Fas-associated factor 1, FAF1, is a member of Fas death-inducing signaling complex.
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.
Ryu
Seung-Wook
SW
Research Center for Biomedicinal Resources and Division of Life Science, PaiChai University, Daejeon 302-735, Korea.
Lee
Soo-Jin
SJ
Park
Min-Young
MY
Jun
Joon-Il
JI
Jung
Yong-Keun
YK
Kim
Eunhee
E
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D053418:Death Domain Receptor Signaling Adaptor Proteins; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D053418:Death Domain Receptor Signaling Adaptor Proteins; C505533:FADD protein, human; C490780:FAF1 protein, human; D053200:Fas-Associated Death Domain Protein; D018124:Receptors, Tumor Necrosis Factor; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases