schematic representations of wild-type and deletion mutants of ASC fused to GFP or Flag. GFP-ASC-WT is a full-length ASC fused to an N-terminal GFP. GFP-ASC-⌬1-100(CARD) is the N-terminal 100-amino-acid truncated-form of ASC consisting of CARD fused to an N-terminal GFP. GFP-ASC-⌬101-195(PYD) is the C-terminal 95-amino-acid truncated-form of ASC consisting of a PYD fused to an N-terminal GFP. Flag-ASC-⌬1-100(CARD) is the N-terminal 100-amino-acid truncated-form of ASC consisting of a CARD tagged with an N-terminal Flag. Flag-ASC-⌬101-195(PYD) is the C-terminal 95-amino-acid truncated-form of ASC consisting of a PYD tagged with an N-terminal Flag.
min, air dried, and rinsed with PBS. Then, cells were incubated with anti-ASC monoclonal antibody, and following secondary FITCconjugated rabbit polyclonal antibody against mouse IgG (Dako). FITC-signals were detected by immunofluorescence microscopy (Zeiss Axioskop). Localizations of GFP-ASC, GFP-ASC-PYD and GFP-ASC-CARD in transfected COS-7 cells were also analyzed by immunofluorescence microscopy. Transfection, expression and immunoprecipitation of tagged proteins. 1 ⫻ 10 6 COS-7 cells were transfected with expression plasmids using LipofectAMINE-PLUS reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Transfected COS-7 cells were lysed and incubated in 0.8 ml of lysis buffer (0.1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl [pH 7.6], 1 mM PMSF, 10 g/ml leupeptin, 10 g/ml pepstatin A, 10 g/ml antipain, 10 g/ml aprotinin) for 5 min. The lysate was removed from the dish after any remaining cellular debris was fully dislodged from the plate surface with a rubber policeman. The lysate from one dish was incubated in a 1.5-ml tube on ice for an additional 10 min, and clarified by centrifugation at 12000g for 15 min. The supernatant was immunoprecipitated with 20 l of protein G-Sepharose 4B (Amersham Pharmacia Biotech) conjugated with anti-Flag monoclonal antibody M2 (Sigma). Immunoprecipitated proteins were subjected to 16% SDSpolyacrylamide electrophoresis and detected by Western blotting using anti-GFP monoclonal antibody (Clontech). Detailed procedures were described previously (9).
RESULTS AND DISCUSSION ASC Formed Filament-like Aggregates in COS-7 Cells We have previously shown that ASC forms insoluble speck-like aggregates during apoptosis of promyelocytic leukemia HL-60 cells (8). Closer examination revealed the speck appeared to be a filament-like aggregate with a hollow center (8). Originally, ASC was identified as a cytoskeletal and/or nuclear matrix molecule (8). ASC expressed transiently in COS-7 cells formed filament-like aggregates (Fig. 2Aa–2Ac) that are similar to CARD-dependent filaments of bcl-10, RAIDD, and the prodomain of caspase-2, or deatheffector filaments including FADD and procaspase-8
FIG. 2. Subcellular localization of ASC, ASC-CARD and ASCPYD. ASC forms filament-like aggregates in transiently transfected COS-7 cells, and not only the CARD of ASC but also the PYD of ASC forms filament-like aggregates in transiently transfected living COS-7 cells. (Aa) COS-7 cells were transiently transfected with pcDNA3-ASC. After 24 h, cells were fixed with 70% ethanol at ⫺20°C for 30 min and immunostained in anti-ASC monoclonal antibody followed by anti-mouse FITC-conjugated secondary antibody. The FITC-fluorescence signals of ASC appeared as aggregates (green). (Ab) Overlay with blue nuclei stained by DAPI of the same field. (Ac) High power view of the same field indicated that ASC has the appearance of filament-like aggregates (green). (Ba) COS-7 cells were transiently transfected with pEGFP-⌬1-100(CARD). After 24 h, green fluorescence signals in the living COS-7 cells were detected by immunofluorescence microscopy. Aggregates of the ASC-CARD were seen in transfected cells (green). (Bb) A phase contrast image of the same field. (Bc) High power view of the same field indicated that the ASC-CARD has the appearance of filament-like aggregates (green). (Ca) COS-7 cells were transfected with pEGFP-⌬101-195(PYD). After 24 h, green fluorescence signals in the living COS-7 cells were detected by immunofluorescence microscopy. Aggregates of the GFPASC-PYD were seen in transfected cells (green). (Cb) A phase contrast image of the same field. (Cc) High power view of the same field indicated that the GFP-ASC-PYD has the appearance of filamentlike aggregates (green). (Da) Coexpression of the GFP-ASC-CARD and the Flag-ASC-PYD. COS-7 cells were transiently cotransfected with pEGFP-ASC-⌬1-100(CARD) and pFLAG-CMV-4-ASC-⌬101195(PYD). After 24 h, cells were fixed with 70% ethanol. Signals were detected by immunofluorescence microscopy using anti-Flag monoclonal antibody followed by rhodamine-conjugated rabbit polyclonal antibody against mouse IgG. Immunofluorescent signals of GFPASC-CARD were detected as filament-like aggregates (green). (Db) Immunofluorescent signals of the rhodamine-Flag-ASC-PYD were also detected in the same aggregates (red). (Dc) Overlay of the same field. Overlapping fluorescence was yellow. (Ea) COS-7 cells were transiently transfected with pEGFP-C2 as a vector control. Diffuse green fluorescent signals were detected but no filament-like aggregates. (Eb) A phase contrast image of the same field. Scale bars are 10 m (Aa, Ab, Ba, Bc, Ca, Cb, Da, Db, Ea, Eb) and 5 m (Ac, Bc, Cc).
