Ataxia telangiectasia mutated (ATM) is a serine–threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65–S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM–PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear–cytoplasmic shuttling mediated by the nuclear export receptor CRM1.

共济失调毛细血管扩张症 (ATM) 是一种丝氨酸-苏氨酸蛋白激酶，是 DNA 损伤反应 (DDR) 的重要调节剂。一个关键的 ATM 靶标是蛋白磷酸酶 2A (PP2A) 的结构亚基 A (PR65–S401)，已知它可以调节多种细胞过程，例如有丝分裂和细胞生长，以及在从 DDR 恢复过程中使许多蛋白质去磷酸化。我们生成了表达 PR65-WT、-S401A（不能被磷酸化）和-S401D（拟磷酸化）转基因的小鼠胚胎成纤维细胞。值得注意的是，S401 突变体表现出广泛的染色体畸变、DNA 双链断裂 (DSB) 修复受损以及辐射后有丝分裂灾难增加。S401A 和 S401D 细胞均显示受损的 DSB 修复（非同源末端连接和同源重组修复）并表现出延迟的 DNA 损伤恢复，这反映在辐射存活率降低上。此外，S401D 细胞显示出增加的 ERK 和 AKT 信号，导致生长速度加快，进一步强调了 ATM–PP2A 信号在调节促生存反应中的多重作用。延时视频和细胞定位实验表明，PR65 在 CRM1（一种核输出蛋白）辐射后被输出到细胞质，这与观察到的非常快速的多效性一致。确定了接近 S401 的假定核输出序列 (NES)，当发生突变时会导致异常的 PR65 穿梭。我们的研究表明，单个的磷酸化，