Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
J Biol Chem. 2019 May 24;294(21):8395-8402. doi: 10.1074/jbc.RA119.008154. Epub 2019 Apr 2.
DNA damage response Ser/Thr kinases, including ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR), control cell cycle progression, DNA repair, and apoptosis. ATR is activated by ETAA1 activator of ATR kinase (ETAA1) or DNA topoisomerase II binding protein 1 (TOPBP1). Both ETAA1 and TOPBP1 contain experimentally defined ATR activation domains (AADs) that are mostly unstructured and have minimal sequence similarity. A tryptophan residue in both AADs is required for ATR activation, but the other features of these domains and the mechanism by which they activate ATR are unknown. In this study, using bioinformatic analyses, kinase assays, co-immunoprecipitation, and immunofluorescence measures of signaling, we more specifically defined the TOPBP1 and ETAA1 AADs and identified additional features of the AADs needed for ATR activation. We found that both ETAA1 and TOPBP1 contain a predicted coiled-coil motif that is required for ATR activation and in cells. Mutation of the predicted coiled coils does not alter AAD oligomerization but does impair binding of the AADs to ATR. These results suggest that TOPBP1 and ETAA1 activate ATR using similar motifs and mechanisms.
DNA 损伤反应丝氨酸/苏氨酸激酶，包括共济失调毛细血管扩张突变（ATM）和 Rad3 相关（ATR），控制细胞周期进程、DNA 修复和细胞凋亡。ATR 被 ATR 激酶激活物（ETAA1）或 DNA 拓扑异构酶 II 结合蛋白 1（TOPBP1）激活。ETAA1 和 TOPBP1 都含有实验定义的 ATR 激活结构域（AAD），这些结构域大部分是非结构化的，序列相似性最小。两个 AAD 中的色氨酸残基是 ATR 激活所必需的，但这些结构域的其他特征以及它们激活 ATR 的机制尚不清楚。在这项研究中，我们使用生物信息学分析、激酶测定、共免疫沉淀和信号的免疫荧光测量，更具体地定义了 TOPBP1 和 ETAA1 的 AAD，并确定了 ATR 激活所需的 AAD 的其他特征。我们发现，ETAA1 和 TOPBP1 都含有一个预测的卷曲螺旋基序，该基序是 ATR 激活所必需的，也是在细胞中激活 ATR 所必需的。预测的卷曲螺旋的突变不会改变 AAD 寡聚化，但会损害 AAD 与 ATR 的结合。这些结果表明，TOPBP1 和 ETAA1 可能使用相似的基序和机制激活 ATR。
