Stat1 as a Component of Tumor Necrosis Factor Alpha Receptor 1-TRADD Signaling Complex To Inhibit NF-kB Activation

Activated tumor necrosis factor alpha (TNF-a) receptor 1 (TNFR1) recruits TNFR1-associated death domain protein (TRADD), which in turn triggers two opposite signaling pathways leading to caspase activation for apoptosis induction and NF-kB activation for antiapoptosis gene upregulation. Here we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as determined by employing a novel antibody array screening method. In HeLa cells, Stat1 was associated with TNFR1 and this association was increased with TNF-a treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-a-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRADD but not with FADD. Interaction between Stat1 and receptor-interacting protein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examination of Stat1-deficient cells showed an apparent increase in TNF-a-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-a-mediated I-kB degradation and NF-kB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-kB activation by TNF-a. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-kB activation.