Campbell Amy E, Ferraz Franco Catarina, Su Ling-I, Corbin Emma K, Perkins Simon, Kalyuzhnyy Anton, Jones Andrew R, Brownridge Philip J, Perkins Neil D, Eyers Claire E
Centre for Proteome Research, Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool L69 7ZB, U.K.
Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle Upon Tyne NE2 4HH, U.K.
Biochem J. 2021 Feb 12;478(3):533-551. doi: 10.1042/BCJ20200627.
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.
不同类型的DNA损伤可引发磷酸化介导的信号级联反应，从而导致刺激特异性的促凋亡或抗凋亡细胞反应。在其众多作用中，核因子κB转录因子RelA是这些DNA损伤反应途径的核心。然而，我们仍然缺乏对协同信号机制的理解，这些机制允许不同的DNA损伤剂通过RelA诱导不同的细胞结果。在这里，我们使用无标记定量磷酸化蛋白质组学，通过监测RelA及其蛋白质结合伙伴的磷酸化状态，来研究U2OS细胞暴露于依托泊苷（ETO）或羟基脲（HU）后的时间效应。尽管在暴露于这些DNA损伤剂后，磷酸化RelA相互作用组的组成成分中几乎没有发现刺激特异性差异，但我们观察到它们的磷酸化状态存在细微但显著的变化，这是治疗类型和持续时间的函数。诱导DNA双链断裂（DSB）的ETO引发的反应比HU更快、更持久，其调控靶点主要参与转录、细胞分裂和经典DSB修复。对ETO调控的磷酸化位点进行激酶底物预测表明，除了已知的ATM/ATR诱导外，CDK和ERK1信号也被消除。相比之下，HU诱导的复制应激介导了时间动态调控，磷酸化的RelA结合伙伴在rRNA/mRNA加工和翻译起始中发挥作用，其中许多含有14-3-3ε结合基序，并且是双特异性激酶CLK1的推定底物。因此，我们的数据表明关键细胞过程的差异调控以及不同信号通路参与调节RelA的DNA损伤特异性功能。
