Mechanism of Dronc activation in Drosophila cells

Proteolytic processing is required for the activation of most caspases. However, recent reports have suggested that the activation of the mammalian initiator caspase caspase-9 occurs during dimerization rather than after processing. Previously, we reported that, in normal living Drosophila S2 cells, the initiator caspase Dronc is continuously processed to a 40 kDa form we called Pr1 and that, during apoptosis, a second processed form of 37 kDa is also observed, which we called Pr2. In this study, we determined that Dronc Pr1 is the result of Dronc autoprocessing at amino acid E352, whereas Pr2 results from Drice cleaving full-length Dronc at amino acid D135. By using purified recombinant proteins and expressing Dronc cleavage mutants in S2 cells, we determined that autoprocessing at E352 is crucial for Dronc caspase activity, whereas Drice cleavage at D135 has little effect on Dronc activity. Suppression of the oligomerizing factor Dark by RNA interference revealed that Dark is required for Dronc autoprocessing at E352, whereas RNA interference of the effector caspase Drice revealed that Drice is also required for apoptosis in S2 cells. These results provide the first details of the mechanisms regulating initiator caspase activation in an invertebrate organism.