Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter
Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter
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DOI：
10.1038/s41598-020-66349-3
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发表时间：
2020-06-09
影响因子：
4.6
通讯作者：
Inoue, Takanari
中科院分区：
文献类型：
--
作者：
Kim, Allen K.;Wu, Helen D.;Inoue, Takanari
蛋白激酶A（Protein Kinase A，PKA）是一种四聚体的全酶，随着cAMP的增加而激活，在心脏生理、神经元发育和脂肪细胞功能等许多生理过程中发挥重要作用。虽然这种激酶已被许多生物传感器设计的主题，一个单一的荧光团报告，进行荧光共振能量转移（FRET）尚未被报道。在这里，我们已经使用PKA底物识别机制和核定位序列基序的静电相互作用的基本观察，设计了一个磷酸化开关，细胞质和细胞核之间的穿梭，一个策略，应该是概括到所有嗜碱性激酶。所得到的报告基因产生了与PKA FRET报告基因AKAR3EV相当的动力学和动态范围。我们还进行了基本的表征，并证明了其在使用基本荧光显微镜监测细胞内多种信号分子中的潜在用途。由于这种报告的单荧光团性质，我们设想，这可能会发现广泛的应用在涉及PKA活性的单细胞分析的研究。
Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Forster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.