A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates
A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates
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DOI：
10.1038/nmeth747
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发表时间：
2005-04-01
期刊：
影响因子：
48
通讯作者：
Imperiali, B
中科院分区：
文献类型：
--
作者：
Shults, MD;Janes, KA;Imperiali, B
直接从复杂细胞混合物中定量蛋白激酶活性的新方法对于理解生物调控途径至关重要。在这里，描述了一种基于荧光的化学传感器策略，用于直接测量粗制哺乳动物细胞裂解物中的激酶活性。我们首先为每个靶蛋白激酶设计了一种新的荧光肽报告底物。这些激酶化学传感器很容易被重组靶酶磷酸化，并在磷酸化后经历几倍的荧光增强。然后，使用未分离的细胞裂解物，建立了一种可重复性、线性和高度选择性地监测目标激酶细胞活性变化的均相激酶分析方法。一般的方法是为蛋白激酶Akt开发的，然后很容易扩展到测量蛋白激酶A(PKA)和丝裂原活化的蛋白激酶相关蛋白激酶2(MK2)的活性。该分析平台可立即用于研究细胞粗提物中的蛋白激酶信号。
New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a severat-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell Lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase-associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.