¹Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, Kyoto 606

²Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606, Japan

³Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL 32610-0266

⁴Division of Oncology Research, Mayo Clinic, Rochester, MN 55905, USA

⁵Pharmaceutical Basic Research Laboratories, Japan Tobacco Inc., Yokohama, Kanagawa 236

⁶College of Medical Technology, Kyoto University, Kyoto 606, Japan

^aAuthor for correspondence

The activation of multiple interleukin-1 converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an affinity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identified six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.

apoptosis; apoptotic cell death; YV(bio)KD-aomk; serpin; Jurkat T cells