Quantitative phosphoproteomic analysis of RIP3-dependent protein phosphorylation in the course of TNF-induced
Chuan-Qi Zhong1, Yuanyue Li, Daowei Yang
1State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, P. R. China.
Abstract:
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.
Insights
This study identifies novel RIP3-dependent phosphorylation sites crucial for tumor necrosis factor (TNF)-induced necroptosis in L929 cells. These findings advance our understanding of programmed cell death mechanisms.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Tumor necrosis factor (TNF) induces programmed cell death, known as necroptosis.
- Receptor interacting protein 3 (RIP3) is essential for TNF-induced necroptosis, but its downstream phosphorylation events remain largely uncharacterized.
Purpose of the Study:
- To investigate the global phosphoproteome changes induced by TNF in L929 cells with and without RIP3.
- To identify RIP3-dependent phosphorylation sites involved in TNF-induced necroptosis.
Main Methods:
- Quantitative mass spectrometry (MS)-based phosphoproteomic analysis.
- Comparison of wild-type (RIP3(+/+)) and RIP3-knockdown L929 cells treated with TNF over time (0.5, 2, and 4 hours).
Main Results:
- Identification of 8058 phosphopeptides, 6892 phosphorylation sites, and 2762 proteins.
- Discovery of 174, 167, and 177 distinct RIP3-dependent phosphorylation sites at 0.5, 2, and 4 hours post-TNF treatment, respectively.
- Most identified sites were novel compared to previous studies in macrophages and fibroblasts.
Conclusions:
- This study provides a comprehensive map of RIP3-dependent phosphorylation in TNF-induced necroptosis in L929 cells.
- The identified phosphorylation sites offer new targets for understanding necroptosis regulation and potential therapeutic interventions.
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