Rapid turnover of c-FLIP short is determined by its unique C-terminal tail

The caspase-8 inhibitor c-FLIP exists as two splice variants, c-FLIPL and c-FLIPS, 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-FLIPS was more prone to ubiquitylation and had a considerably shorter half-life. Analysis of the c-FLIPS-specific ubiquitylation revealed two lysines, 192 and 195, C-terminal to the death effector domains, as principal ubiquitin acceptors in c-FLIPS but not in c-FLIPL. Furthermore the c-FLIPS-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-FLIPS. 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-FLIPS was not affected by the tail deletion, the antiapoptotic activity and ubiquitin-mediated degradation of c-FLIPS are functionally and structurally independent processes. The presence of a small destabilizing sequence in c-FLIPS constitutes an important determinant of c-FLIPS/c-FLIPL ratios by allowing differential degradation of c-FLIP isoforms. The conformation-based predisposition of c-FLIPS to ubiquitin-mediated degradation introduces a novel concept to the regulation of the death-inducing signaling complex.