Temporal modulation of the NF-kappa B ReIA network in response to different types of DNA damage

Campbell, Amy E ORCID: 0000-0001-8390-4360, Franco, Catarina Ferraz, Ling-I, Su, Corbin, Emma K, Perkins, Simon, Kalyuzhnyy, Anton, Jones, Andrew R ORCID: 0000-0001-6118-9327, Brownridge, Philip J, Perkins, Neil D and Eyers, Claire E ORCID: 0000-0002-3223-5926
(2021) Temporal modulation of the NF-kappa B ReIA network in response to different types of DNA damage. Biochemical Journal, 478 (3). pp. 533-551.

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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.

Item Type: Article
Uncontrolled Keywords: Cell Line, Tumor, Humans, Osteosarcoma, Bone Neoplasms, DNA Damage, Hydroxyurea, Etoposide, Protein Kinases, DNA, Neoplasm, Chromatography, Liquid, Proteomics, Apoptosis, DNA Replication, Protein Processing, Post-Translational, Amino Acid Sequence, Amino Acid Motifs, Consensus Sequence, Phosphorylation, Time Factors, Transcription Factor RelA, DNA Breaks, Double-Stranded, Tandem Mass Spectrometry, Protein Interaction Maps
Divisions: Faculty of Health and Life Sciences
Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 24 Jun 2021 08:04
Last Modified: 18 Jan 2023 23:00
DOI: 10.1042/BCJ20200627
Related URLs:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3115510