Analysis of the death-domain containing protein FADD: Novel functions, interactions and mechanisms of regulation

Vertebrate innate immunity provides a first line of defense against invading pathogens. Virus infection can activate a potent innate immune response by mechanisms thought to be triggered by intracellular double-stranded (ds) RNA, produced during viral replication, resulting in the production of interferon (IFN). The IFN-inducible, dsRNA-dependent protein kinase (PKR) plays a role in innate immunity by triggering apoptosis in response to intracellular dsRNA and infection with certain viruses. Our lab has previously shown that this apoptotic cell death occurs through a Fas-associated death domain protein (FADD) dependent pathway. FADD, a cytoplasmic adapter molecule essential for embryonic development, links the family of death receptors to the activation of caspases in cells undergoing ligand-induced cell death.In order to identify proteins that may regulate FADD function and possibly elucidate novel roles through the discovery of new interactions, a yeast two-hybrid screen was performed using the death effector domain (DED) of FADD as bait. Subsequently, we identified a novel interaction between FADD and a newly described inhibitor of the signal transducer and activator of transcription 1 (STAT1) referred to as protein inhibitor of activated STAT1 (PIAS1). Both of these proteins have been implicated in diverse cellular processes such as apoptosis and cytokine signaling.While examining the interaction between FADD and PIAS1, we observed that FADD-deficient mammalian cells are very susceptible to virus infection. Subsequent analysis indicated that although IFN-mediated Janus kinase (JAK)/STAT pathways were functional in FADD-deficient mouse embryonic fibroblasts (MEFs), intracellular dsRNA/virus-activated gene expression, including production of type I (alpha/beta) IFNs was severely defective. The signaling pathway incorporating FADD is largely independent of toll-like receptor (TLR) 3, tumour necrosis factor receptor associate factor (TRAF) 6, PKR and caspase activation but appears to require receptor interacting protein (RIP) 1 and tank binding kinase (TBK)-1/I-kappaB kinase (IKK)delta-mediated activation of the interferon regulatory factor (IRF) 3.The requirement for FADD in TLR-independent mammalian innate immune responses is evocative of the IMD innate immune signaling pathway in Drosophila. This pathway is dependent upon the Drosophila homologues of FADD- and RIP1/IMD and encompasses a signaling cascade that responds to bacterial infection by activating the transcription of anti-microbial genes. In conclusion, our data demonstrates the existence of a conserved FADD-dependent virus recognition pathway in mammalian cells that is essential for the optimal induction of type I IFNs and other genes important for host defense