Nyati Shyam, Young Grant, Ross Brian Dale, Rehemtulla Alnawaz
Center for Molecular Imaging, University of Michigan, Ann Arbor, MI, 48109, USA.
Department of Radiation Oncology, University of Michigan, 109 Zina Pitcher place, AAT-BSRB, Level A, Room # 628, Ann Arbor, MI, 48109 2200, USA.
Methods Mol Biol. 2017;1596:131-145. doi: 10.1007/978-1-4939-6940-1_9.
Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.
共济失调毛细血管扩张症突变基因（ATM）是一种丝氨酸/苏氨酸激酶，对细胞的DNA损伤反应至关重要，包括DNA双链断裂（DSB）。ATM的激活会引发一系列复杂的事件，促进DNA损伤修复、细胞周期检查点控制和细胞存活。传统上，蛋白激酶是在体外使用生化方法（使用纯化蛋白的激酶测定或免疫测定）进行分析的，这些方法需要大量细胞并进行细胞裂解。基于光学分子成像（如荧光或生物发光）的基因编码生物传感器已被开发出来，以能够在活细胞中以高信噪比询问激酶活性。我们通过基因工程构建了一种杂合蛋白，其生物发光活性依赖于ATM介导的底物磷酸化。该工程蛋白由基于分裂荧光素酶的蛋白互补对组成，带有CHK2（ATM激酶活性的底物）靶序列和源自酵母Rad53的磷酸丝氨酸/苏氨酸结合结构域FHA2。ATM对靶序列内丝氨酸残基的磷酸化会导致其与磷酸丝氨酸结合结构域相互作用，从而阻止分裂荧光素酶对的互补并导致报告基因活性丧失。对培养板中或作为小鼠异种移植物的报告基因表达细胞进行生物发光成像，以无创、动态的方式提供了ATM激酶活性以及因此细胞DNA损伤反应的定量替代指标。
