Kinoshita-Kikuta Emiko, Aoki Yuri, Kinoshita Eiji, Koike Tohru
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Kasumi 1-2-3, Hiroshima University, Hiroshima 734-8553, Japan.
Mol Cell Proteomics. 2007 Feb;6(2):356-66. doi: 10.1074/mcp.T600044-MCP200. Epub 2006 Nov 5.
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-3beta, 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 Abl 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 microM in the presence of 0.10 mM ATP.
在此，我们描述了使用一种新型的磷酸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。磷酸亲和位点是一种与聚丙烯酰胺结合的双核Mn2+复合物，它能够从非磷酸化对应物中检测磷酸化蛋白的迁移率变化。第一种应用是体外激酶活性分析，用于分析不同磷酸化状态的磷蛋白同种型。使用具有多个磷酸化位点的底物蛋白Tau，测定了六种激酶（糖原合酶激酶-3β、细胞周期蛋白依赖性激酶5/p35、蛋白激酶A、丝裂原活化蛋白激酶（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，每种激酶都显示出从非磷酸化Tau向上迁移的特征性多个电泳迁移带。第二种应用是体内激酶活性分析，用于分析参与细胞内信号转导的蛋白质磷酸化。通过随后用抗-Shc和抗-MAPK抗体进行免疫印迹，将A431细胞中表皮生长因子诱导的Shc和MAPK磷酸化水平的时间进程变化显示为高度向上迁移的条带。第三种应用是体外激酶抑制分析，用于激酶特异性抑制剂的定量筛选。使用底物Abltide-GST（一种由Abl的特异性底物肽和谷胱甘肽S-转移酶组成的融合蛋白）和已批准的药物格列卫（一种ATP竞争性抑制剂），测定了酪氨酸激酶Abl（一种组氨酸标签的重组小鼠Abl激酶）的抑制谱。在激酶测定中，迁移较慢的条带，单磷酸化的Abltide-GST，随时间依赖性增加，而迁移较快的条带，非磷酸化的Abltide-GST，则减少。格列卫的剂量依赖性抑制通过较快和较慢迁移条带比例的变化来确定，在存在0.10 mM ATP的情况下，其IC50值为1.6 microM。
