Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3ε binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA.
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Research Article|
January 13 2021
Temporal modulation of the NF-κB RelA network in response to different types of DNA damage
Amy E. Campbell;
Amy E. Campbell
University of Liverpool, Liverpool, United Kingdom
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Caterina Ferraz Franco;
Caterina Ferraz Franco
University of Liverpool, Liverpool, United Kingdom
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Ling-I Su;
Ling-I Su
Newcastle University, Newcastle Upon Tyne, United Kingdom
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Emma K Corbin;
Emma K Corbin
Newcastle University, Newcastle Upon Tyne, United Kingdom
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Simon Perkins;
Simon Perkins
Institute of Integrative Biology, Liverpool, United Kingdom
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Anton Kalyuzhnyy;
Anton Kalyuzhnyy
University of Liverpool, Liverpool, United Kingdom
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Andrew R Jones;
Andrew R Jones
University of Liverpool, Liverpool, United Kingdom
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Philip J. Brownridge;
Philip J. Brownridge
University of Liverpool, Liverpool, United Kingdom
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Neil D. Perkins;
Neil D. Perkins
Newcastle University, Newcastle Upon Tyne, United Kingdom
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Claire E Eyers
University of Liverpool, Liverpool, United Kingdom
* Corresponding Author; email: ceyers@liverpool.ac.uk
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Biochem J (2021) BCJ20200627.
Article history
Received:
August 10 2020
Revision Received:
January 04 2021
Accepted:
January 12 2021
Citation
Amy E. Campbell, Caterina Ferraz Franco, Ling-I Su, Emma K Corbin, Simon Perkins, Anton Kalyuzhnyy, Andrew R Jones, Philip J. Brownridge, Neil D. Perkins, Claire E Eyers; Temporal modulation of the NF-κB RelA network in response to different types of DNA damage
. Biochem J 2021; BCJ20200627. doi: https://doi.org/10.1042/BCJ20200627Download citation file:
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