Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis

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.