Temporal modulation of the NF-κB RelA network in response to different types of DNA damage

Abstract

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.

Profiling the Human Phosphoproteome to Estimate the True Extent of Protein Phosphorylation

“…As a general rule, phosphorylation sites with <5 independent observations should be treated with caution, and those with only one observation in a database are likely to be false positives. In a recent phosphoproteomics study from our group, we demonstrated the utility of the classification presented here, by matching the sites identified by LC-MS/MS to their evidence categories from PSP and PA. 73 For new phosphoproteomics studies, it will be common for some ambiguity to remain regarding phosphosite localization, and many sites will be observed that would not pass a 1 or 5% false localization rate cutoff from a single dataset, but for which there may be some supporting evidence. By evaluating new datasets in combination with all of the evidence collated from a large number of previous studies, greater confidence can be assigned to “borderline” significant phosphosites that may indeed be correct, or conversely, sites with weak evidence that have never been reported can be rejected.…”

Class 1 Histone Deacetylases and Ataxia-Telangiectasia Mutated Kinase Control the Survival of Murine Pancreatic Cancer Cells upon dNTP Depletion

“…We measured this as p-KAP1 because our antibodies were not able to detect p-ATM in murine PDAC cells (data not shown). The ATM-related checkpoint kinase ATR is one of the first molecules that are activated upon dNTP depletion by hydroxyurea [58,65,66]. Activated ATR and the single strand DNA binding protein RPA protect stalled replication forks and prevent DNA collapse and double strand breaks [58].…”

Systems approaches to understand oxygen sensing: how multi-omics has driven advances in understanding oxygen-based signalling

“…Interestingly, more than 50% of the mapped phosphorylation sites on the HIF-α subunits were differentially observed at different oxygen tensions [ 85 ]. Further analysis may reveal that the dynamic nature of this combinatory PTM code of HIF can alter its activity in response to specific stimuli or timing, as seen in the combinatorial modification of the NF-κB transcription factor in response to different stimuli [ 88 ].…”