653
Vol. 280, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(10 –13), although, the COS-7 cells with aggregates of ASC exhibited no apoptotic changes. Under physiological conditions, ASC was cytosolic soluble protein and the aggregates appeared only in apoptotic cells (8). Since overexpression experiments in transformed-cells are under limited-conditions where the effecter molecules interacting with ASC may not be expressed, these results do not role out a relationship between ASC aggregation and apoptosis. Indeed, overexpression experiments of other CARD-containing proteins including bcl10 and RAIDD were reported to appear filament-like structures without any apoptotic stimulation (10, 11). Both the GFP-ASC-CARD and GFP-ASC-PYD Domains Formed Filament-like Aggregates in Transfected COS-7 Cells We have demonstrated that the murine ortholog of ASC oligomerizes via self-association and forms specklike aggregates (14). Hence, we examined which domain is involved in the oligomerization of ASC. GFPASC-WT, GFP-ASC-CARD or GFP-ASC-PYD was transiently expressed in COS-7 cells, and detected by immunofluorescence microscopy. GFP-ASC-WT appeared as speck-like aggregates in COS-7 cells (data not shown), and not only GFP-ASC-CARD but also GFP-ASC-PYD appeared as filament-like aggregates (Figs. 2Ba–2Bc, 2Ca–2Cc). The CARD-dependent filaments and the PYD-dependent filaments were similar. Then, we tested whether the CARD-dependent filaments colocalize on PYD-dependent filaments. Clear overlapping of GFP-ASC-CARD (Fig. 2Da, green) and rhodamine-Flag-ASC-PYD (Fig. 2Db, red) was seen in an overlay of the two channels (Fig. 2Dc, yellow). It was noted that these filament-like aggregates were not seen in COS-7 cells transfected with pEGFP-C2 (vector control) (Figs. 2Ea and 2Eb). Since CARD is an oligomerization domain, we expected that ASC might oligomerize in a CARD-dependent manner. However, unexpectedly, the filament-like aggregates were also observed in the COS-7 cells transfected with pEGFPASC-⌬101-195(PYD) (Fig. 2Ca–2Cc), and GFP-ASCCARD signals overlapped on rhodamine-Flag-ASCPYD signals (Figs. 2Da–2Dc). These results suggest that ASC may oligomerize in a PYD-dependent manner as well as the CARD. Such filaments were similar to the CARD-dependent filaments of bcl-10, RAIDD or death-effector filaments including FADD (10 –13). In the case of bcl-10, were it has been suggested that the filaments act as a scaffold for recruitment of NF-Bactivating downstream signal transduction molecules (10). Thus, ASC may provide a scaffold for ASCbinding partners acting in specific signal transduction pathways.
FIG. 3. ASC self-associates in a manner dependent on PYD-PYD, CARD-CARD, and PYD-CARD interactions. 1 ⫻ 10 6 COS-7 cells co-transfected with pEGFP-ASC-⌬101-195(PYD) (2.4 g) or pEGFPASC-⌬1-100(CARD) (2.4 g) and with pFLAG-CMV-4-ASC-⌬101195(PYD) (2.4 g), pFLAG-CMV-4-ASC-⌬1-100(CARD) (2.4 g) or pFLAG-CMV-4(vector) (2.4 g) were lysed, and immunoprecipitated with anti-Flag monoclonal antibody (top panel). GFP-fusion protein and Flag-tagged protein expressions were confirmed by Western blotting with anti-GFP monoclonal antibody (middle panel), and anti-Flag monoclonal antibody (bottom panel). Size markers in kilodaltons are on the left. WB, Western blotting; IP, immunoprecipitation. The asterisks indicate truncated products from GFP-fused proteins.
Homophilic and Heterophilic Interactions of Both the PYD and CARD Domains Were Involved in the Oligomerization of ASC A recent study showed that the death-inducing adaptor molecules including DD, DED or CARD form homodimers (4). In our study, we examined the possible homo- or heterophilic interactions of the PYD and CARD domains by immunoprecipitation. COS-7 cells cotransfected with pEGFP-ASC-⌬101-195(PYD) or pEGFP-ASC-⌬1-100(CARD) and pFLAG-CMV-4-ASC⌬101-195(PYD), pFLAG-CMV-4-ASC-⌬1-100(CARD) or pFLAG-CMV-4(empty vector) were lysed, and immunoprecipitated with anti-Flag monoclonal antibody. Then, coimmunoprecipitated proteins were detected by immunoblotting with anti-GFP monoclonal antibody (Fig. 3, top panel). As shown in Fig. 3, the GFP-ASCPYD was coimmunoprecipitated with the Flag-ASCPYD, and the GFP-ASC-CARD was coimmunoprecipitated with either the Flag-ASC-CARD or the FlagASC-PYD. These results indicate that the homophilic and heterophilic interactions of both the PYD and CARD domains are involved in self-association and filament-like aggregation of ASC, suggesting that the PYD domain is the novel homophilic interaction motif.
654
Vol. 280, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
At present, we hypothesize that the heterophilic interaction between PYD and CARD may be involved in an intramolecular interaction of ASC that prevent the aggregation of ASC under normal condition. ASC consists of two domains, the PYD and CARD domains that are involved in homophilic interaction like FADD, a DED- and DD-containing molecule, or RAIDD/CRADD, a CARD- and DD-containing molecule (6, 11, 13, 15–18). In the apoptosis signaling pathway, FADD, one of the components of the deathinducing signaling complex (DISC) (19, 20), binds to Fas via its DD and recruits caspase-8 through its DED, resulting in locally high concentrations of caspase-8 zymogens (21). RAIDD binds to TNF-R55 to form a signaling complex via its DD, and recruits caspase-2 (16). It was reported that RAIDD-CARD and RAIDD-DD are associated with each other (11). Thus self-association of ASC as an adaptor with two homodimerization domains, the PYD and CARD domains may result in the formation of ASC-containing higherorder complexes. Although the signaling pathway involving ASC is not clear, our study suggests that the dimarization of ASC caused by both the PYD and CARD domains is essential for oligomerization of ASC. The possible interaction between ASC and other PYD-containing molecules through the PYD domain remains to future studies.
10.
ACKNOWLEDGMENTS
15.
We thank Drs. Hiroshi Zenda and Shin Ohta (Department of Pharmacy, Shinshu University Hospital) for encouragement during this work, and Koichi Ayukawa, Yukiko Hattori, Taro Yokoyama and Guan Xin (Department of Molecular Oncology and Angiology) for discussions.
16.
7.
8.
9.
11.
12.
13.
14.
17.
REFERENCES 1. Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239 –257. 2. Arends, M. J., and Wyllie, A. H. (1991) Apoptosis: Mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32, 223–254. 3. Nu´n˜ez, G., Benedict, M. A., Hu, Y., and Inohara, N. (1998) Caspases: The proteases of the apoptotic pathway. Oncogene 17, 3237–3245. 4. Hofmann, K. (1999) The modular nature of apoptotic signaling proteins. Cell. Mol. Life Sci. 55, 1113–1128. 5. Hofmann, K., Bucher, P., and Tschopp, J. (1997) The CARD domain: A new apoptotic signalling motif. Trends Biochem. Sci. 22, 155–156. 6. Chinnaiyan, A. M., O’Rourke, K., Tewari, M., and Dixit, V. M. (1995) FADD, a novel death domain-containing protein, inter-
18.
19.
20.
21.
