IAP antagonist Grim mediates cell death through divergent pathways

Abstract

Apoptosis is an evolutionarily conserved process, carried out through proteolytic cascades that lead to activation of cysteinyl aspartate-specific proteases (caspases), which in turn cause cell self-destruction. While caspases are activated upon various death stimuli or developmental cues, inhibitor of apoptosis (IAPs) can prevent caspase-mediated cell death, not only through direct binding and inhibition, but also by directly ubiquitinating caspases, which inactivates them or targets them for degradation. In flies and mammals, cell death is facilitated or enhanced by the accumulation of “IAP antagonists”, which promote cell death by utilizing their N-terminal IAP binding motifs (IBMs) to interact with IAPs, to displace caspases from IAP-binding, and to promote IAPs autoubiquitination. Herein, we demonstrate that a Drosophila IAP antagonist, Grim, induces cell death through divergent mechanisms: an IBM-dependent pathway, requiring a crosstalk with Drosophila IAP1 (DIAP1), and an IBM-independent manner, involving Grim’s C-terminus for translational repression. We discovered that Grim is a target for caspase-mediated cleavage at Grim’s C-terminus (Asp132). Grim loses its only lysine (Lys 136) after cleavage, but acquires a longer half-life due to inability of DIAP1 to ubiquitinate it, which in turn leads to greater cell death. This novel regulation allows increased levels of Grim to efficiently kill through an IBM-dependent mechanism that derepresses caspase inhibition. However, in addition to IBM-dependent cell death, Grim is able to induce caspase activation independent of its IBM. In our studies, we have demonstrated that IBM-independent cell death requires Grim’s C-terminal domain as well as the characteristic Grim Helix 3 (GH3) domain. Importantly, Grim’s C-terminus localizes Grim to distinct cytoplasmic granules, i.e. P-bodies, where translation is repressed due to the accumulation of protein components that regulate mRNA translation or stability. Knockdown of a P-body-localized translational repressor, Me31B, largely reduced IBM-independent cell death. Taken together, IBM-independent cell death occurs, at least in part, through Me31B-mediated translational repression, requiring the localization of Grim to P-bodies via Grim’s C-terminus. Overall, we have discovered that the interplay between caspases and the proteasome system creates an intricate regulatory system for maintaining Grim at low levels in the cell under normal conditions. Once needed, Grim levels accumulate rapidly and induce cell death efficiently through IBM-dependent destruction of DIAP1 and derepression of caspases as well as through IBM-independent translational repression.