Abstract
The long FLIP splice form FLIP(L) can act as both an inhibitor and promoter of caspase‐8 at death‐inducing signalling complexes (DISCs) formed by death receptors such as TRAIL‐R2 and related intracellular complexes such as the ripoptosome. Herein, we describe a revised DISC assembly model that explains how FLIP(L) can have these opposite effects by defining the stoichiometry (with respect to caspase‐8) at which it converts from being anti‐ to pro‐apoptotic at the DISC. We also show that in the complete absence of FLIP(L), procaspase‐8 activation at the TRAIL‐R2 DISC has significantly slower kinetics, although ultimately the extent of apoptosis is significantly greater. This revised model of DISC assembly also explains why FLIP's recruitment to the TRAIL‐R2 DISC is impaired in the absence of caspase‐8 despite showing that it can interact with the DISC adaptor protein FADD and why the short FLIP splice form FLIP(S) is the more potent inhibitor of DISC‐mediated apoptosis.
Highlights
IntroductionApoptosis can be activated through the intrinsic (mitochondrialmediated) and extrinsic (death receptor-mediated) pathways and is frequently dysfunctional in cancer [1,2]
Apoptosis can be activated through the intrinsic and extrinsic pathways and is frequently dysfunctional in cancer [1,2]
We quantified the relative levels of FLIP, caspase-8 and FADD at the TRAIL-R2 death-inducing signalling complexes (DISCs) using recombinant protein standards [20]
Summary
Apoptosis can be activated through the intrinsic (mitochondrialmediated) and extrinsic (death receptor-mediated) pathways and is frequently dysfunctional in cancer [1,2]. Engagement of the FADD DD by the activated receptor enables its other domain, the death effector domain (DED), to engage in homotypic protein– protein interactions with other DED-containing proteins, most importantly the tandem DED-containing procaspase-8 (FADD-like interleukin-1b-converting enzyme, FLICE) and its regulator FLIP (FLICE-inhibitory protein) [8,9,10]. At this complex, known as the death-inducing signalling complex (DISC), procaspase-8 forms homodimers or heterodimers with FLIP via homotypic interactions between their DEDs. Homo-dimerisation is required for activation and processing of procaspase-8, which occurs in a two-step manner: rate-limiting inter-dimer cleavage between adjacent dimers recruited to the DISC that cleaves the linker between its large (p18) and small (p10) catalytic domains, followed by intra-dimer cleavage between the p18subunit and the DED-containing pro-domain. The 2nd cleavage step releases the active enzyme made up of a hetero-tetramer of two p18 and two p10 subunits from the DISC, which can propagate an apoptotic signal by cleaving downstream substrates such as BID and procaspases-3/7 [11,12,13,14,15,16]
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