title: Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail.
abstract: The caspase-8 inhibitor c-FLIP exists as two splice variants, c-FLIP(L) and c-FLIP(S), with distinct roles in death receptor signaling. The mechanisms determining their turnover have not been established. We found that in differentiating K562 erythroleukemia cells both c-FLIP isoforms were inducibly degraded by the proteasome, but c-FLIP(S) was more prone to ubiquitylation and had a considerably shorter half-life. Analysis of the c-FLIP(S)-specific ubiquitylation revealed two lysines, 192 and 195, C-terminal to the death effector domains, as principal ubiquitin acceptors in c-FLIP(S) but not in c-FLIP(L). Furthermore the c-FLIP(S)-specific tail of 19 amino acids, adjacent to the two target lysines, was demonstrated to be the key element determining the isoform-specific instability of c-FLIP(S). Molecular modeling in combination with site-directed mutagenesis demonstrated that the C-terminal tail is required for correct positioning and subsequent ubiquitylation of the target lysines. Because the antiapoptotic operation of c-FLIP(S) was not affected by the tail deletion, the antiapoptotic activity and ubiquitin-mediated degradation of c-FLIP(S) are functionally and structurally independent processes. The presence of a small destabilizing sequence in c-FLIP(S) constitutes an important determinant of c-FLIP(S)/c-FLIP(L) ratios by allowing differential degradation of c-FLIP isoforms. The conformation-based predisposition of c-FLIP(S) to ubiquitin-mediated degradation introduces a novel concept to the regulation of the death-inducing signaling complex.
authors.affiliation: Turku Centre for Biotechnology, University of Turku and Abo Akademi University, FIN-20521 Turku.
authors.forename: Minna
authors.initials: M
authors.lastname: Poukkula
authors.forename: Aura
authors.initials: A
authors.lastname: Kaunisto
authors.forename: Ville
authors.initials: V
authors.lastname: Hietakangas
authors.forename: Konstantin
authors.initials: K
authors.lastname: Denessiouk
authors.forename: Tuire
authors.initials: T
authors.lastname: Katajamäki
authors.forename: Mark S
authors.initials: MS
authors.lastname: Johnson
authors.forename: Lea
authors.initials: L
authors.lastname: Sistonen
authors.forename: John E
authors.initials: JE
authors.lastname: Eriksson
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D001665:Binding Sites; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D045744:Cell Line, Tumor; D006207:Half-Life; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008660:Metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D047908:Intracellular Signaling Peptides and Proteins; D020033:Protein Isoforms; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex