Radiation-induced phosphorylation of DNA-PK at 2638/47

辐射诱导的 DNA-PK 2638/47 磷酸化

• 批准号: 6949922
• 负责人: GREGORY T LANGLAND
• 金额: $ 2.5万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6949922
• 关键词: CHO cells; DNA repair; HeLa cells; X ray; active sites; cell cycle; enzyme activity; enzyme induction /repression; flow cytometry; genetic recombination; helicase; immunoprecipitation; ionizing radiation; mass spectrometry; phosphorylation; polymerase chain reaction; postdoctoral investigator; posttranslational modifications; protein kinase; radiation sensitivity; western blottings

DESCRIPTION (provided by applicant): The DNA-dependent protein kinase (DNA-PK) is a key component of the non-homologous end-joining pathway of DNA double-strand break (DSB) repair in mammalian cells. DNA-PK activity is essential for DSB repair and VDJ recombination. DNA-PK phosphorylates a variety of substrates including its self, in vitro; however its in vivo targets remain unclear. DNA-PK is phosphorylated in vitro in response to free DNA ends. Mass spectrometry has revealed several potential phosphorylation sites present in DNA-PKcs. Previous studies in our laboratory have demonstrated that DNA-PKcs is phosphorylated at T2609 in response to DSBs and mutation of that site results in radiation sensitivity and an end-joining defect. We propose to study T2638 and T2647 phosphorylation in DNA-Pkcs and determine the role of these specific post-translational events in DNA repair and cell viability. The hypotheses to be tested are (1) DNA-PKcs will be phosphorylated at sites T2638 and T2647 in response to DSBs. (2) These sites will only be phosphorylated in G1 and early S phase and (3) the mutation of these sites will result in radiation sensitivity, an end-joining defect and a longer half-life of phospho-specific (ala) mutants at these sites.

描述（由申请人提供）：DNA依赖性蛋白激酶（DNA-PK）是哺乳动物细胞中DNA双链断裂（DSB）修复的非同源末端连接途径的关键组成部分。 DNA-PK 活性对于 DSB 修复和 VDJ 重组至关重要。 DNA-PK 在体外磷酸化多种底物，包括其自身；然而其体内目标仍不清楚。 DNA-PK 在体外因游离 DNA 末端而被磷酸化。质谱分析揭示了 DNA-PKcs 中存在的几个潜在磷酸化位点。我们实验室之前的研究表明，DNA-PKcs 在 T2609 处被磷酸化以响应 DSB，并且该位点的突变会导致辐射敏感性和末端连接缺陷。我们建议研究 DNA-Pkcs 中的 T2638 和 T2647 磷酸化，并确定这些特定翻译后事件在 DNA 修复和细胞活力中的作用。要测试的假设是 (1) DNA-PKc 将响应 DSB 在位点 T2638 和 T2647 处磷酸化。 (2) 这些位点仅在 G1 期和早期 S 期被磷酸化，(3) 这些位点的突变将导致辐射敏感性、末端连接缺陷和磷酸化特异性 (ala) 突变体的半衰期更长。这些网站。