Quantitative phosphoproteomic analysis of RIP3-dependent protein phosphorylation in the course of TNF-induced necroptosis

Proteomics. 2014 Mar;14(6):713-24. doi: 10.1002/pmic.201300326. Epub 2014 Feb 20.


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.

Keywords: Cell biology; Necroptosis; Phosphoproteomics; iTRAQ.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line, Tumor
  • Gene Knockdown Techniques
  • Lipopolysaccharides / immunology
  • Macrophages / cytology
  • Macrophages / immunology
  • Mass Spectrometry
  • Mice
  • Necrosis*
  • Phosphopeptides / analysis*
  • Phosphopeptides / immunology
  • Phosphoproteins / analysis*
  • Phosphoproteins / immunology
  • Phosphorylation
  • Proteome / analysis*
  • Proteome / immunology
  • Receptor-Interacting Protein Serine-Threonine Kinases / genetics
  • Receptor-Interacting Protein Serine-Threonine Kinases / immunology*
  • Tumor Necrosis Factor-alpha / immunology*


  • Lipopolysaccharides
  • Phosphopeptides
  • Phosphoproteins
  • Proteome
  • Tumor Necrosis Factor-alpha
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk3 protein, mouse