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

Abstract

Inhibitors of apoptosis proteins (IAPs) suppress cell death by inactivating proapoptotic regulators, and therefore play important roles in controlling apoptosis in normal and malignant cells. Many IAPs are ubiquitin ligases, and their activity is mediated via ubiquitination and subsequent degradation of their targets. Here we corroborate a previous observation that DIAP1 (Drosophila IAP1) can be degraded via a two-step mechanism: (i) limited caspase-mediated cleavage and (ii) degradation of the released fragment via the ubiquitin N-end rule pathway. Yet, we demonstrate that this pathway is not the only one involved in DIAP1 degradation, and the intact protein can be degraded independent of prior caspase cleavage. Importantly, this mode of degradation does not require the RING-finger-mediated autoubiquitinating activity of DIAP1, believed to target many RING-finger E3s for self-destruction. Our preliminary data suggest that DIAP2 mediates DIAP1 degradation, suggesting a novel regulatory loop within the apoptotic pathway. Studying the role of the autoubiquitinating activity of DIAP1, we demonstrate that it does not involve formation of Lys48-based polyubiquitin chains, but probably chains linked via Lys63. Our preliminary data suggest that the autoubiquitination serves to attenuate the ligase activity of DIAP1 towards its exogenous substrates. Recent studies indicate that ubiquitination plays an important role in regulating apoptosis. This is partially mediated by the inhibitors of apoptosis proteins (IAPs), a centrally important group of cell death regulators, many of them are ubiquitin ligases. IAPs suppress cell death by inactivating different proapoptotic factors, some by targeting them for ubiquitination and subsequent proteasomal degradation. At least eight IAPs have been identified in mammals. In Drosophila melanogaster, expression of DIAP1 and DIAP2 1 can suppress apoptosis that occurs both during normal development as well as following overexpression of proapoptotic factors such as Reaper (Rpr). 2 Loss of DIAP1 function results in early embryonic death as a consequence of massive apoptosis. [3][4][5] The IAP family is characterized structurally by one-three Nterminal copies of Baculovirus IAP Repeat (BIR) domains. DIAP1 contains two copies of BIR that bind effector and initiator caspases. In some cases, this binding leads to ubiquitination and subsequent degradation of the caspase. 2 IAP-mediated inactivation of caspases is effectively inhibited by a family of proapoptotic proteins that share an IAP-binding tetra-peptide motif at their N-termini. Among those proteins are Smac/Diablo in mammals, and Rpr, Hid and Grim (RHG) in Drosophila. It was shown that RHG proteins abrogate effectively DIAP1-mediated inactivation of Dronc and DrICE. 6 DIAP1 contains also a C-terminal RING-finger motif that serves to recruit the E2 component of the ubiquitin conjugation machinery. For RING-finger E3s, their best-characterized activity is self-ubiquitination thought to regulate their cellular level, but it is assumed th...

Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms

“…43 This targeting of one IAP by another appears to be conserved in humans as well: XIAP was shown to be targeted for degradation by cIAP1. 44 An additional mechanism of degradation of Diap1 is provided by caspase-catalyzed processing followed by the proteasomal degradation of the C-terminally released fragment by the N-end rule pathway.…”

“…It therefore seems that long polyubiquitin chains are required for this effect, suggesting that it may result from steric hindrance preventing binding to, or efficient conjugation of the substrate. 43 Self-ubiquitination can also serve as a mechanism to recruit substrates with ubiquitin-binding properties, as has been shown for TRAF6 and NEDD4. TRAFs are RING domaincontaining E3 ligases that have crucial roles in the initial activation of several signaling cascades including the NF-kB, JNK, and p38 kinase pathways.…”

“…44 An additional mechanism of degradation of Diap1 is provided by caspase-catalyzed processing followed by the proteasomal degradation of the C-terminally released fragment by the N-end rule pathway. 43,45 Degradation of E3 ligases both through self-targeting and through external ligase(s). For several ligases that mediate their own degradation, it has also been shown that they can be targeted by alternative mechanisms involving external ligases.…”

Inhibitor of Apoptosis (IAP) Proteins-Modulators of Cell Death and Inflammation

“…Disruption of drICE ubiquitylation or NEDDylation, either by loss of DIAP1's E3 activity or generation of a nonmodifyable form of drICE, renders this effector caspase resistant to DIAP1-mediated inactivation (Ditzel et al 2008;Broemer et al 2010). In addition to its own RING finger domain, DIAP1 recruits a second Ub-E3 ligase that belongs to the N-end-rule pathway (UBR), to effectively block caspase activity (Ditzel et al 2003;Herman-Bachinsky et al 2007;Tenev et al 2007). Recruitment of the N-end rule E3 ligase requires caspase-mediated cleavage of DIAP1 and exposure of a docking site for the UBR at the neo-NH 2 terminus of cleaved DIAP1 .…”

Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila

“…11 Previous reports have shown that DIAP1 can be ubiquitinated by UBR family proteins (also called N-recognins) and degraded by proteasome. 33 To test this possibility, first we examined the DIAP1 protein levels in ubr3 mutant cells using a DIAP1-specific antibody (Figures 4a-a''), whose specificity is verified in Supplementary Figures S2a-a''. The fact that protein levels of DIAP1 were slightly reduced only in some but not all ubr3 mutant cells excludes the possibility that Ubr3 targets DIAP1 for degradation.…”