Regulation of FLIP(L) and TRAIL-R2 signalling by the SCF^Skp2Ubiquitin Ligase Complex

Abstract

Depending on its expression levels, the long splice form of the pseudo-caspase FLIP (FLIP(L)) can act as an inhibitor (high expression) or activator (low expression) of apoptosis induction by the TRAIL-R2 death-inducing signalling complex (DISC); its expression levels are therefore tightly regulated. Here, we demonstrate that the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCF Skp2 ) regulates the stability of FLIP(L) (but not the short splice form FLIP(S)), and, unusually, this is mediated by direct binding of FLIP(L) to Cullin-1 rather than via Skp2. By fine mapping the interaction of FLIP(L) with Cullin-1 to the large subunit of its pseudo-caspase domain, we found that the interaction is significantly stronger with FLIP(L)'s DISC-processed p43-form.Importantly, this interaction disrupts the ability of p43-FLIP to interact with FADD, caspase-8 and another DISC component, TRAF2. Moreover, we find that SCF Skp2 associates with TRAIL-R2 constitutively and does so independently of FLIP(L) and other canonical DISC components. Inhibition of Cullin-1 expression (using siRNA) or activity (using a NEDDylation inhibitor, MLN4924) enhanced FLIP(L) and TRAF2 levels at the TRAIL-R2 DISC and enhanced caspase-8 processing. This suggests that processing of FLIP(L) to p43-FLIP at the TRAIL-R2 DISC enhances its interaction with co-localised SCF Skp2 , leading to disruption of p43-FLIP's association with the DISC thereby altering caspase-8 processing. These findings provide important new insights into how FLIP(L) expression and TRAIL-R2 signaling is controlled. Regulation of FLIP(L) and TRAIL signalling by SCF Skp2JZ Roberts et al. 3 IntroductionProgrammed cell death (apoptosis) plays a key role in maintaining normal tissue homeostasis and preventing disease 1 . Apoptosis is orchestrated by a family of cysteine proteases, the caspases 2 . The multi-protein complexes formed following activation of the CD95 (Fas) and TRAIL-R1/R2 (DR4/DR5) death receptors by their ligands (FasL and TRAIL) expressed by immune effector cells and by 2 nd generation therapeutic agonists (ABBV621 and MEDI3039) is called the death-inducing signalling complex (DISC), consisting of the receptors, the adaptor molecule FADD, procaspase-8 and FLIP 3 .Recruitment into these complexes exposes FADD's N-terminal death effector domain (DED), which recruits procaspase-8 by interacting with its N-terminal tandem DEDs 4 . Procaspase-8 homodimerization results in conformational changes in its catalytic domains that lead to its activation 5 . Two main splice forms of FLIP have been identified in humans: a long form (FLIP(L)) and a short form (FLIP(S)), both of which contain tandem DEDs and can be recruited to the DISC and related complexes (such as TNFR1 Complex II and the ripoptosome) where they form heterodimers with procaspase-8 4 . The relative amounts of FLIP(S) and FLIP(L) recruited to these complexes is a key determinant of cell fate (survival, apoptosis or necroptosis) and whether FLIP(L) acts as an inhibitor or activator o...