Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
The Proteomics and Metabolomics Core Facility (PROMEC) at NTNU, Trondheim, Norway.
Sci Rep. 2017 Jul 24;7(1):6322. doi: 10.1038/s41598-017-06770-3.
XRCC1 is a scaffold protein involved in base excision repair and single strand break repair. It is a phosphoprotein that contains more than 45 phosphorylation sites, however only a few of these have been characterized and connected to specific kinases and functions. Mitogen activated protein kinases (MAPK) are mediators of cellular stress responses, and here we demonstrate that p38 MAPK signaling is involved in phosphorylation of XRCC1 and regulation of recruitment to oxidative stress. Inhibition of p38 MAPK caused a marked pI shift of XRCC1 towards a less phosphorylated state. Inhibition of p38 also increased the immediate accumulation of XRCC1 at site of DNA damage in a poly(ADP)-ribose (PAR) dependent manner. These results suggest a link between PARylation, p38 signaling and XRCC1 recruitment to DNA damage. Additionally, we characterized two phosphorylation sites, T358 and T367, located within, or close to, the phosphate-binding pocket of XRCC1, which is important for interaction with PAR. Mutation of these sites impairs recruitment of XRCC1 to DNA damage and binding to PARP1/PAR. Collectively, our data suggest that phosphorylation of T358 and T367 and p38 signaling are important for proper regulation of XRCC1 recruitment to DNA damage and thereby avoidance of potential toxic and mutagenic BER-intermediates.
XRCC1 是一种参与碱基切除修复和单链断裂修复的支架蛋白。它是一种磷酸化蛋白，含有超过 45 个磷酸化位点，但只有少数几个被鉴定并与特定的激酶和功能相关。丝裂原活化蛋白激酶（MAPK）是细胞应激反应的介质，在这里我们证明 p38 MAPK 信号参与 XRCC1 的磷酸化和对氧化应激募集的调节。p38 MAPK 的抑制导致 XRCC1 的 pI 向更非磷酸化状态发生明显的偏移。p38 的抑制也以多聚（ADP-核糖）（PAR）依赖性方式增加 XRCC1 在 DNA 损伤部位的即刻积累。这些结果表明 PAR 化、p38 信号和 XRCC1 募集到 DNA 损伤之间存在联系。此外，我们还鉴定了两个磷酸化位点，T358 和 T367，位于 XRCC1 的磷酸结合口袋内或附近，这对于与 PAR 的相互作用很重要。这些位点的突变会损害 XRCC1 向 DNA 损伤的募集和与 PARP1/PAR 的结合。总之，我们的数据表明，T358 和 T367 的磷酸化和 p38 信号对于 XRCC1 募集到 DNA 损伤的适当调节以及避免潜在的毒性和诱变 BER 中间体非常重要。
