title: Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly.
abstract: Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: J
authors.initials: J
authors.lastname: Majkut
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.|Now at Department of Bioengineering and Therapeutic Sciences, University of California.
authors.forename: M
authors.initials: M
authors.lastname: Sgobba
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: C
authors.initials: C
authors.lastname: Holohan
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: N
authors.initials: N
authors.lastname: Crawford
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: A E
authors.initials: AE
authors.lastname: Logan
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: E
authors.initials: E
authors.lastname: Kerr
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: C A
authors.initials: CA
authors.lastname: Higgins
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: K L
authors.initials: KL
authors.lastname: Redmond
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: J S
authors.initials: JS
authors.lastname: Riley
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: I
authors.initials: I
authors.lastname: Stasik
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.|Now at Cancer Studies and Molecular Medicine, University of Leicester, UK.
authors.forename: D A
authors.initials: DA
authors.lastname: Fennell
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: S
authors.initials: S
authors.lastname: Van Schaeybroeck
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.|Now at School of Pharmacy, University College London.
authors.forename: S
authors.initials: S
authors.lastname: Haider
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: P G
authors.initials: PG
authors.lastname: Johnston
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: D
authors.initials: D
authors.lastname: Haigh
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK.
authors.forename: D B
authors.initials: DB
authors.lastname: Longley
mesh_terms: D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism*; D002850:Chromatography, Gel; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism*; D045325:HCT116 Cells; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D053181:Caspase 8