title: Investigation of receptor interacting protein (RIP3)-dependent protein phosphorylation by quantitative phosphoproteomics.
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 phosphopeptides in 4306 proteins from the macrophages and 4732 phosphopeptides in 1785 proteins from the MEFs. Analysis of amino acid sequence motifs among the phosphopeptides identified a potential motif of RIP3 phosphorylation. Among the phosphopeptides identified, 73 were found exclusively in RIP3(+/+) macrophages, 121 were detected exclusively from RIP3(+/+) MEFs, 286 phosphopeptides were induced more in RIP3(+/+) macrophages than in RIP3(-/-) macrophages and 26 phosphopeptides had higher induction in RIP3(+/+) MEFs than in RIP3(-/-) cells. Many of the RIP3 regulated phosphoproteins from the macrophages and MEF cells are functionally associated with the cell cycle; the rest, however, appear to have diverse functions in that a number of metabolism related proteins were phosphorylated in macrophages and development related phosphoproteins were induced in MEFs. The results of our phosphoproteomic analysis suggest that RIP3 might function beyond necrosis and that cell type specific function of RIP3 exists.
authors.affiliation: State Key Laboratory of Cellular Stress Biology and School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.
authors.forename: Xiurong
authors.initials: X
authors.lastname: Wu
authors.forename: Lili
authors.initials: L
authors.lastname: Tian
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Victor
authors.initials: V
authors.lastname: Han
authors.forename: Yuanyue
authors.initials: Y
authors.lastname: Li
authors.forename: Xiaozheng
authors.initials: X
authors.lastname: Xu
authors.forename: Hanjie
authors.initials: H
authors.lastname: Li
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Jinan
authors.initials: J
authors.lastname: Chen
authors.forename: Wenhai
authors.initials: W
authors.lastname: Jin
authors.forename: Yongming
authors.initials: Y
authors.lastname: Xie
authors.forename: Jiahuai
authors.initials: J
authors.lastname: Han
authors.forename: Chuan-Qi
authors.initials: CQ
authors.lastname: Zhong
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D007553:Isotope Labeling; D008070:Lipopolysaccharides; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009336:Necrosis / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D015398:Signal Transduction; D013194:Staining and Labeling; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D008070:Lipopolysaccharides; D010748:Phosphopeptides; D020543:Proteome; D014409:Tumor Necrosis Factor-alpha; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse