Regulation of FLIP(L) and TRAIL-R2 signalling by the SCFSkp2 Ubiquitin Ligase Complex

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 (SCFSkp2) 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 SCFSkp2 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 SCFSkp2, leading to disruption of p43-FLIPs 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.