Abstract
Although loss of the inhibitor of apoptosis (IAP) protein DIAP1 has been shown to result in caspase activation and spontaneous cell death in Drosophila cells and embryos, the point at which DIAP1 normally functions to inhibit caspase activation is unknown. Depletion of the DIAP1 protein in Drosophila S2 cells or the Sf-IAP protein in Spodoptera frugiperda Sf21 cells by RNA interference (RNAi) or cycloheximide treatment resulted in rapid and widespread caspase-dependent apoptosis. Co-silencing of dronc or dark largely suppressed this apoptosis, indicating that DIAP1 is normally required to inhibit an activity dependent on these proteins. Silencing of dronc also inhibited DRICE processing following stimulation of apoptosis, demonstrating that DRONC functions as an apical caspase in S2 cells. Silencing of diap1 or treatment with UV light induced DRONC processing, which occurred in two steps. The first step appeared to occur continuously even in the absence of an apoptotic signal and to be dependent on DARK, because full-length DRONC accumulated when dark was silenced in non-apoptotic cells. In addition, treatment with the proteasome inhibitor MG132 resulted in accumulation of this initially processed form of DRONC, but not full-length DRONC, in non-apoptotic cells. The second step in DRONC processing was observed only in apoptotic cells. These results indicate that the initial step in DRONC processing occurs continuously via a DARK-dependent mechanism in Drosophila cells and that DIAP1 is required to prevent excess accumulation of this first form of processed DRONC, presumably through its ability to act as a ubiquitin-protein ligase.
