The SCFSkp2 ubiquitin ligase complex modulates TRAIL-R2-induced apoptosis by regulating FLIP(L)

Roberts, J. Z. et al. (2020) The SCFSkp2 ubiquitin ligase complex modulates TRAIL-R2-induced apoptosis by regulating FLIP(L). Cell Death and Differentiation, 27(9), pp. 2726-2741. (doi: 10.1038/s41418-020-0539-7) (PMID:32313199) (PMCID:PMC7429845)

Abstract

TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2’s pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1’s DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1’s recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)’s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.

• Item Type: Articles
• Additional Information: This work was funded by grants from The Wellcome Trust (110371/Z/15/Z), Cancer Research UK (C11884/A24387), Northern Ireland Department for the Economy (NI DfE) (SFI-DEL 14/1 A/2582) and a NI DfE studentship (JZR).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Riley, Dr Joel

Creator Roles

Riley, J. S.: Investigation

• Authors: Roberts, J. Z., Holohan, C., Sessler, T., Fox, J., Crawford, N., Riley, J. S., Khawaja, H., Majkut, J., Evergren, E., Humphreys, L. M., Ferris, J., Higgins, C., Espona-Fiedler, M., Moynagh, P., McDade, S. S., and Longley, D. B.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: Cell Death and Differentiation
• Publisher: Nature Research
• ISSN: 1350-9047
• ISSN (Online): 1476-5403
• Published Online: 20 April 2020
• Copyright Holders: Copyright © 2020 The Authors
• First Published: First published in Cell Death and Differentiation 27(9): 2726-2741
• Publisher Policy: Reproduced under a Creative Commons License