Rational design of homogenous protein kinase assay platforms that allow both fluorometric and colorimetric signal readouts.
Rational design of homogenous protein kinase assay platforms that allow both fluorometric and colorimetric signal readouts.
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DOI：
10.1039/b609529a
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发表时间：
2006-10
影响因子：
--
通讯作者：
K. Tomizaki;Hisakazu Mihara
中科院分区：
文献类型：
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作者：
K. Tomizaki;Hisakazu Mihara
蛋白激酶在调节许多细胞生物学过程的信号通路中发挥重要作用，包括细胞凋亡、细胞生长和细胞外刺激的分化。设计均一的蛋白激酶分析平台，包括设计有效的蛋白激酶底物，对于探索磷酸化蛋白质组是必不可少的。在这里，我们描述了一种独特的基于显色的分析(CHROBA)技术，用于直接测量蛋白激酶活性。CHROBA是一种新型的化学传感器系统，它根据引入到多肽底物中的螺吡喃衍生物的光致变色和热力学性质产生信号。检测蛋白激酶活性的ChROBA技术包括以下五个步骤：(I)磷酸化，(Ii)反应混合物的光漂白，(Iii)加入离子聚合物(S)，(Iv)黑暗孵育，(V)信号读出。这种简单的“终点”分析方法允许定量测量蛋白激酶A、Src蛋白酪氨酸激酶、c-Abl蛋白酪氨酸激酶和蛋白激酶Calpha的活性，即使有过量的ATP也是如此。我们的结果表明，净电荷在+2到0之间的螺吡喃多肽底物适合于本方法。这些信息应该有助于合理设计不同的蛋白激酶分析平台。目前的ChROBA技术可以适应具有荧光和比色读数的微板格式，并将用于高通量药物发现和磷蛋白质组的分析。
Protein kinases play important roles in signaling pathways that regulate many cellular biological processes, including apoptosis, cell growth, and differentiation in response to extracellular stimuli. Design of homogenous protein kinase assay platforms including design of potent protein kinase substrates is essential for exploration of the phosphoproteome. Here, we describe a unique chromism-based assay (CHROBA) technique for the direct measurement of protein kinase activities. The CHROBA is a novel chemosensor system that produces signals based on the photochromic and thermodynamic properties of a spiropyran derivative incorporated into peptide substrates. The CHROBA technique for detecting protein kinase activities involves the following five steps: (i) phosphorylation, (ii) photobleaching of the reaction mixture, (iii) addition of ionic polymer(s), (iv) incubation in the dark, and (v) signal readout. This simple 'end-point' assay method allows quantitative measurements of protein kinase A, Src protein tyrosine kinase, c-Abl protein tyrosine kinase, and protein kinase Calpha activities even with excess ATP. Our results showed that spiropyran-containing peptide substrates with net charges between +2 and 0 are suitable for the present CHROBA method. This information should aid in the rational design of diverse protein kinase assay platforms. The present CHROBA technique can be adapted to a microplate format with both fluorometric and colorimetric readouts and would be useful for high-throughput drug discovery and analysis of the phosphoproteome.