Two novel synthetic tetrapeptides, VEID-CHO and DMQD-CHO, could selectively inhibit caspase-6 and caspase-3, respectively. We used these inhibitors to dissect the pathway of caspase activation in Fas-stimulated Jurkat cells and identify the roles of each active caspase in apoptotic processes. Affinity labeling techniques revealed a branched protease cascade in which caspase-8 activates caspase-3 and -7, and caspase-3, in turn, activates caspase-6. Both caspase-6 and -3 have major roles in nuclear apoptosis. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of nuclei. Caspase-3 cleaves NuMA at sites distinct from caspase-6, and mediates DNA fragmentation and chromatin condensation. It is also involved in extranuclear apoptotic events: cleavage of PAK2, formation of apoptotic bodies, and exposure of phosphatidylserine on the cell surface. In contrast, a caspase(s) distinct from caspase-3 or -6 mediates the disruption of mitochondrial membrane potential (permeability transition) and the shrinkage of cytoplasm. These findings demonstrate that caspases are organized in a protease cascade, and that each activated caspase plays a distinct role(s) in the execution of Fas-induced cell death.
Caspases Are Activated in a Branched Protease Cascade and Control Distinct Downstream Processes in Fas-induced Apoptosis
1Abbreviations used in this paper: DAPI, 4′,6-diamidino-2-phenylindole; DFF, DNA fragmentation factor; DISC, death-inducing signaling complex; DMQD-CHO, acetyl-Asp-Met-Gln-Asp-aldehyde; FSC, forward light scatter; ICE, IL-1β converting enzyme; NuMA, nuclear mitotic apparatus protein; PAK, p21-activated kinase; PARP, poly (ADP-ribose) polymerase; PKCδ, protein kinase C δ; PS, phosphatidylserine; PT, permeability transition; VEID-CHO, acetyl-Val-Ileu-Asp-aldehyde; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone.
We thank Emad S. Alnemri for the generous gift of cDNAs for caspases; Nancy A. Thornberry for providing YV(bio)KD-aomk; Gerald M. Cohen for anti–caspase-7 Ab; Charles H. Yang for anti-NuMA Ab; Alastair Mackay for critical review of the manuscript and creative suggestions; Yuri A. Lazebnik, Scott H. Kaufmann, William C. Earnshaw, Eisuke Nishida, Fumiko Toyoshima, and Katsumi Takada for helpful discussion and thoughtful suggestions; and Akira Komoriya, Kuniko Takano, Akinori Maeda, and Kouhei Yamashita for help in flow cytometric analysis.
A. Takahashi is a Research Resident of the Japanese Foundation of Aging and Health.
Address correspondence to Atsushi Takahashi, The First Division, Department of Internal Medicine, Kyoto University Hospital, 54 Shogoin Kawara-cho, Sakyo-ku, Kyoto 606, Japan. Phone: 81-75-751-4290; Fax: 81-75-751-4221; E-mail: [email protected]
Hirokazu Hirata, Atsushi Takahashi, Susumu Kobayashi, Shin Yonehara, Hirofumi Sawai, Toshiro Okazaki, Kokichi Yamamoto, Masataka Sasada; Caspases Are Activated in a Branched Protease Cascade and Control Distinct Downstream Processes in Fas-induced Apoptosis . J Exp Med 16 February 1998; 187 (4): 587–600. doi: https://doi.org/10.1084/jem.187.4.587
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