Department of Biochemistry and Systems Biology, ISMIB, University of Liverpool, Liverpool, UK.
Department of Pharmacology, University of Washington, Seattle, WA, USA.
Methods Mol Biol. 2022;2483:297-317. doi: 10.1007/978-1-0716-2245-2_19.
Generation of the prototypic second messenger cAMP instigates numerous signaling events. A major intracellular target of cAMP is Protein kinase A (PKA), a Ser/Thr protein kinase. Where and when this enzyme is activated inside the cell has profound implications on the functional impact of PKA. It is now well established that PKA signaling is focused locally into subcellular signaling "islands" or "signalosomes." The A-Kinase Anchoring Proteins (AKAPs) play a critical role in this process by dictating spatial and temporal aspects of PKA action. Genetically encoded biosensors, small molecule and peptide-based disruptors of PKA signaling are valuable tools for rigorous investigation of local PKA action at the biochemical level. This chapter focuses on approaches to evaluate PKA signaling islands, including a simple assay for monitoring the interaction of an AKAP with a tunable PKA holoenzyme. The latter approach evaluates the composition of PKA holoenzymes, in which regulatory subunits and catalytic subunits can be visualized in the presence of test compounds and small-molecule inhibitors.
原型第二信使 cAMP 的产生引发了许多信号事件。cAMP 的主要细胞内靶标是蛋白激酶 A（PKA），一种丝氨酸/苏氨酸蛋白激酶。这种酶在细胞内何时何地被激活对 PKA 的功能影响有深远的影响。现在已经确定，PKA 信号集中在细胞内的亚细胞信号“岛”或“信号体”中。A-激酶锚定蛋白（AKAPs）通过决定 PKA 作用的空间和时间方面在这个过程中起着关键作用。遗传编码的生物传感器、小分子和基于肽的 PKA 信号破坏剂是在生化水平上严格研究局部 PKA 作用的有价值的工具。本章重点介绍评估 PKA 信号岛的方法，包括一种简单的测定法，用于监测 AKAP 与可调节 PKA 全酶的相互作用。后一种方法评估 PKA 全酶的组成，其中可以在存在测试化合物和小分子抑制剂的情况下可视化调节亚基和催化亚基。
