Regulation of Fas-associated death domain interactions by the death effector domain identified by a modified reverse two-hybrid screen.
The adapter protein FADD consists of two protein interaction domains and is an essential component of the death inducing signaling complex (DISC) that is formed by activated death receptors of the tumor necrosis factor (TNF) receptor family. The FADD death domain binds to activated receptors such as Fas or other adapters such as TRADD, whereas the FADD death effector domain binds to procaspase 8. Each domain can interact with its target in the absence of the other domain, and this has led to the idea that the two domains function independently. FADD death domain interactions with Fas and TRADD are thought to occur on the same surface; however, the regulation of these interactions is poorly understood. We developed a modified reverse two-hybrid method that can identify mutations, which inhibit some protein-protein interactions without affecting other interactions. Using this method, we identified mutations in FADD that prevent binding to Fas but do not affect binding to TRADD. Surprisingly, these mutations were in the death effector domain rather than the death domain. To test whether the mutants function in mammalian cells, we expressed wild type or mutant FADD molecules in FADD-deficient cells. Wild type FADD rescued both Fas ligand- and TNF-dependent signaling, whereas the FADD death effector domain mutants rescued only TNF signaling. These data indicate that in contrast to current models, the death effector domain of FADD is involved in interaction with Fas.
Thomas
Lance R
LR
Department of Cancer Biology, Comprehensive Cancer Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA.
Stillman
David J
DJ
Thorburn
Andrew
A
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D019169:Jurkat Cells; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology; D047888:Receptors, Tumor Necrosis Factor, Type I; D013329:Structure-Activity Relationship; D047990:TNF Receptor-Associated Factor 1; D020798:Two-Hybrid System Techniques; D019014:fas Receptor / Q000737:chemistry* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D002352:Carrier Proteins; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D011506:Proteins; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047990:TNF Receptor-Associated Factor 1; D019014:fas Receptor