Steady State and Time-Dependent Fluorescent Peptide Assays for Protein Kinases.
Steady State and Time-Dependent Fluorescent Peptide Assays for Protein Kinases.
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蛋白激酶的稳态和时间依赖性荧光肽测定。
DOI：
10.1002/cpz1.998
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Dalby,KevinN
中科院分区：
文献类型：
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作者：
Sammons,RaeM;Devkota,AshwiniK;Kaoud,TamerS;Warthaka,Mangalika;Cho,EunJeong;Dalby,KevinN
蛋白激酶催化蛋白质的磷酸化，最常见的是在Ser、Thr和Tyr残基上，并调节真核细胞中的许多细胞事件，如细胞周期进程、转录、代谢和凋亡。蛋白激酶各自具有保守的ATP结合位点和一个或多个底物结合位点，其表现出对不同蛋白底物的识别特征。通过使ATP和底物接近，每个蛋白激酶可以将ATP分子的γ磷酸转移到底物上靶残基的羟基。以这种方式，可以基于底物的磷酸化与去磷酸化状态来调节底物下游的信号传导途径。虽然有许多方法来测定蛋白激酶的活性，但它们中的大多数在技术上是繁琐的和/或是间接的或基于淬灭反应。该方案描述了一种采用荧光肽底物来真实的实时检测蛋白激酶磷酸化的测定法。该测定基于肽底物的磷酸化导致附加荧光团的荧光发射强度增加的原理。我们将该测定的应用扩展到如何评估激酶的时间依赖性共价抑制的示例。© 2024 Wiley Periodicals LLC.基本方案1：使用荧光肽测量蛋白激酶活性替代方案：使用荧光酶标仪测量蛋白激酶活性支持方案：使用sox荧光团标记肽基本方案2：使用荧光肽测量蛋白激酶的时间依赖性ATP竞争性抑制
Protein kinases catalyze the phosphorylation of proteins most commonly on Ser, Thr, and Tyr residues and regulate many cellular events in eukaryotic cells, such as cell cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP‐binding site and one or more substrate‐binding site(s) that exhibit recognition features for different protein substrates. By bringing ATP and a substrate into proximity, each protein kinase can transfer theγphosphate of the ATP molecule to a hydroxyl group of the target residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated based on the phosphorylated versus dephosphorylated status of the substrate. Although there are a number of ways to assay the activity of protein kinases, most of them are technically cumbersome and/or are indirect or based on quenched reactions. This protocol describes an assay employing a fluorescent peptide substrate to detect phosphorylation by protein kinases in real time. The assay is based on the principle that the phosphorylation of the peptide substrate leads to an increase in the fluorescence emission intensity of an appended fluorophore. We extend the application of this assay to an example of how to assess time‐dependent covalent inhibition of kinases as well. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Measuring protein kinase activity using fluorescent peptidesAlternate Protocol: Measuring protein kinase activity using a fluorescence plate readerSupport Protocol: Labeling peptides with sox fluorophoreBasic Protocol 2: Measuring time‐dependent ATP‐competitive inhibition of protein kinases using fluorescent peptides