Sensitive fluorescent biosensor reveals differential subcellular regulation of PKC

IF 12.9 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Qi Su, Jing Zhang, Wei Lin, Jin-Fan Zhang, Alexandra C. Newton, Sohum Mehta, Jing Yang, Jin Zhang
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引用次数: 0

Abstract

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.

Abstract Image

灵敏的荧光生物传感器揭示了 PKC 的亚细胞调控差异
丝氨酸和苏氨酸激酶的蛋白激酶 C(PKC)家族由三个不同的亚家族组成,已被证实对各种细胞功能至关重要。然而,PKC 酶在不同亚细胞位置,特别是在新出现的信号枢纽是如何被调控的还不清楚。在这里,我们展示了一种灵敏的激发比率测量 C 激酶活性报告物(ExRai-CKAR2),它能检测亚细胞 PKC 活性的微小变化(相当于最大刺激的 0.2%)。利用带有增强型二酰甘油(DAG)生物传感器的 ExRai-CKAR2,我们发现 G 蛋白偶联受体刺激会在内质网和溶酶体引发持续的 PKC 活性,这些活性分别由对 Ca2+ 敏感的传统 PKC 和对 DAG 敏感的新型 PKC 介导。ExRai-CKAR2 的灵敏度很高,可以靶向细胞质或分区缺陷复合物,这进一步使我们能够在三维器官组织中检测到以前无法获得的内源性非典型 PKC 活性。综上所述,ExRai-CKAR2 是研究 PKC 在生理刺激下调控的有力工具。
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来源期刊
Nature chemical biology
Nature chemical biology 生物-生化与分子生物学
CiteScore
23.90
自引率
1.40%
发文量
238
审稿时长
12 months
期刊介绍: Nature Chemical Biology stands as an esteemed international monthly journal, offering a prominent platform for the chemical biology community to showcase top-tier original research and commentary. Operating at the crossroads of chemistry, biology, and related disciplines, chemical biology utilizes scientific ideas and approaches to comprehend and manipulate biological systems with molecular precision. The journal embraces contributions from the growing community of chemical biologists, encompassing insights from chemists applying principles and tools to biological inquiries and biologists striving to comprehend and control molecular-level biological processes. We prioritize studies unveiling significant conceptual or practical advancements in areas where chemistry and biology intersect, emphasizing basic research, especially those reporting novel chemical or biological tools and offering profound molecular-level insights into underlying biological mechanisms. Nature Chemical Biology also welcomes manuscripts describing applied molecular studies at the chemistry-biology interface due to the broad utility of chemical biology approaches in manipulating or engineering biological systems. Irrespective of scientific focus, we actively seek submissions that creatively blend chemistry and biology, particularly those providing substantial conceptual or methodological breakthroughs with the potential to open innovative research avenues. The journal maintains a robust and impartial review process, emphasizing thorough chemical and biological characterization.
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