Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate

The death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIP(L) only) or anti-apoptotic (c-FLIP(L)/c-FLIP(S)) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIP(L/S) binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIP(L/S) via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIP(L/S) to procaspase-8, which determines composition of the procaspase-8:c-FLIP(L/S) heterodimer. Thus, procaspase-8:c-FLIP(L) exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIP(S) lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIP(L) and crucially defines how c-FLIP isoforms differentially control cell fate.