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.

丝氨酸和苏氨酸激酶的蛋白激酶 C （PKC） 家族由三个明显调节的亚家族组成，已被确定为各种细胞功能的关键。然而，PKC 酶如何在不同的亚细胞位置进行调节，特别是在新兴的信号传导枢纽，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%）。使用带有增强型甘油二酯 （DAG） 生物传感器的 ExRai-CKAR2，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⁺ 敏感的常规 PKC 和 DAG 敏感的新型 PKC 介导。ExRai-CKAR2 的高灵敏度，靶向胞质溶胶或分配缺陷复合物，进一步使我们能够在三维类器官中检测以前无法接近的内源性非典型 PKC 活性。综上所述，ExRai-CKAR2 是询问 PKC 对生理刺激的调节的强大工具。