Fas-associated Factor 1, FAF1, is a member of fas death-inducing signaling complex

Authors:
Ryu SW, Lee SJ, Park MY, Jun JI, Jung YK and Kim E
In:
J Biol Chem (2003) 278(26): 24003-24010
Experiment
The authors studied Fas-receptor mediated apoptosis in JURKAT cells. The role of FAF1 as a Fas-binding protein in apoptotic execution is not established. It was demonstrated that FAF1 overexpression in JURKAT cells caused significant apoptotic death. To know if the DEDID domain (death effector domain-interacting domain) performs critical functions in Fas-induced apoptosis JURKAT cells were nucleofected with a FAF1-deltaDEDID deletion mutant. After 30 hours cells were treated with anti-Fas antibody and stained with DAPI. The number of apoptotic cells was determined by evaluating nuclear morphology. The deletion mutant failed to induce apoptosis indicating that DEDID is essential in mediating Fas-induced apoptosis.
Abstract
FAF1 has been introduced as a Fas-binding protein. However, the function of FAF1 in apoptotic execution is not established. Based on the fact that FAF1 is a Fas-binding protein, we asked if FAF1 interacted with other members of the Fas-death-inducing signaling complex (Fas-DISC) such as Fas-associated death domain protein (FADD) and caspase-8. FAF1 could interact with caspase-8 and FADD in vivo as well as in vitro. The death effector domains (DEDs) of caspase-8 and FADD interacted with the amino acid 181-381 region of FAF1, previously known to have apoptotic potential. Considering that FAF1 directly binds to Fas and caspase-8, FAF1 shows similar protein-interacting characteristics to that of FADD. In the coimmunoprecipitation with an anti-Fas antibody (APO-1) in Jurkat cells, endogenous FAF1 was associated with the precipitates in which caspase-8 was present. By confocal microscopic analysis, both Fas and FAF1 were detected in the cytoplasmic membrane before Fas activation, and in the cytoplasm after Fas activation. FADD and caspase-8 colocalized with Fas in Jurkat cells validating the presence of FAF1 in the authentic Fas-DISC. Overexpression of FAF1 in Jurkat cells caused significant apoptotic death. In addition, the FAF1 deletion mutant lacking the N terminus where Fas, FADD, and caspase-8 interact protected Jurkat cells from Fas-induced apoptosis demonstrating dominant-negative phenotype. Cell death by overexpression of FAF1 was suppressed significantly in both FADD- and caspase-8-deficient Jurkat cells when compared with that in their parental Jurkat cells. Collectively, our data show that FAF1 is a member of Fas-DISC acting upstream of caspase-8.