Quantitative phosphoproteomic analysis of RIP3-dependent protein phosphorylation in the course of TNF-induced necroptosis.
Tumor necrosis factor (TNF) induced cell death in murine fibrosarcoma L929 cells is a model system in studying programed necrosis (also known as necroptosis). Receptor interacting protein 3 (RIP3), a serine-threonine kinase, is known to play an essential role in TNF-induced necroptosis; however, the phosphorylation events initiated by RIP3 activation in necroptotic process is still largely unknown. Here, we performed a quantitative MS based analysis to compare TNF-induced changes in the global phosphoproteome of wild-type (RIP3(+/+) ) and RIP3-knockdown L929 cells at different time points after TNF treatment. A total of 8058 phosphopeptides spanning 6892 phosphorylation sites in 2762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 h. By comparing the phosphorylation sites in wild-type and RIP3-knockdown L929 cells, 174, 167, and 177 distinct phosphorylation sites were revealed to be dependent on RIP3 at the 0.5, 2, and 4 h time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3-dependent phosphorylation in lipopolysaccharide-stimulated peritoneal macrophages and TNF-treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report are highly relevant to the study of TNF-induced necroptosis of L929 cells.
State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, P. R. China.
Chuan-Qi
CQ
Zhong
Yuanyue
Y
Li
Daowei
D
Yang
Na
N
Zhang
Xiaozheng
X
Xu
Yaying
Y
Wu
Jinan
J
Chen
Jiahuai
J
Han
D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques; D008070:Lipopolysaccharides / Q000276:immunology; D008264:Macrophages / Q000166:cytology / Q000276:immunology; D013058:Mass Spectrometry; D051379:Mice; D009336:Necrosis; D010748:Phosphopeptides / Q000032:analysis* / Q000276:immunology; D010750:Phosphoproteins / Q000032:analysis* / Q000276:immunology; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis* / Q000276:immunology; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D008070:Lipopolysaccharides; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; D014409:Tumor Necrosis Factor-alpha; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse
Cell biology; Necroptosis; Phosphoproteomics; iTRAQ