Department of Pharmacology, University of California San Diego, La Jolla, CA, USA.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA.
Nat Commun. 2024 Sep 6;15(1):7804. doi: 10.1038/s41467-024-51270-4.
Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into "permanent" fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA activity distribution in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of Gα-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.
蛋白激酶是调节基本生物和疾病过程的关键信号节点。从多个角度阐明激酶信号可以深入了解疾病机制并改善治疗靶点。虽然荧光生物传感器是实时可视化活细胞激酶活性动力学的强大工具，但需要新的分子工具来记录瞬时信号活动，以便进行事后分析和靶向操作。在这里，我们开发了一种光门控激酶活性偶联转录整合器（KINACT），它将动态激酶信号转换为“永久”荧光标记。KINACT 能够在多个尺度上进行强大的激酶活性监测，准确记录细胞内 PKA 活性，突出 3D 培养物中 PKA 活性分布，并在高通量筛选中识别 PKA 激活剂和抑制剂。我们进一步利用 KINACT 驱动信号效应物表达的能力，允许对 Gα 诱导的 PKA 和 ERK 激活的平衡进行反馈操作，并剖析致癌 G 蛋白信号的机制。
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