Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappa B activation

Activated tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) recrui ts TNFR1-associated death domain protein (TRADD), which in turn triggers tw o opposite signaling pathways leading to caspase activation for apoptosis i nduction and NF-kappa B activation for antiapoptosis gene upregulation. Her e we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as d etermined by employing a novel antibody array screening method. In HeLa cel ls, Stat1 was associated with TNFR1 and this association was increased with TNF-alpha treatment. TNFR1 signaling factors TRADD and Fas-associated deat h domain protein (FADD) were also found to interact with Stat1 in a TNF-alp ha-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRAD D but not with FADD. Interaction between Stat1 and receptor-interactimg pro tein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examinatio n of Stat1-deficient cells showed an apparent increase in TNF-alpha-induced TRADD-RIP and TRADD-TRAF2, complex formation, while interaction between TR ADD and FADD was unaffected. As a consequence, TNF-alpha-mediated I-kappa B degradation and NF-kappa B activation were markedly enhanced in Stat1-defi cient cells, whereas overexpression of Stat1 in 293T cells blocked NF-kappa B activation by TNF-alpha. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-kappa B activation.