Temporal and spatial profiling of nuclei-associated proteins upon TNF-alpha/NF-kappaB signaling.
The tumor necrosis factor (TNF)-alpha/NF-kappaB-signaling pathway plays a pivotal role in various processes including apoptosis, cellular differentiation, host defense, inflammation, autoimmunity and organogenesis. The complexity of the TNF-alpha/NF-kappaB 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-alpha/NF-kappaB 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-alpha-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-alpha/NF-kappaB 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-alpha stimulation, which was further verified by western blotting and immunofluorescence analysis. This indicates that FANCD2 might be involved in TNF-alpha/NF-kappaB 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-alpha/NF-kappaB-signaling pathway, but also provided a systematic strategy for monitoring temporal and spatial changes in cell signaling.
Ma
Dan-jun
DJ
Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
Li
Su-Jun
SJ
Wang
Lian-Shui
LS
Dai
Jie
J
Zhao
Shi-lin
SL
Zeng
Rong
R
D000595:Amino Acid Sequence; D002460:Cell Line; D002853:Chromatography, Liquid; D030541:Databases, Genetic; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D040901:Proteomics; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D052236:Fanconi Anemia Complementation Group D2 Protein; D016328:NF-kappa B; D009687:Nuclear Proteins; D014409:Tumor Necrosis Factor-alpha