655
acts with the death domain of Fas and initiates apoptosis. Cell 81, 505–512. Feinstein, E., Kimchi, A., Wallach, D., Boldin, M., and Varfolomeev, E. (1995) The death domain: A module shared by proteins with diverse cellular functions. Trends Biochem. Sci. 20, 342–344. Masumoto, J., Taniguchi, S., Ayukawa, K., Sarvotham, H., Kishino, T., Niikawa, N., Hidaka, E., Katsuyama, T., Higuchi, T., and Sagara, J. (1999) ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J. Biol. Chem. 274, 33835–33838. Masumoto, J., Sagara, J., Hayama, M., Hidaka, E., Katsuyama, T., and Taniguchi, S. (1998) Differential expression of moesin in cells of hematopoietic lineage and lymphatic systems. Histochem. Cell Biol. 110, 33– 41. Guiet, C., and Vito, P. (2000) Caspase recruitment domain (CARD)-dependent cytoplasmic filaments mediate bcl10-induced NF-B activation. J. Cell Biol. 148, 1131–1140. Shearwin-Whyatt, L. M., Harvey, N. L., and Kumar, S. (2000) Subcellular localization and CARD-dependent oligomerization of the death adaptor RAIDD. Cell Death Differ. 7, 155–165. Perez, D., and White, E. (1998) E1B 19K inhibits Fas-mediated apoptosis through FADD-dependent sequestration of FLICE. J. Cell Biol. 141, 1255–1266. Siegel, R. M., Martin, D. A., Zheng, L., Ng, S. Y., Bertin, J., Cohen, J., and Lenardo, M. J. (1998) Death-effector filaments: Novel cytoplasmic structures that recruit caspases and trigger apoptosis. J. Cell Biol. 141, 1243–1253. Masumoto, J., Taniguchi, S., Nakayama, K., Ayukawa, K., and Sagara, J. (2000) Murine ortholog of ASC, a CARD-containing protein, self-associates and exhibits restricted distribution in developing mouse embryos. Exp. Cell Res. In press doi:10.1006/ excr.2000.5078. Boldin, M. P., Varfolomeev, E. E., Pancer, Z., Mett, I. L., Camonis, J. H., and Wallach, D. (1995) A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J. Biol. Chem. 270, 7795–7798. Duan, H., and Dixit, V. M. (1997) RAIDD is a new ‘death’ adaptor molecule. Nature 385, 86 – 89. Ahmad, M., Srinivasula, S. M., Wang, L., Talanian, R. V., Litwack, G., Fernandes-Alnemri, T., and Alnemri, E. S. (1997) CRADD, a novel human apoptotic adaptor molecule for caspase-2, and FasL/tumor necrosis factor receptor-interacting protein RIP. Cancer Res. 57, 615– 619. Chou, J. J., Matsuo, H., Duan, H., and Wagner, G. (1998) Solution structure of the RAIDD CARD and model for CARD/CARD interaction in caspase-2 and caspase-9 recruitment. Cell 94, 171–180. Muzio, M., Stockwell, B. R., Stennicke, H. R., Salvesen, G. S., and Dixit, V. M. (1998) An induced proximity model for caspase-8 activation. J. Biol. Chem. 273, 2926 –2930. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Involvement of MACH, a novel MORT1/FADDinteracting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85, 803– 815. Salvesen, G. S., and Dixit, V. M. (1999) Caspase activation: The induced-proximity model. Proc. Natl. Acad. Sci. USA 96, 10964 – 10967.
Pyrin N-Terminal Homology Domain- and Caspase Recruitment Domain-Dependent Oligomerization of ASC Junya Masumoto, Shun’ichiro Taniguchi, and Junji Sagara 1 Department of Molecular Oncology and Angiology, Research Center on Aging and Adaptation, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Nagano, Japan
Received December 3, 2000
ASC was first identified as a caspase recruitment domain (CARD)-containing proapoptotic molecule that forms insoluble aggregates during apoptosis. Here, we report both the pyrin N-terminal homology domain (PYD) and CARD domains are involved in the aggregation of ASC. Preliminary experiments indicated that overexpression of ASC formed filament-like aggregates in COS-7 cells. Expression experiments using green fluorescent protein (GFP) constructs showed that not only the GFP-ASC-CARD but also the GFP-ASC-PYD formed filament-like aggregates in COS-7 cells. We confirmed these filament-like aggregates of both the ASC-PYD and the ASC-CARD due to homophilic interaction by immunoprecipitation method. We also demonstrated that the ASC-PYD associated with the ASC-CARD by heterophilic interaction. These observations suggest that the dimerization of the PYD as well as the CARD plays an important role in the oligomerization of ASC as an adaptor molecule. © 2001 Academic Press Key Words: ASC; PYD; CARD; adaptor; homophilic interaction; heterophilic interaction; aggregation; oligomerization; filament formation.
