Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes

ATM is a protein kinase that initiates a well-characterized
signaling cascade in cells exposed to ionizing radiation (IR). However,
the role for ATM in coordinating critical protein interactions and
subsequent exchanges within DNA damage response (DDR) complexes is
unknown. We combined SILAC-based tandem mass spectrometry and a subcellular
fractionation protocol to interrogate the proteome of irradiated cells
treated with or without the ATM kinase inhibitor KU55933. We developed
an integrative network analysis to identify and prioritize proteins
that were responsive to KU55933, specifically in chromatin, and that
were also enriched for physical interactions with known DNA repair
proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as
strong candidates. Using fluorescence recovery after photobleaching,
we found that the exchange of GFP-53BP1 in DDR complexes decreased
with KU55933. Further, we found that ANXA1 knockdown sensitized cells
to IR via a mechanism that was not potentiated by KU55933. Our study
reveals a role for ATM kinase activity in the dynamic exchange of
proteins in DDR complexes and identifies a role for ANXA1 in cellular
radioprotection.

共济失调突变蛋白激酶（ATM）是一种蛋白激酶，可在受电离辐射（ionizing radiation, IR）暴露的细胞中触发已被广泛阐明的信号级联反应。然而，ATM在DNA损伤应答（DNA damage response, DDR）复合物内协调关键蛋白质相互作用及后续蛋白交换过程中的作用尚不明确。本研究结合基于稳定同位素标记细胞培养（Stable Isotope Labeling with Amino Acids in Cell Culture, SILAC）的串联质谱（tandem mass spectrometry）技术与亚细胞分级分离（subcellular fractionation）方案，对经或未经ATM激酶抑制剂KU55933处理的受辐照细胞的蛋白质组进行了解析。我们开发了一套整合网络分析方法，用于识别并优先筛选在染色质中对KU55933产生响应、且同时富集于与已知DNA修复蛋白质物理相互作用的蛋白质。该分析鉴定出53BP1与膜联蛋白A1（annexin A1, ANXA1）为极具潜力的候选蛋白。借助荧光漂白后恢复（fluorescence recovery after photobleaching）实验，我们发现DDR复合物中绿色荧光蛋白（Green Fluorescent Protein, GFP）标记的53BP1的交换速率随KU55933的处理而显著降低。进一步研究表明，敲低ANXA1会增加细胞对电离辐射的敏感性，且该效应无法被KU55933进一步增强。本研究揭示了ATM激酶活性在DDR复合物内蛋白质动态交换过程中的关键作用，并明确了ANXA1在细胞辐射防护中的功能。