Su Qi, Zhang Jing, Lin Wei, Zhang Jin-Fan, Newton Alexandra C, Mehta Sohum, Yang Jing, Zhang Jin
Department of Pharmacology, School of Medicine, University of California San Diego, La Jolla, CA, USA.
Moores Cancer Center, University of California San Diego, La Jolla, CA, USA.
bioRxiv. 2024 Mar 30:2024.03.29.587373. doi: 10.1101/2024.03.29.587373.
The protein kinase C (PKC) family of serine/threonine kinases, which consist of three distinctly regulated subfamilies, have long been established as critical for a variety of cellular functions. However, how PKC enzymes are regulated at different subcellular locations, particularly at emerging signaling hubs such as the ER, lysosome, and Par signaling complexes, is unclear. Here, we present a sensitive Excitation Ratiometric (ExRai) C Kinase Activity Reporter (ExRai-CKAR2) that enables the detection of minute changes in subcellular PKC activity. Using ExRai-CKAR2 in conjunction with an enhanced diacylglycerol (DAG) biosensor capable of detecting intracellular DAG dynamics, we uncover the differential regulation of PKC isoforms at distinct subcellular locations. We find that G-protein coupled receptor (GPCR) stimulation triggers sustained PKC activity at the ER and lysosomes, primarily mediated by Ca sensitive conventional PKC (cPKC) and novel PKC (nPKC), respectively, with nPKC showing high basal activity due to elevated basal DAG levels on lysosome membranes. The high sensitivity of ExRai-CKAR2, targeted to either the cytosol or Par-complexes, further enabled us to detect previously inaccessible endogenous atypical PKC (aPKC) activity in 3D organoids. Taken together, ExRai-CKAR2 is a powerful tool for interrogating PKC regulation in response to physiological stimuli.
丝氨酸/苏氨酸激酶的蛋白激酶C（PKC）家族由三个调控方式截然不同的亚家族组成，长期以来一直被认为对多种细胞功能至关重要。然而，PKC酶如何在不同的亚细胞位置受到调控，尤其是在内质网、溶酶体和Par信号复合物等新兴信号枢纽处，目前尚不清楚。在此，我们展示了一种灵敏的激发比率（ExRai）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。将ExRai-CKAR2与一种能够检测细胞内二酰甘油（DAG）动态变化的增强型二酰甘油生物传感器结合使用，我们揭示了不同亚细胞位置PKC亚型的差异调控。我们发现，G蛋白偶联受体（GPCR）刺激在内质网和溶酶体处触发持续的PKC活性，分别主要由钙敏感的传统PKC（cPKC）和新型PKC（nPKC）介导，由于溶酶体膜上基础DAG水平升高，nPKC表现出较高的基础活性。靶向细胞质或Par复合物的ExRai-CKAR2的高灵敏度，进一步使我们能够在三维类器官中检测到以前无法检测到的内源性非典型PKC（aPKC）活性。综上所述，ExRai-CKAR2是一种用于研究PKC对生理刺激反应调控的强大工具。