Apoptosis plays important roles in regulating the growth and development of organisms, and also mediates normal and neoplastic tissue growth by removing This work was supported by a Grant-in-aid 12670109 from the Ministry of Education, Science and Culture, Japan. Abbreviations used: ASC, apoptosis-associated speck-like protein containing a CARD; CARD, caspase recruitment domain; DD, death domain; DED, death effector domain; PYD, pyrin N-terminal homology domain; bcl, B-cell lymphoma/leukemia; RAIDD, RIP-associated ICH-1/Ced-3-homologous protein with a death domain; CRADD, caspase and RIP adaptor with death domain; PCR, polymerase chain reaction; GFP, green fluorescent protein; FADD, Fas-associated protein with death domain; DISC, death-inducing signaling complex. 1 To whom correspondence should be addressed. Fax: 81-263-372724. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
excess cells (1, 2). Apoptosis is implemented by death machinery linked to signaling pathways in which death adaptor domains act together in the effectorproximal part of apoptotic signaling components (3, 4). The caspase recruitment domain (CARD) was proposed as the third example of a heterodimerization domain involved in apoptosis signaling pathways in addition to the death domain (DD) and the death effector domain (DED) (5–7). ASC is a CARD-containing molecule composed of an N-terminal pyrin N-terminal homology domain (PYD) and a C-terminal CARD. Under physiological conditions, ASC is a cytosolic soluble protein that aggregates during apoptosis (8). While the aggregation of ASC is thought to reflect the nature of the CARD of ASC as an oligomerization domain, the function of the PYD of ASC remains unknown. In this study, we show that not only the CARD but also the PYD is involved in the aggregation of ASC. Finally, by immunoprecipitation analysis, we demonstrate the direct homophilic and heterophilic interactions of the ASC-PYD and ASC-CARD domains, suggesting that the PYD domain is a novel homophilic interaction motif. MATERIALS AND METHODS Construction of expression plasmids. The entire open reading frame of ASC was inserted into the EcoRI and SalI sites of pEGFP-C2 (Clontech) to produce pEGFP-ASC-WT. Deletion mutants of pEGFP-ASC-1-100(CARD) and pEGFP-ASC-⌬101195(PYD) were constructed by polymerase chain reaction (PCR) using the entire open reading frame of ASC-including gt11 clone (8) as a template following insertion into the EcoRI and SalI sites of pEGFP-C2. Deletion mutants of pFLAG-CMV-4-ASC-⌬1-100(CARD) and pFLAG-CMV-4-ASC-⌬101-195(PYD) were constructed as described above by insertion into the EcoRI and KpnI sites of pFLAGCMV-4 (Sigma). pcDNA3-ASC and anti-ASC monoclonal antibody were described previously (8). Schematic structures of these expression products are presented in Fig. 1. Immunofluorescence analysis. After COS-7 cells were transfected with pcDNA3-ASC, cells were fixed with 70% ethanol at ⫺20°C 30
652
Vol. 280, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Schematic representations of wild-type and deletion mutants of ASC fused to GFP or Flag. GFP-ASC-WT is a full-length ASC fused to an N-terminal GFP. GFP-ASC-⌬1-100(CARD) is the N-terminal 100-amino-acid truncated-form of ASC consisting of CARD fused to an N-terminal GFP. GFP-ASC-⌬101-195(PYD) is the C-terminal 95-amino-acid truncated-form of ASC consisting of a PYD fused to an N-terminal GFP. Flag-ASC-⌬1-100(CARD) is the N-terminal 100-amino-acid truncated-form of ASC consisting of a CARD tagged with an N-terminal Flag. Flag-ASC-⌬101-195(PYD) is the C-terminal 95-amino-acid truncated-form of ASC consisting of a PYD tagged with an N-terminal Flag.
min, air dried, and rinsed with PBS. Then, cells were incubated with anti-ASC monoclonal antibody, and following secondary FITCconjugated rabbit polyclonal antibody against mouse IgG (Dako). FITC-signals were detected by immunofluorescence microscopy (Zeiss Axioskop). Localizations of GFP-ASC, GFP-ASC-PYD and GFP-ASC-CARD in transfected COS-7 cells were also analyzed by immunofluorescence microscopy. Transfection, expression and immunoprecipitation of tagged proteins. 1 ⫻ 10 6 COS-7 cells were transfected with expression plasmids using LipofectAMINE-PLUS reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Transfected COS-7 cells were lysed and incubated in 0.8 ml of lysis buffer (0.1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl [pH 7.6], 1 mM PMSF, 10 g/ml leupeptin, 10 g/ml pepstatin A, 10 g/ml antipain, 10 g/ml aprotinin) for 5 min. The lysate was removed from the dish after any remaining cellular debris was fully dislodged from the plate surface with a rubber policeman. The lysate from one dish was incubated in a 1.5-ml tube on ice for an additional 10 min, and clarified by centrifugation at 12000g for 15 min. The supernatant was immunoprecipitated with 20 l of protein G-Sepharose 4B (Amersham Pharmacia Biotech) conjugated with anti-Flag monoclonal antibody M2 (Sigma). Immunoprecipitated proteins were subjected to 16% SDSpolyacrylamide electrophoresis and detected by Western blotting using anti-GFP monoclonal antibody (Clontech). Detailed procedures were described previously (9).
