Cleavage of the Apoptosis Inhibitor DIAP1 by the Apical Caspase DRONC in Both Normal and Apoptotic Drosophila Cells

Abstract

In Drosophila S2 cells, the apical caspase DRONC undergoes a low level of spontaneous autoprocessing. Unintended apoptosis is prevented by the inhibitor of apoptosis DIAP1, which targets the processed form of DRONC for degradation through its E3 ubiquitin protein ligase activity. Recent reports have demonstrated that shortly after the initiation of apoptosis in S2 cells, DIAP1 is cleaved following aspartate residue Asp-20 by the effector caspase DrICE. Here we report a novel caspase-mediated cleavage of DIAP1 in S2 cells. In both living and dying S2 cells, DIAP1 is cleaved by DRONC after glutamate residue Glu-205, located between the first and second BIR domains. The mutation of Glu-205 prevented the interaction of DIAP1 and processed DRONC but had no effect on the interaction with full-length DRONC. The mutation of Glu-205 also had a negative effect on the ability of overexpressed DIAP1 to prevent apoptosis stimulated by the proapoptotic protein Reaper or by UV light. These results expand our knowledge of the events that occur in the Drosophila apoptosome prior to and after receiving an apoptotic signal. IAP1 proteins were first discovered in baculoviruses and have now been found in a wide range of organisms (1). IAPs are identified by the presence of at least one baculovirus IAP repeat (BIR), a roughly 70-amino acid domain shown to be important for protein-protein interactions. Many IAPs also contain a C-terminal RING finger domain, and several of these have been shown to possess E3 ubiquitin ligase activity (2-8).Although all IAP proteins contain a BIR domain, not all BIR-containing proteins can inhibit apoptosis, and some BIRcontaining proteins have been implicated in cell signaling and cell cycle regulation (9). Several IAPs have been shown to directly bind and inhibit the activity of a family of proapoptotic proteins known as caspases (10, 11). Caspases are cysteine proteases and are synthesized as zymogens, which must usually be proteolytically cleaved to become activated (12). They are divided into two types depending on the size of an Nterminal prodomain found on all caspases. Initiator caspases are identified by a long prodomain, whereas a short prodomain is indicative of an effector caspase. Upon reception of a death signal, upstream initiator caspases are first activated and in turn cleave and activate downstream effector caspases leading to apoptosis.In mammalian cells, caspase-9 functions as the initiator caspase for the intrinsic death pathway and upon induction of apoptosis associates with APAF-1, the mammalian CED-4 homolog. Caspase-9 and APAF-1 binding occur through their mutual CARD domains and along with the cofactors cytochrome c and dATP form the apoptosome. The apoptosome is a large complex that induces dimerization and activation of caspase-9 (13). Activated caspase-9 then activates effector caspases including caspase-3 and caspase-7, which bring about the morphological and biochemical events associated with apoptosis. In mammalian cells, apoptosome formation is thought to occur only afte...

Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways

“…Needless to say that the double mutant I393A,D20A was also unstable (lanes 10-12), and its degradation was proteasome-dependent ( Figure 3cii). Assuming that other, yet to be discovered cleavages can also lead to subsequent degradation of the cleaved product (see for example, Muro et al 34 ), it was important to test whether the RING finger domain can be involved in the degradation of Figure 3 Processing of DIAP1 is not essential for its degradation and does not require its RING finger domain. (a) DIAP1 is rapidly degraded in the presence or absence of caspase inhibitor.…”

A biochemical analysis of the activation of the Drosophila caspase DRONC

“…35,36 Treatment of Drosophila SL2 cells with apoptotic stimuli results in rapid removal of full-length DIAP1 protein 35,37 and this degradation is delayed by addition of the proteosome inhibitor MG132. 37 We have used BG2 cells and confirmed that following treatment with cycloheximide, endogenous DIAP1 protein is degraded in 4 h, concurrent with cleavage of DRONC and DRICE processing at 6 h ( Figure 1b). We used MG132 to assess the effect of sustained DIAP1 protein levels on caspase activation.…”

Activation mechanism and substrate specificity of the Drosophila initiator caspase DRONC

“…This has been demonstrated previously on synthetic peptidyl substrates, 16 and also in terms of its autolytic cleavage and cleavage of Drosophila IAP1. 14,16,25 Interestingly, as shown in Figure 4a, caspase-9 also tolerates Glu in its primary specificity pocket, and can cleave its own inter-chain segment following a Glu residue, although this cleavage is subservient to the natural Asp cleavage sites in the caspase-9 linker. 26,27 We demonstrate, using a matched set of substrates, that DRONC may have almost equal activity on tetrapeptides containing Asp and Glu in P1, but this does not hold for a downstream substrate.…”