Rapid Turnover of c-FLIPshort Is Determined by Its Unique C-terminal Tail

J Biol Chem. 2005 Jul 22;280(29):27345-55. doi: 10.1074/jbc.M504019200. Epub 2005 May 10.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Apoptosis
  • Binding Sites
  • CASP8 and FADD-Like Apoptosis Regulating Protein
  • Cell Line, Tumor
  • Half-Life
  • Humans
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Kinetics
  • Metabolism
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Isoforms / metabolism
  • Transfection
  • Ubiquitin / metabolism

Substances

  • CASP8 and FADD-Like Apoptosis Regulating Protein
  • CFLAR protein, human
  • Intracellular Signaling Peptides and Proteins
  • Protein Isoforms
  • Ubiquitin
  • Proteasome Endopeptidase Complex