RESULTS AND DISCUSSION ASC Formed Filament-like Aggregates in COS-7 Cells We have previously shown that ASC forms insoluble speck-like aggregates during apoptosis of promyelocytic leukemia HL-60 cells (8). Closer examination revealed the speck appeared to be a filament-like aggregate with a hollow center (8). Originally, ASC was identified as a cytoskeletal and/or nuclear matrix molecule (8). ASC expressed transiently in COS-7 cells formed filament-like aggregates (Fig. 2Aa–2Ac) that are similar to CARD-dependent filaments of bcl-10, RAIDD, and the prodomain of caspase-2, or deatheffector filaments including FADD and procaspase-8
FIG. 2. Subcellular localization of ASC, ASC-CARD and ASCPYD. ASC forms filament-like aggregates in transiently transfected COS-7 cells, and not only the CARD of ASC but also the PYD of ASC forms filament-like aggregates in transiently transfected living COS-7 cells. (Aa) COS-7 cells were transiently transfected with pcDNA3-ASC. After 24 h, cells were fixed with 70% ethanol at ⫺20°C for 30 min and immunostained in anti-ASC monoclonal antibody followed by anti-mouse FITC-conjugated secondary antibody. The FITC-fluorescence signals of ASC appeared as aggregates (green). (Ab) Overlay with blue nuclei stained by DAPI of the same field. (Ac) High power view of the same field indicated that ASC has the appearance of filament-like aggregates (green). (Ba) COS-7 cells were transiently transfected with pEGFP-⌬1-100(CARD). After 24 h, green fluorescence signals in the living COS-7 cells were detected by immunofluorescence microscopy. Aggregates of the ASC-CARD were seen in transfected cells (green). (Bb) A phase contrast image of the same field. (Bc) High power view of the same field indicated that the ASC-CARD has the appearance of filament-like aggregates (green). (Ca) COS-7 cells were transfected with pEGFP-⌬101-195(PYD). After 24 h, green fluorescence signals in the living COS-7 cells were detected by immunofluorescence microscopy. Aggregates of the GFPASC-PYD were seen in transfected cells (green). (Cb) A phase contrast image of the same field. (Cc) High power view of the same field indicated that the GFP-ASC-PYD has the appearance of filamentlike aggregates (green). (Da) Coexpression of the GFP-ASC-CARD and the Flag-ASC-PYD. COS-7 cells were transiently cotransfected with pEGFP-ASC-⌬1-100(CARD) and pFLAG-CMV-4-ASC-⌬101195(PYD). After 24 h, cells were fixed with 70% ethanol. Signals were detected by immunofluorescence microscopy using anti-Flag monoclonal antibody followed by rhodamine-conjugated rabbit polyclonal antibody against mouse IgG. Immunofluorescent signals of GFPASC-CARD were detected as filament-like aggregates (green). (Db) Immunofluorescent signals of the rhodamine-Flag-ASC-PYD were also detected in the same aggregates (red). (Dc) Overlay of the same field. Overlapping fluorescence was yellow. (Ea) COS-7 cells were transiently transfected with pEGFP-C2 as a vector control. Diffuse green fluorescent signals were detected but no filament-like aggregates. (Eb) A phase contrast image of the same field. Scale bars are 10 m (Aa, Ab, Ba, Bc, Ca, Cb, Da, Db, Ea, Eb) and 5 m (Ac, Bc, Cc).
653
Vol. 280, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(10 –13), although, the COS-7 cells with aggregates of ASC exhibited no apoptotic changes. Under physiological conditions, ASC was cytosolic soluble protein and the aggregates appeared only in apoptotic cells (8). Since overexpression experiments in transformed-cells are under limited-conditions where the effecter molecules interacting with ASC may not be expressed, these results do not role out a relationship between ASC aggregation and apoptosis. Indeed, overexpression experiments of other CARD-containing proteins including bcl10 and RAIDD were reported to appear filament-like structures without any apoptotic stimulation (10, 11). Both the GFP-ASC-CARD and GFP-ASC-PYD Domains Formed Filament-like Aggregates in Transfected COS-7 Cells We have demonstrated that the murine ortholog of ASC oligomerizes via self-association and forms specklike aggregates (14). Hence, we examined which domain is involved in the oligomerization of ASC. GFPASC-WT, GFP-ASC-CARD or GFP-ASC-PYD was transiently expressed in COS-7 cells, and detected by immunofluorescence microscopy. GFP-ASC-WT appeared as speck-like aggregates in COS-7 cells (data not shown), and not only GFP-ASC-CARD but also GFP-ASC-PYD appeared as filament-like aggregates (Figs. 2Ba–2Bc, 2Ca–2Cc). The CARD-dependent filaments and the PYD-dependent filaments were similar. Then, we tested whether the CARD-dependent filaments colocalize on PYD-dependent filaments. Clear overlapping of GFP-ASC-CARD (Fig. 2Da, green) and rhodamine-Flag-ASC-PYD (Fig. 2Db, red) was seen in an overlay of the two channels (Fig. 2Dc, yellow). It was noted that these filament-like aggregates were not seen in COS-7 cells transfected with pEGFP-C2 (vector control) (Figs. 2Ea and 2Eb). Since CARD is an oligomerization domain, we expected that ASC might oligomerize in a CARD-dependent manner. However, unexpectedly, the filament-like aggregates were also observed in the COS-7 cells transfected with pEGFPASC-⌬101-195(PYD) (Fig. 2Ca–2Cc), and GFP-ASCCARD signals overlapped on rhodamine-Flag-ASCPYD signals (Figs. 2Da–2Dc). These results suggest that ASC may oligomerize in a PYD-dependent manner as well as the CARD. Such filaments were similar to the CARD-dependent filaments of bcl-10, RAIDD or death-effector filaments including FADD (10 –13). In the case of bcl-10, were it has been suggested that the filaments act as a scaffold for recruitment of NF-Bactivating downstream signal transduction molecules (10). Thus, ASC may provide a scaffold for ASCbinding partners acting in specific signal transduction pathways.
