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.
Highlights
This page was generated automatically upon download from the ETH Zurich Research Collection
The cellular response to genotoxic stress is signaling networks of checkpoint and repair pathways largely governed by three members of the family of Abbreviations ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; CDK, cell cycle-dependent kinase; CHEK1, checkpoint kinase 1; CHEK2, checkpoint kinase 2; DDA, DNA-damaging agent; DDR, DNA damage response; DNA-PKcs, catalytic subunit of the DNA-dependent protein kinase; ERK, extracellular signal-regulated kinase; Gene ontology (GO), gene ontology; IHC, immunohistochemistry; IR, ionizing radiation; KSRs, kinase–substrate relationships; MAPK, mitogen-activated protein kinase; METi, MET inhibitor EMD1214063; MS, mass spectrometry; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; PIKKs, phosphatidylinositol 3-kinase-related kinases; RSKs, ribosomal protein S6 kinases; RTK, receptor tyrosine kinase; Selected reaction monitoring (SRM), selected reaction monitoring; WB, western blotting
In a discovery experiment, using immunoaffinity enrichment followed by MS, we identified hundreds of modulated phosphopeptides, on par with such experiments (Kirkpatrick et al, 2013; Matsuoka et al, 2007), including two METi-modulated subsets with potential roles in DDR signaling: a subset of IR-induced phosphorylations which are upregulated by prolonged prior METi treatment, and another subset of phosphorylations which are downregulated by METi regardless of IR
Summary
This page was generated automatically upon download from the ETH Zurich Research Collection. We explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). The cellular response to genotoxic stress is signaling networks of checkpoint and repair pathways largely governed by three members of the family of Abbreviations ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; CDK, cell cycle-dependent kinase; CHEK1, checkpoint kinase 1; CHEK2, checkpoint kinase 2; DDA, DNA-damaging agent; DDR, DNA damage response; DNA-PKcs, catalytic subunit of the DNA-dependent protein kinase; ERK, extracellular signal-regulated kinase; GO, gene ontology; IHC, immunohistochemistry; IR, ionizing radiation; KSRs, kinase–substrate relationships; MAPK, mitogen-activated protein kinase; METi, MET inhibitor EMD1214063; MS, mass spectrometry; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; PIKKs, phosphatidylinositol 3-kinase-related kinases; RSKs, ribosomal protein S6 kinases; RTK, receptor tyrosine kinase; SRM, selected reaction monitoring; WB, western blotting
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
