Temporal and spatial profiling of nuclei-associated proteins upon TNF-α/NF-κB signaling

The tumor necrosis factor (TNF)-α/NF-κB-signaling pathway plays a pivotal role in various processes including apoptosis, cellular differentiation, host defense, inflammation, autoimmunity and organogenesis. The complexity of the TNF-α/NF-κB signaling is in part due to the dynamic protein behaviors of key players in this pathway. In this present work, a dynamic and global view of the signaling components in the nucleus at the early stages of TNF-α/NF-κB signaling was obtained in HEK293 cells, by a combination of subcellular fractionation and stable isotope labeling by amino acids in cell culture (SILAC). The dynamic profile patterns of 547 TNF-α-induced nuclei-associated proteins were quantified in our studies. The functional characters of all the profiles were further analyzed using that Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. Additionally, many previously unknown effectors of TNF-α/NF-κB signaling were identified, quantified and clustered into differential activation profiles. Interestingly, levels of Fanconi anemia group D2 protein (FANCD2), one of the Fanconi anemia family proteins, was found to be increased in the nucleus by SILAC quantitation upon TNF-α stimulation, which was further verified by western blotting and immunofluorescence analysis. This indicates that FANCD2 might be involved in TNF-α/NF-κB signaling through its accumulation in the nucleus. In summary, the combination of subcellular proteomics with quantitative analysis not only allowed for a dissection of the nuclear TNF-α/NF-κB-signaling pathway, but also provided a systematic strategy for monitoring temporal and spatial changes in cell signaling.