FIG. 3. ASC self-associates in a manner dependent on PYD-PYD, CARD-CARD, and PYD-CARD interactions. 1 ⫻ 10 6 COS-7 cells co-transfected with pEGFP-ASC-⌬101-195(PYD) (2.4 g) or pEGFPASC-⌬1-100(CARD) (2.4 g) and with pFLAG-CMV-4-ASC-⌬101195(PYD) (2.4 g), pFLAG-CMV-4-ASC-⌬1-100(CARD) (2.4 g) or pFLAG-CMV-4(vector) (2.4 g) were lysed, and immunoprecipitated with anti-Flag monoclonal antibody (top panel). GFP-fusion protein and Flag-tagged protein expressions were confirmed by Western blotting with anti-GFP monoclonal antibody (middle panel), and anti-Flag monoclonal antibody (bottom panel). Size markers in kilodaltons are on the left. WB, Western blotting; IP, immunoprecipitation. The asterisks indicate truncated products from GFP-fused proteins.
Homophilic and Heterophilic Interactions of Both the PYD and CARD Domains Were Involved in the Oligomerization of ASC A recent study showed that the death-inducing adaptor molecules including DD, DED or CARD form homodimers (4). In our study, we examined the possible homo- or heterophilic interactions of the PYD and CARD domains by immunoprecipitation. COS-7 cells cotransfected with pEGFP-ASC-⌬101-195(PYD) or pEGFP-ASC-⌬1-100(CARD) and pFLAG-CMV-4-ASC⌬101-195(PYD), pFLAG-CMV-4-ASC-⌬1-100(CARD) or pFLAG-CMV-4(empty vector) were lysed, and immunoprecipitated with anti-Flag monoclonal antibody. Then, coimmunoprecipitated proteins were detected by immunoblotting with anti-GFP monoclonal antibody (Fig. 3, top panel). As shown in Fig. 3, the GFP-ASCPYD was coimmunoprecipitated with the Flag-ASCPYD, and the GFP-ASC-CARD was coimmunoprecipitated with either the Flag-ASC-CARD or the FlagASC-PYD. These results indicate that the homophilic and heterophilic interactions of both the PYD and CARD domains are involved in self-association and filament-like aggregation of ASC, suggesting that the PYD domain is the novel homophilic interaction motif.
654
Vol. 280, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
At present, we hypothesize that the heterophilic interaction between PYD and CARD may be involved in an intramolecular interaction of ASC that prevent the aggregation of ASC under normal condition. ASC consists of two domains, the PYD and CARD domains that are involved in homophilic interaction like FADD, a DED- and DD-containing molecule, or RAIDD/CRADD, a CARD- and DD-containing molecule (6, 11, 13, 15–18). In the apoptosis signaling pathway, FADD, one of the components of the deathinducing signaling complex (DISC) (19, 20), binds to Fas via its DD and recruits caspase-8 through its DED, resulting in locally high concentrations of caspase-8 zymogens (21). RAIDD binds to TNF-R55 to form a signaling complex via its DD, and recruits caspase-2 (16). It was reported that RAIDD-CARD and RAIDD-DD are associated with each other (11). Thus self-association of ASC as an adaptor with two homodimerization domains, the PYD and CARD domains may result in the formation of ASC-containing higherorder complexes. Although the signaling pathway involving ASC is not clear, our study suggests that the dimarization of ASC caused by both the PYD and CARD domains is essential for oligomerization of ASC. The possible interaction between ASC and other PYD-containing molecules through the PYD domain remains to future studies.
10.
ACKNOWLEDGMENTS
15.
