Sep 4, 2012

Investigation of receptor interacting protein (RIP3)-dependent protein phosphorylation by quantitative phosphoproteomics

Molecular & Cellular Proteomics : MCP
Xiurong WuChuan-qi Zhong

Abstract

Receptor interacting protein 3 (RIP3) is a protein kinase that plays a key role in programmed necrosis. Despite the importance of RIP3-dependent necrosis in many pathological processes, current knowledge on the function of RIP3 is very limited. Here we present the results of a proteome-wide analysis of RIP3-regulated phosphorylation sites using cells from wildtype (RIP3(+/+)) and RIP3 knockout (RIP3(-/-)) mice. Because the activation of RIP3 requires stimulation by certain extracellular stimuli such as ligands of death receptors or Toll-like receptors, we compared the phosphorylation sites of lipopolysaccharide (LPS)-treated peritoneal macrophages from RIP3(+/+) and RIP3(-/-) mice and the phosphorylation sites of tumor necrosis factor (TNF)-treated RIP3(+/+) and RIP3(-/-) mouse embryonic fibroblast (MEF) cells. Stable isotope labeling by amino acids in cell culture and spike-in stable isotope labeling by amino acids in cell culture were used in the analyses of the MEFs and macrophages, respectively. Proteomic analyses using stable isotope labeling by amino acids in cell culture coupled with immobilized metal affinity chromatography-hydrophilic interaction liquid chromatography fractionation and nanoLC MS/MS identified 14,057 ph...Continue Reading

Mentioned in this Paper

Metabolic Process, Cellular
Macrophages, Peritoneal
Necrosis
Tumor Necrosis Factor-alpha
Extracellular
Amino Acids, I.V. solution additive
Isotope Labeling, Stable
Cell Culture Techniques
Protein Phosphorylation
MPRIP gene

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