Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response
Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response
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DOI：
10.1038/emboj.2012.86
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发表时间：
2012-05-16
期刊：
影响因子：
11.4
通讯作者：
Chowdhury, Dipanjan
中科院分区：
文献类型：
--
作者：
Lee, Dong-Hyun;Goodarzi, Aaron A.;Chowdhury, Dipanjan
蛋白磷酸酶PP 4C与DNA损伤反应（DDR）有关，但其在DDR中的底物仍不清楚。我们设计了一种新的蛋白质组学策略，用于系统鉴定由PP 4C脱磷酸化的蛋白质，并鉴定KRAB结构域相关蛋白1（KAP-1）作为底物。电离辐射导致KAP-1在S824（通过ATM）和S473（通过CHK 2）磷酸化。PP 4C/R3 β复合物与KAP-1相互作用，沉默该复合物导致磷酸-S824和磷酸-S473的持续存在。我们通过显示S473的磷酸化影响G2/M检查点来确定KAP-1在DDR中的新作用。PP 4 R3 β的消耗或磷酸化模拟物KAP-1 S473突变体（S473 D）的表达导致延长的G2/M检查点。S824的磷酸化对于异染色质DNA损伤的修复是必需的，并且与表达拟磷酸化KAP-1 S824突变体（S824 D）的细胞或PP 4 R3 β-沉默的细胞类似，显示染色质的延长松弛，同时释放染色质重塑蛋白质CHD 3。我们的研究结果定义了PP 4介导的去磷酸化在DDR中的新作用，包括KAP-1在检查点反应中以前未描述的功能的调节。The EMBO Journal（2012）31，2403-2415. doi：10.1038/daj.2012.86; 2012年4月10日在线发布
Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB-domain-associated protein 1 (KAP-1) as a substrate. Ionizing radiation leads to phosphorylation of KAP-1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3 beta complex interacts with KAP-1 and silencing this complex leads to persistence of phospho-S824 and phospho-S473. We identify a new role for KAP-1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3 beta or expression of the phosphomimetic KAP-1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP-1 S824 mutant (S824D), or PP4R3 beta-silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4-mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP-1 in checkpoint response. The EMBO Journal (2012) 31, 2403-2415. doi: 10.1038/emboj.2012.86; Published online 10 April 2012