Ligand binding to tumor necrosis factor receptor 1 (TNFR1) stimulates production of reactive oxygen species (ROS) through the activation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1), which contains the cofactor FAD (flavin adenine dinucleotide). Because the mechanism by which TNFR1 couples to Nox1 had not been completely elucidated, Yazdanpanah et al. performed a yeast two-hybrid screen using a portion of the death domain (DD) of TNFR1 as bait and identified riboflavin kinase (RFK), which converts riboflavin to flavin mononucleotide (FMN) and FAD. RFK coimmunoprecipitated with TNFR1 from HeLa cells treated with TNF, an interaction that required the death domain of TNFR1 and was facilitated by the adaptor protein TRADD (TNF receptor–associated death domain). ROS production in response to TNF was reduced in HeLa cells stably expressing a short hairpin RNA (shRNA) against RFK (HeLashRFK) compared with those that expressed a control shRNA (HeLashScr). TNF-induced ROS production required the kinase activity of RFK, because decreased ROS production in HeLashRFK cells was rescued with wild-type RFK but not a kinase-dead RFK mutant. Furthermore, HeLashRFK cells supplemented with FMN and FAD produced similar amounts of ROS to TNF-stimulated HeLashScr cells. In TNF-treated HeLashScr cells, various components of the NADPH oxidase complex, such as p22phox, Nox1, and Nox2, as well as the guanosine triphosphatase (GTPase) Rac1 (an activator of NADPH oxidase), were recruited to TNFR1 receptosomes; this recruitment was not observed in HeLashRFK cells. Because the activity of RFK increased in cells exposed to TNF, TNF might prime NADPH oxidase by inducing FAD synthesis. Accordingly, HeLashScr cells produced more ROS in response to the NADPH oxidase activator TPA (tetradecanoylphorbol-13-acetate) when pre-incubated with TNF, and the production of ROS in TPA-treated HeLashRFK cells was maximal when the cells were supplemented with FMN or FAD. Thus, RFK plays two key roles in the production of ROS by the TNFR1 receptosome: It produces FAD and FMN in a TNF-dependent manner and physically links NADPH oxidase to TNFR1.
B. Yazdanpanah, K. Wiegmann, V. Tchikov, O. Krut, C. Pongratz, M. Schramm, A. Kleinridders, T. Wunderlich, H. Kashkar, O. Utermöhlen, J. C. Brüning, S. Schütze, M. Krönke, Riboflavin kinase couples TNF receptor 1 to NADPH oxidase. Nature 460, 1159–1163 (2009). [PubMed]