Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.

蛋白激酶几乎控制着细胞功能的各个方面。这些关键的信号传导节点整合了多种途径输入，以调节复杂的生理过程，并且异常的激酶信号传导与多种病理相关。尽管基于荧光蛋白的生物传感器通过在活细胞中进行直接，时空精确的激酶活性测量，已经彻底革新了激酶信号传导的研究，但仍需要强大的新型分子工具，能够在各种实验环境中稳健地跟踪激酶活性动态，以充分剖析激酶的作用。生理和疾病中的信号传递。在这里，我们报告了通过高通量接头文库筛选获得的超灵敏第二代兴奋性计量蛋白激酶A（PKA）活性报告物（ExRai-AKAR2）的开发，能够跨多种荧光检测方式灵敏，快速地监测活细胞PKA活性，包括板读数，细胞分选和单光子或双光子成像。值得注意的是，清醒小鼠体内的视觉皮层成像显示通过强迫运动迅速募集的高动态神经元PKA活性。