F1Aα, a death receptor-binding protein homologous to the Caenorhabditis elegans sex-determining protein, FEM-1, is a caspase substrate that mediates apoptosis

Abstract

Apoptosis is an evolutionarily conserved process that is critical for tissue homeostasis and development including sex determination in essentially all multicellular organisms. Here, we report the cloning of an ankyrin repeat-containing protein, termed F1Aα, in a yeast two-hybrid screen using the cytoplasmic domain of Fas (CD95/APO-1) as bait. Amino acid sequence analysis indicates that F1Aα has extensive homology to the sex-determining protein FEM-1 of the Caenorhabditis elegans, which is required for the development of all aspects of the male phenotype. F1Aα associates with the cytoplasmic domains of Fas and tumor necrosis factor receptor 1, two prototype members of the 'death receptor' family. The F1Aα protein also oligomerizes. Overexpression of F1Aα induces apoptosis in mammalian cells, and co-expression of Bcl-XL or the dominant negative mutants of either FADD or caspase-9 blocks this effect. Deletion analysis revealed the center region of F1Aα, including a cluster of five ankyrin repeats to be necessary and sufficient for maximum apoptotic activity, and the N-terminal region appears to regulate negatively this activity. Furthermore, F1Aα is cleaved by a caspase-3-like protease at Asp342, and the cleavage-resistant mutant is unable to induce apoptosis upon overexpression. F1Aα is therefore a member of a growing family of death receptor-associated proteins that mediates apoptosis.