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-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-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-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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.