title: Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate.
abstract: 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-FLIPL only) or anti-apoptotic (c-FLIPL/c-FLIPS) 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-FLIPL/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-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Hughes
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Powley
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Rebekah
authors.initials: R
authors.lastname: Jukes-Jones
authors.affiliation: Department of Dermatology, Venereology and Allergology, Medical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Horn
authors.affiliation: Department of Dermatology and Allergology, Medical Faculty of the RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Feoktistova
authors.affiliation: Henry Wellcome Laboratories of Structural Biology, Department of Molecular and Cell Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Louise
authors.initials: L
authors.lastname: Fairall
authors.affiliation: Henry Wellcome Laboratories of Structural Biology, Department of Molecular and Cell Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: John W R
authors.initials: JW
authors.lastname: Schwabe
authors.affiliation: Department of Dermatology and Allergology, Medical Faculty of the RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Leverkus
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, UK. Electronic address: kc5@le.ac.uk.
authors.forename: Kelvin
authors.initials: K
authors.lastname: Cain
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, UK. Electronic address: mm21@le.ac.uk.
authors.forename: Marion
authors.initials: M
authors.lastname: MacFarlane
mesh_terms: D017209:Apoptosis / Q000235:genetics; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics* / Q000378:metabolism; D053181:Caspase 8 / Q000235:genetics* / Q000378:metabolism; D019070:Cell Lineage / Q000235:genetics*; D053200:Fas-Associated Death Domain Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D020033:Protein Isoforms; D053181:Caspase 8