Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria.
Proteomics. 2010 May;10(10):2015-25. doi: 10.1002/pmic.200900749.
Signaling networks regulate cellular responses to external stimuli through post-translational modifications such as protein phosphorylation. Phosphoproteomics facilitate the large-scale identification of kinase substrates. Yet, the characterization of critical connections within these networks and the identification of respective kinases remain the major analytical challenge. To address this problem, we present a novel approach for the identification of direct kinase substrates using chemical genetics in combination with quantitative phosphoproteomics. Quantitative identification of kinase substrates (QIKS) is a novel-screening platform developed for the proteome-wide substrate-analysis of specific kinases. Here, we aimed to identify substrates of mitogen-activated protein kinase/Erk kinase (Mek1), an essential kinase in the mitogen-activated protein kinase cascade. An ATP analog-sensitive mutant of Mek1 (Mek1-as) was incubated with a cell extract from Mek1 deficient cells. Phosphorylated proteins were analyzed by LC-MS/MS of IMAC-enriched phosphopeptides, labeled differentially for relative quantification. The identification of extracellular regulated kinase 1/2 as the sole cytoplasmic substrates of MEK1 validates the applicability of this approach and suggests that QIKS could be used to identify substrates of a wide variety of kinases.
信号转导网络通过蛋白质磷酸化等翻译后修饰来调节细胞对外界刺激的反应。磷酸蛋白质组学有助于大规模鉴定激酶底物。然而，这些网络中的关键连接的描述以及相应激酶的鉴定仍然是主要的分析挑战。为了解决这个问题，我们提出了一种使用化学遗传学结合定量磷酸蛋白质组学来鉴定直接激酶底物的新方法。定量鉴定激酶底物（QIKS）是一种新的筛选平台，用于特定激酶的蛋白质组范围的底物分析。在这里，我们旨在鉴定丝裂原激活的蛋白激酶/细胞外信号调节激酶（Mek1）的底物，Mek1 是丝裂原激活的蛋白激酶级联中的必需激酶。用 Mek1 缺陷细胞的细胞提取物孵育 Mek1 的 ATP 类似物敏感突变体（Mek1-as）。通过 IMAC 富集磷酸肽的 LC-MS/MS 分析磷酸化蛋白，并进行相对定量标记。鉴定细胞外调节激酶 1/2 为 MEK1 的唯一细胞质底物验证了这种方法的适用性，并表明 QIKS 可用于鉴定各种激酶的底物。
