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

Depending on its expression levels, FLIP(L) can act as an activator or inhibitor of caspase-8 at the TRAIL-R2 death-inducing signalling complex (DISC). Here, we demonstrate that the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2) regulates FLIP(L) protein expression by regulating its ubiquitination, and, unusually, this is mediated by direct binding of FLIP(L) to Cullin-1 rather than via Skp2. The interaction of FLIP(L) with SCFSkp2 is significantly stronger with its DISC-processed p43-form, and 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 independently of 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 activation and apoptosis induction. These findings provide new insights into how FLIP(L) expression and TRAIL receptor signaling is controlled.