We thank Drs. Hiroshi Zenda and Shin Ohta (Department of Pharmacy, Shinshu University Hospital) for encouragement during this work, and Koichi Ayukawa, Yukiko Hattori, Taro Yokoyama and Guan Xin (Department of Molecular Oncology and Angiology) for discussions.
16.
7.
8.
9.
11.
12.
13.
14.
17.
REFERENCES 1. Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239 –257. 2. Arends, M. J., and Wyllie, A. H. (1991) Apoptosis: Mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32, 223–254. 3. Nu´n˜ez, G., Benedict, M. A., Hu, Y., and Inohara, N. (1998) Caspases: The proteases of the apoptotic pathway. Oncogene 17, 3237–3245. 4. Hofmann, K. (1999) The modular nature of apoptotic signaling proteins. Cell. Mol. Life Sci. 55, 1113–1128. 5. Hofmann, K., Bucher, P., and Tschopp, J. (1997) The CARD domain: A new apoptotic signalling motif. Trends Biochem. Sci. 22, 155–156. 6. Chinnaiyan, A. M., O’Rourke, K., Tewari, M., and Dixit, V. M. (1995) FADD, a novel death domain-containing protein, inter-
18.
19.
20.
21.
655
acts with the death domain of Fas and initiates apoptosis. Cell 81, 505–512. Feinstein, E., Kimchi, A., Wallach, D., Boldin, M., and Varfolomeev, E. (1995) The death domain: A module shared by proteins with diverse cellular functions. Trends Biochem. Sci. 20, 342–344. Masumoto, J., Taniguchi, S., Ayukawa, K., Sarvotham, H., Kishino, T., Niikawa, N., Hidaka, E., Katsuyama, T., Higuchi, T., and Sagara, J. (1999) ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J. Biol. Chem. 274, 33835–33838. Masumoto, J., Sagara, J., Hayama, M., Hidaka, E., Katsuyama, T., and Taniguchi, S. (1998) Differential expression of moesin in cells of hematopoietic lineage and lymphatic systems. Histochem. Cell Biol. 110, 33– 41. Guiet, C., and Vito, P. (2000) Caspase recruitment domain (CARD)-dependent cytoplasmic filaments mediate bcl10-induced NF-B activation. J. Cell Biol. 148, 1131–1140. Shearwin-Whyatt, L. M., Harvey, N. L., and Kumar, S. (2000) Subcellular localization and CARD-dependent oligomerization of the death adaptor RAIDD. Cell Death Differ. 7, 155–165. Perez, D., and White, E. (1998) E1B 19K inhibits Fas-mediated apoptosis through FADD-dependent sequestration of FLICE. J. Cell Biol. 141, 1255–1266. Siegel, R. M., Martin, D. A., Zheng, L., Ng, S. Y., Bertin, J., Cohen, J., and Lenardo, M. J. (1998) Death-effector filaments: Novel cytoplasmic structures that recruit caspases and trigger apoptosis. J. Cell Biol. 141, 1243–1253. Masumoto, J., Taniguchi, S., Nakayama, K., Ayukawa, K., and Sagara, J. (2000) Murine ortholog of ASC, a CARD-containing protein, self-associates and exhibits restricted distribution in developing mouse embryos. Exp. Cell Res. In press doi:10.1006/ excr.2000.5078. Boldin, M. P., Varfolomeev, E. E., Pancer, Z., Mett, I. L., Camonis, J. H., and Wallach, D. (1995) A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J. Biol. Chem. 270, 7795–7798. Duan, H., and Dixit, V. M. (1997) RAIDD is a new ‘death’ adaptor molecule. Nature 385, 86 – 89. Ahmad, M., Srinivasula, S. M., Wang, L., Talanian, R. V., Litwack, G., Fernandes-Alnemri, T., and Alnemri, E. S. (1997) CRADD, a novel human apoptotic adaptor molecule for caspase-2, and FasL/tumor necrosis factor receptor-interacting protein RIP. Cancer Res. 57, 615– 619. Chou, J. J., Matsuo, H., Duan, H., and Wagner, G. (1998) Solution structure of the RAIDD CARD and model for CARD/CARD interaction in caspase-2 and caspase-9 recruitment. Cell 94, 171–180. Muzio, M., Stockwell, B. R., Stennicke, H. R., Salvesen, G. S., and Dixit, V. M. (1998) An induced proximity model for caspase-8 activation. J. Biol. Chem. 273, 2926 –2930. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Involvement of MACH, a novel MORT1/FADDinteracting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85, 803– 815. Salvesen, G. S., and Dixit, V. M. (1999) Caspase activation: The induced-proximity model. Proc. Natl. Acad. Sci. USA 96, 10964 – 10967.