Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis
Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis
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DOI：
10.1074/mcp.t600044-mcp200
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发表时间：
2007-02-01
影响因子：
7
通讯作者：
Koike, Tohru
中科院分区：
文献类型：
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作者：
Kinoshita-Kikuta, Emiko;Aoki, Yuri;Koike, Tohru
在此，我们描述了使用新型磷酸盐亲和聚丙烯酰胺凝胶电泳进行无标记激酶分析的三种应用。磷酸盐亲和位点是聚丙烯酰胺结合的双核 Mn2+ 复合物，可实现磷酸化蛋白质与其非磷酸化蛋白质的迁移率变化检测。第一个应用是体外激酶活性分析，用于分析磷酸化状态下的各种磷蛋白同种型。使用具有多个磷酸化位点的底物蛋白 Tau 测定六种激酶（糖原合成酶激酶 3 beta、细胞周期蛋白依赖性激酶 5/p35、蛋白激酶 A、丝裂原激活蛋白激酶 (MAPK)、酪蛋白激酶 II 和钙调蛋白依赖性蛋白激酶 II）的活性谱。由于磷酸化位点和化学计量的差异，每种激酶都表现出比非磷酸化 Tau 上移的特征性多电泳迁移带。第二个应用是体内激酶活性分析，用于分析细胞内信号转导中涉及的蛋白质磷酸化。通过随后使用抗 Shc 和抗 MAPK 抗体进行免疫印迹，A431 细胞中表皮生长因子诱导的 Shc 和 MAPK 磷酸化水平的时间过程变化可视化为高度上移的迁移带。第三个应用是体外激酶抑制分析，用于定量筛选激酶特异性抑制剂。使用底物 Abltide-GST（一种由 Abl 的特定底物肽和谷胱甘肽 S-转移酶组成的融合蛋白）和已批准的药物格列卫（ATP 竞争剂）测定酪氨酸激酶 Abl（一种组氨酸标记的重组小鼠 AN 激酶）的抑制谱。在激酶测定中，较慢迁移带（单磷酸化 Abltide-GST）随时间依赖性增加，而较快迁移带（非磷酸化 Abltide-GST）则减少。格列卫的剂量依赖性抑制是通过较快和较慢迁移带的比率变化来确定的，在 0.10 mm ATP 存在下，其 IC50 值为 1.6 μm。
Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3 beta, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse AN kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 mu m in the presence of 0.10 mm ATP.