Global profiling of protein kinase activities in cancer cells by mass spectrometry
Global profiling of protein kinase activities in cancer cells by mass spectrometry
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DOI：
10.1016/j.jprot.2012.09.029
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发表时间：
2012-12-21
影响因子：
3.3
通讯作者：
Cutillas, Pedro R.
中科院分区：
文献类型：
--
作者：
Beltran, Luisa;Casado, Pedro;Cutillas, Pedro R.
蛋白激酶在细胞生物学中具有重要的控制功能，并与包括癌症在内的多种疾病有关。在这里，我们描述了一种以全球方式量化蛋白激酶活性的技术，而不存在对被研究细胞或组织中可能活跃的激酶的先入为主的概念。在全球酶活性图谱(GKAP)中，实验细胞裂解产物中存在的蛋白激酶在特定条件下磷酸化内源底物，也存在于裂解产物中。然后使用基于LC-MS/MS的标准磷酸蛋白质组学技术对反应产物进行定量。因此，该技术允许测量针对共同底物的激酶的结合活性，这些底物被LC-MS/MS检测为磷酸肽。在一个人上皮细胞系中，几乎400个激酶反应可以被定量，其中177个反应因EGF处理而增加，而在暴露于激酶抑制剂LY294002或U0126的细胞中，其他的减少。GKAP还检测到对激酶抑制剂敏感性不同的人类白血病细胞株的激酶活性模式存在显著差异。这些结果表明，GICAP检测并量化了数百种受生长因子或药物抑制剂调节的激酶活性，这些活性与癌细胞的表型和它们对激酶抑制剂的反应有关。(C)2012爱思唯尔B.V.保留所有权利。
Protein kinases have important functions in the control of cell biology and are implicated in several diseases including cancer. Here we describe a technique to quantify protein kinase activity in a global fashion and without preconception of the kinases that may be active in the cell or tissue under investigation. In Global Kinase Activity Profiling (GKAP), protein kinases present in experimental cell lysates phosphorylate endogenous substrates, also present in the lysate, under defined conditions. Reaction products are then quantified using standard phosphoproteomic techniques based on LC-MS/MS. The technique thus allows measuring the combined activities of kinases targeting common substrates, which are detected as phosphopeptides by LC-MS/MS. Almost four hundred kinase reactions could be quantified in a human epithelial cell line, 177 of which increased in response to EGF treatment while others decreased in cells exposed to the kinase inhibitors LY294002 or U0126. GKAP also detected marked differences in the patterns of kinase activities in human leukemia cell lines with different sensitivities to kinase inhibitors. These results reveal that GICAP detects and quantifies hundreds of kinase activities modulated by growth factors or pharmacological inhibitors, and that these activities correlate with the phenotypes of cancer cells and their responses to kinase inhibitors. (C) 2012 Elsevier B.V. All rights reserved.