Dataset Information

Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics.

ABSTRACT: Increasing evidence suggests that interference with growth factor receptor tyrosine kinase (RTK) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA-damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). Following an immunoaffinity-based phosphoproteomic discovery survey, we selected candidate phosphorylation sites for extensive characterization by targeted proteomics focusing on phosphorylation sites in both signaling networks. Several substrates of the DDR were confirmed to be modulated by sequential MET inhibition and IR, or MET inhibition alone. Upon combined treatment, for two substrates, NUMA1 S395 and CHEK1 S345, the gain and loss of phosphorylation, respectively, were recapitulated using invivo tumor models by immunohistochemistry, with possible utility in future translational research. Overall, we have corroborated phosphorylation sites at the intersection between MET and the DDR signaling networks, and suggest that these represent a class of proteins at the interface between oncogene-driven proliferation and genomic stability.

SUBMITTER: Bensimon A

PROVIDER: S-EPMC7266272 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics.

Bensimon Ariel A Koch Jonas P JP Francica Paola P Roth Selina M SM Riedo Rahel R Glück Astrid A AA Orlando Eleonora E Blaukat Andree A Aebersold Daniel M DM Zimmer Yitzhak Y Aebersold Ruedi R Medová Michaela M

Molecular oncology 20200513 6

Increasing evidence suggests that interference with growth factor receptor tyrosine kinase (RTK) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA-damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radi ...[more]

PMID: 32336009

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Project description: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.

Dataset Information

Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics.

Publications

Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics.

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