Abstract
Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses1,2. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components3. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase4) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22 phox , the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22 phox is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.
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Acknowledgements
We thank M. Pasparakis for TRADD-deficient and TNFR1-deficient MEFs, R. Brandes for gp91-deficient mice and helpful discussions, and D. Männel for TNFR1 and TNFR2 doubly deficient MEFs and for recombinant TNF. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; SFB670 to O.K., J.C.B., O.U. and M.K., and SFB415 to S.S.), DFG grant 733/7-1 to S.S., and a grant from the DFG Leibniz programme to J.C.B. and M.K.
Author Contributions Experiments were performed by B.Y. (Figs 2 and 4, and Supplementary Figs 2b, 3a, b, 5, 6a, b and 8–11). K.W. originally cloned the RFK cDNA (Figs 1c, 2f–h and 3c, d, and Supplementary Figs 2b, 4, 7 and 9). V.T. and S.S. analysed TNF receptosomes (Figs 1a–e and 3a, b, and Supplementary Fig. 3c). O.K. planned experimental approaches (Supplementary Fig. 1). C.P. (Fig. 2c), M.S. and O.U. (Supplementary Figs 6c and 7) performed ROS and NO measurements in RFK-deficient cells. H.K. supervised the apoptosis assays. A.K., T.W. and J.C.B. contributed to the knockout strategy for the generation of RFK-deficient strain of mice. M.K. conceived the study, evaluated the experimental results and wrote the paper.
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Yazdanpanah, B., Wiegmann, K., Tchikov, V. et al. Riboflavin kinase couples TNF receptor 1 to NADPH oxidase. Nature 460, 1159–1163 (2009). https://doi.org/10.1038/nature08206
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DOI: https://doi.org/10.1038/nature08206
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