Biochemical Analysis of AKAP-Anchored PKA Signaling Complexes.
Biochemical Analysis of AKAP-Anchored PKA Signaling Complexes.
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DOI：
10.1007/978-1-0716-2245-2_19
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发表时间：
2022
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原型第二信使cAMP的产生引发许多信号传导事件。cAMP的主要细胞内靶标是蛋白激酶A（PKA），一种Ser/Thr蛋白激酶。这种酶在细胞内何时何地被激活对PKA的功能影响有着深远的意义。现在已经确定PKA信号传导局部集中在亚细胞信号传导“岛”或“信号体”中。A激酶介导蛋白（AKAP）通过决定PKA作用的空间和时间方面在这一过程中发挥关键作用。基因编码的生物传感器，小分子和基于肽的PKA信号转导干扰物是在生物化学水平上严格研究PKA局部作用的有价值的工具。本章重点介绍PKA信号岛的评估方法，包括一个简单的检测方法，用于监测AKAP与可调PKA全酶的相互作用。后一种方法评估PKA全酶的组成，其中调节亚基和催化亚基可以在测试化合物和小分子抑制剂的存在下可视化。
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. This 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.