Smolka Marcus, Comstock Will, Navarro Marcos, Maybee Deanna, Rho Yiseo, Wagner Mateusz, Wang Yingzheng
Cornell University.
Res Sq. 2025 Mar 27:rs.3.rs-6220494. doi: 10.21203/rs.3.rs-6220494/v1.
Understanding kinase action requires precise quantitative measurements of their activity . In addition, the ability to capture spatial information of kinase activity is crucial to deconvolute complex signaling networks, interrogate multifaceted kinase actions, and assess drug effects or genetic perturbations. Here we developed a proteomic kinase activity sensor platform (ProKAS) for the analysis of kinase signaling using mass spectrometry. ProKAS is based on a tandem array of peptide sensors with amino acid barcodes that allow multiplexed analysis for spatial, kinetic, and screening applications. We engineered a ProKAS module to simultaneously monitor the activities of the DNA damage response kinases ATR, ATM, and CHK1 in response to genotoxic drugs, while also uncovering differences between these signaling responses in the nucleus, cytosol, and replication factories. Furthermore, we developed an approach for the rational design of specific substrate peptides expandable to other kinases. Overall, ProKAS is a novel versatile system for systematically and spatially probing kinase action in cells.
了解激酶作用需要对其活性进行精确的定量测量。此外，获取激酶活性空间信息的能力对于剖析复杂的信号网络、探究多方面的激酶作用以及评估药物效果或基因扰动至关重要。在此，我们开发了一种蛋白质组学激酶活性传感器平台（ProKAS），用于通过质谱分析激酶信号传导。ProKAS基于带有氨基酸条形码的肽传感器串联阵列，可实现对空间、动力学和筛选应用的多重分析。我们设计了一个ProKAS模块，以同时监测DNA损伤反应激酶ATR、ATM和CHK1对基因毒性药物的反应活性，同时还揭示这些信号反应在细胞核、细胞质和复制工厂之间的差异。此外，我们开发了一种合理设计特定底物肽的方法，该方法可扩展到其他激酶。总体而言，ProKAS是一种新颖的通用系统，用于在细胞中系统地和空间地探究激酶作用。
