丝氨酸和苏氨酸激酶的蛋白激酶 C (PKC) 家族由三个明显调控的亚家族组成，已被确定为对各种细胞功能至关重要。然而，PKC 酶如何在不同的亚细胞位置，特别是在新兴的信号中枢受到调节，尚不清楚。在这里，我们提出了一种灵敏的激发比率 C 激酶活性报告基因 (ExRai-CKAR2)，它能够检测亚细胞 PKC 活性的微小变化（相当于最大刺激的 0.2%）。使用 ExRai-CKAR2 和增强型二酰基甘油 (DAG) 生物传感器，我们发现 G 蛋白偶联受体刺激触发内质网和溶酶体的持续 PKC 活性，分别由 Ca2 敏感的传统 PKC 和 DAG 敏感的新型 PKC 差异介导。 ExRai-CKAR2 针对细胞质或分区缺陷复合物的高灵敏度进一步使我们能够检测三维类器官中以前无法检测到的内源性非典型 PKC 活性。总而言之，ExRai-CKAR2 是一个强大的工具，用于询问 PKC 调节对生理刺激的反应。© 2024。作者获得 Springer Nature America, Inc. 的独家许可。

The protein kinase C (PKC) family of serine and threonine kinases, consisting of three distinctly regulated subfamilies, has been established as critical for various cellular functions. However, how PKC enzymes are regulated at different subcellular locations, particularly at emerging signaling hubs, is unclear. Here we present a sensitive excitation ratiometric C kinase activity reporter (ExRai-CKAR2) that enables the detection of minute changes (equivalent to 0.2% of maximum stimulation) in subcellular PKC activity. Using ExRai-CKAR2 with an enhanced diacylglycerol (DAG) biosensor, we uncover that G-protein-coupled receptor stimulation triggers sustained PKC activity at the endoplasmic reticulum and lysosomes, differentially mediated by Ca2+-sensitive conventional PKC and DAG-sensitive novel PKC, respectively. The high sensitivity of ExRai-CKAR2, targeted to either the cytosol or partitioning defective complexes, further enabled us to detect previously inaccessible endogenous atypical PKC activity in three-dimensional organoids. Taken together, ExRai-CKAR2 is a powerful tool for interrogating PKC regulation in response to physiological stimuli.© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.