In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage.
In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage.
复制标题
DOI：
10.1093/jrr/rrv086
复制
发表时间：
2016-03
影响因子：
2
通讯作者：
Matsumoto Y
中科院分区：
文献类型：
--
作者：
Sharma MK;Imamichi S;Fukuchi M;Samarth RM;Tomita M;Matsumoto Y
XRCC4 是一种与 DNA 连接酶 IV 相关的蛋白质，它被认为在 DNA 双链断裂修复的最后一步通过非同源末端连接连接两个 DNA 末端。为了响应电离辐射或 DNA 损伤剂的治疗，XRCC4 会发生 DNA-PK 依赖性磷酸化。此外，体外纯化的DNA-PK通过质谱法将XRCC4的Ser260和Ser320（或选择性剪接形式的Ser318）鉴定为主要磷酸化位点。然而，尚不清楚这些位点是否在体内因 DNA 损伤而被磷酸化。在本研究中，我们生成了一种与 Ser320 位点磷酸化的 XRCC4 反应的抗体，并检查了 XRCC4 Ser320 位点的纤维素磷酸化状态。 XRCC4 Ser320 的磷酸化是通过 γ 射线照射和 Zeocin 处理诱导的。即使在 1 Gy 照射后也检测到 XRCC4 Ser320 的磷酸化，并且以依赖于辐射剂量的方式增加。照射后立即观察到磷酸化，并且在长达 4 小时内基本保持不变。 DNA-PK 抑制剂 NU7441 抑制磷酸化，并且在 DNA-PKcs 缺陷细胞中检测不到，表明磷酸化主要由 DNA-PK 介导。这些结果表明 XRCC4 Ser320 的磷酸化状态作为活细胞中 DNA-PK 功能的指标具有潜在的用途。
XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells.