The Death Domain of FADD Is Essential for Embryogenesis, Lymphocyte Development, and Proliferation

Hongxia Z. Imtiyaz, Xiaohui Zhou, Haibing Zhang, Dehua Chen, Taishan Hu, Jianke Zhang
2009 Journal of Biological Chemistry  
The Fas-associated death domain-containing protein (FADD) is an adaptor for relaying apoptotic signals initiated by death receptors such as Fas. Whereas a lack of death receptors has no effect on mouse development, FADD deficiency results in early embryonic lethality, indicating that FADD has additional functions independent of death receptors. We have previously shown that conditional deletion of FADD not only impairs apoptosis but also leads to defective lymphocyte proliferation. The
more » ... otic signaling mediated by FADD remains poorly understood. Earlier studies have suggested that FADD carboxyl terminal serine phosphorylation likely plays a role in FADDmediated proliferation signaling in T cells. The FADD death domain is presumably only required for apoptotic signaling, as it interacts with death receptors which are dispensable during embryonic development and lymphocyte proliferation. To test this hypothesis, we have performed mutational analyses of the FADD death domain and identified a mutant, R117Q, which lacks binding to Fas and, thus, is incapable of apoptotic signaling in cell lines. Unexpectedly, this death domain point mutation disrupted mouse embryonic development as shown by in vivo functional reconstitution analyses. Interestingly, a second FADD death domain mutant, V121N, retained normal Fas binding and apoptotic signaling ability but also failed to support mouse development. Furthermore, lymphocyte proliferation responses were impaired by V121N. This reverse genetic study has revealed a previously unappreciated role of the FADD death domain, which likely functions as a molecular switch regulating two distinct signals leading to apoptosis and cell proliferation and is critical for embryogenesis, lymphocyte development, and proliferation. The death domain (DD) 3 was initially identified during mutational studies of the pro-apoptotic receptors Fas and TNF-R1, also known as death receptors (DRs) (1, 2). Amino acid replacements targeted at the DD, a stretch of about 80 amino acids within the intracellular sequences of Fas and TNF-R1, abrogate cell death signaling induced by these two DRs. Sequence homology searches led to the identification of additional members of the DR family, including DR3, DR4 (TRAIL-R1), DR5 (TRAIL-R2), and DR6, which also contain an intracellular DD. The physiological function of DRs has been investigated by analyses of humans and animals carrying mutations in the DR genes. Mutations in the DD of Fas lead to lymphoproliferation (lpr) and autoimmune diseases (3, 4) and also result in predisposition to skin and lymphoid malignancy (5-7). TNF-R1-deficient mice are resistant to endotoxic shock but are highly susceptible to pathogens (8, 9) . DR3 Ϫ/Ϫ mice have no obvious abnormalities except compromised thymic negative selection (10). DR4/5 Ϫ/Ϫ mice have a normal lymphoid system but show enhanced innate immune responses to viral infection (11). Blocking the binding of TRAIL in mice with soluble DR5 enhances autoimmune inflammation and cell cycle progression in lymphocytes (12). TRAIL Ϫ/Ϫ mice are susceptible to induced and spontaneous tumors as well as metastasis of engrafted tumors (13-15). FADD (or Mort1) was initially identified as an adaptor for Fas-induced apoptosis (16 -18) and was later shown to be involved in apoptotic pathways initiated by TNF-R1, DR3, and TRAIL-Rs (19 -28). The adaptor protein, TRADD, is involved in the TNF-R1 signaling pathway and has been shown to associate with FADD (29). There is a DD-like sequence at the carboxyl proximal region of FADD that interacts with the DDs of DRs and TRADD. FADD contains a second protein interaction structure at the NH 2 terminus, called the death effector domain (DED), that binds to the DED present in the pro-domain of caspase 8 (FLICE or MACH) (30, 31). Gene-targeting studies have revealed a novel function of FADD essential for embryonic development (25, 32, 33) . FADD Ϫ/Ϫ and caspase 8 Ϫ/Ϫ mice die midgestation, whereas mice lacking individual DRs have no obvious defects regarding embryonic development. Analyses of conditional mutant mice demonstrated that lymphocytes lacking either FADD or caspase 8 are not only defective in apoptosis induced by Fas but also impaired in proliferative responses induced by the T cell antigen receptor and Toll-like receptors (TLRs) (34 -38). Overexpression of a fragment of FADD that contains the DD but lacks the DED blocks both apoptosis and proliferation in T cells (16, 18, 39 -41). During development cell death is required for proper organogenesis and generation of complex multicellular tissues (42, 43) . In adults, homeostasis in the lymphoid system and other
doi:10.1074/jbc.m900249200 pmid:19203997 pmcid:PMC2665115 fatcat:ay36jhfkgfbdxiscuuzcbhjnvq