The apoptosis-signaling adaptor molecule (FADD) plays multiple roles in the immune system. Although FADD was initially characterized for its critical role in mediating apoptotic signals from the DRs, it has since become obvious that FADD is not a functionally one-dimensional molecule. FADD has been implicated in innate immune responses, proliferation and/or cell cycle regulation of lymphoid and nonlymphoid cells, and necroptotic and autophagic signaling. Therefore, it appears FADD is a multifunctional protein that has various roles that may or may not be dependent on its interaction with the death receptors.
FADD is indispensable for proper lymphocyte proliferation
FADD is expressed at high levels in most tissues and organs and germ line deletion leads to embryonic lethality by day 11.5. Various groups have thus relied on alternate methods to assess the in vivo role of FADD. Surprisingly, these mouse models have suggested an additional, apoptosis-opposing role for FADD: FADD is an important mediator of proliferation.
Cell cycle regulation by FADD
FADD's effect on proliferation is not at the level of proximal receptor signaling. Researchers have found intact NFκB and MAPK signaling pathways, activation marker upregulation, as well as early cytokine production, in mice lacking FADD function in T cells. Although the exact mechanism of FADD's role in lymphocyte proliferation remains elusive, one hypothesis is that FADD regulates proliferation at the level of the cell cycle. Furthermore, phosphorylation and localization may help FADD to compartmentalize its apoptotic and non-apoptotic functions.
Mouse FADD (205 amino acids) and human FADD (208 amino acids) are highly homologous, sharing 68% identity and 80% similarity. Mouse FADD is phosphorylated on serine 191 and currently unknown threonine residues. Human FADD is phosphorylated solely at serine 194, which is the equivalent site to mouse FADD 191. Evidence has been shown for both mouse and human FADD that this particular serine is important for its role in proliferation. FADD knockout mice reconstituted with a transgene that expresses FADD with a serine-to-aspartic acid mutation at amino acid 191 show defects in proliferation and regulation of cell cycle. This mutant form of FADD, FADD-S191D, mimics constitutively phosphorylated FADD. In contrast, transgenic mice harboring a FADD mutation from serine to alanine at this same site (FADD-S191A), mimicking the underphosphorylated form of FADD, are phenotypically normal. FADD-S191D transgenic mice are anemic and exhibit characteristics associated with impairment of proliferation, including decreased thymocyte numbers resulting from a defect at the DN3 to DN4 transition, impaired peripheral T cell expansion, and limited progression into cell cycle after stimulation. Importantly, FADD-S191D T cells are still able to undergo normal Fas-induced death. Thus, the C-terminal tail of FADD, which contains the serine phosphorylation site, may represent a proliferation domain.
Human FADD also appears to be dependent upon this serine site for proper cell cycle regulation in nonlymphoid cells. In cell lines, serine 194 phosphorylation is highest during G2/M phases of cell cycle and lowest during Gl/S, with CKIa being responsible for phosphorylation of FADD at G2/M. In addition, we have shown CKIα to be responsible for phosphorylating mouse FADD at G2/M in primary T cells. Constitutive phosphorylation of FADD at this site, mimicked by a S194E mutation, renders cells more susceptible to G2/M cell cycle arrest by chemotherapeutic agents. Thus, the phosphorylation status of FADD may control the sensitivity of cells to death during cell cycle progression. In support of this, FADD has been shown to interact with MBD4, a DNA repair and genome surveillance protein.
Human FADD contains a nuclear localization sequence (NLS) and a nuclear export sequence (NES) within its DED, both of which we have found to be conserved within mouse FADD. Thus, FADD is not solely a cytoplasmic protein, as being a member of the DISC may otherwise suggest. While mutation of the human FADD NES confines FADD to the nucleus in various cell lines and reduces the efficacy of DR-induced apoptosis, export from the nucleus is dependent upon exportin-5 and requires an intact serine 194 phosphorylation site.
Interestingly, the status of FADD phosphorylation and subcellular localization are associated with various cancers and correlate with their prognosis for treatment. Thus, FADD phosphorylation and localization appear to be coupled and their dissection potentially represents our understanding of FADD's role in proliferation.
FADD and otherforms of cellular death
Necroptosis is induced through death receptor (DR) ligation in the absence of caspase function, suggesting that the apoptotic pathway normally supercedes this process in many systems. However, in the case of damage induced by stroke, necroptosis occurs in the presence of intact apoptotic machinery. In Jurkat cell lines and activated primary T cells which lack caspase function, FasL triggering of Fas can induce necroptosis, which appears to be dependent on FADD. Likewise, TRAIL-R mediated necroptosis also appears to rely on FADD. However, the role for FADD in TNFRl-signaled necroptosis is more controversial. FADD deficiency sensitizes Jurkat T cells to necroptosis induced by TNFα, whereas FADD-/- mouse embryonic fibroblasts require FADD for TNFα induced necroptosis.
FADD has also been implicated has having a role in autophagy. Autophagy is generally a survival mechanism whereby organelles and cytosol are recycled to the lysosome for degradation under various cellular stresses. Under certain circumstances, however, hyperautophagy can result in autophagic cell death, or death that coincides with autophagy. In an IFNγ model of autophagic cell death, Atg5, a critical component for autophagosome formation, was found to induce death through interaction with FADD. More recently, FADD has been suggested to be an inhibitor of ATG5-mediated hyperautophagy during T cell proliferation. However, hyperautophagy has been linked to both apoptosis and necroptosis, and whether it is a cause or a consequence of programmed cell death remains controversial.