Quantitative Phosphoproteomic Analysis of RIP3‐dependent Protein Phosphorylation in the Course of TNF‐induced Necroptosis (LB175)

Abstract

Tumor necrosis factor (TNF)‐induced cell death in murine fibrosarcoma L929 cells is a model system in studying programmed 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 the necroptotic process is still largely unknown. Here, we performed a quantitative mass spectrometry‐based analysis to compare TNF‐induced changes in the global phosphoproteome of wildtype (RIP3+/+) and RIP3‐knockdown L929 cells at different time points after TNF treatment. A total of 8,058 phosphopeptides spanning 6,892 phosphorylation sites in 2,762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 hours, respectively. By comparing the phosphorylation sites in wildtype 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 hour time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3 dependent phosphorylation in LPS‐stimulated peritoneal macrophages and TNF‐treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report is highly relevant to the study of TNF‐induced necroptosis of L929 cells.