Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail.
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.
Poukkula
Minna
M
Turku Centre for Biotechnology, University of Turku and Abo Akademi University, FIN-20521 Turku.
Kaunisto
Aura
A
Hietakangas
Ville
V
Denessiouk
Konstantin
K
Katajamäki
Tuire
T
Johnson
Mark S
MS
Sistonen
Lea
L
Eriksson
John E
JE
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
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