Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle
Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle
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DOI：
10.1016/j.molcel.2008.07.007
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发表时间：
2008-08-08
期刊：
影响因子：
16
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
--
作者：
Daub, Henrik;Olsen, Jesper V.;Mann, Matthias
蛋白激酶是细胞信号传导的关键调节剂，其通过位点特异性磷酸化事件调节彼此的功能和活性。这些关键的调控修饰尚未得到全面研究，因为激酶的低细胞丰度导致其在以前的磷酸化蛋白质组研究中的代表性不足。在这里，我们结合联合收割机激酶选择性亲和纯化与定量质谱分析细胞周期调控的蛋白激酶。这种蛋白质组学方法使我们能够从S期和M期阻滞的人类癌细胞中定量219种蛋白激酶。我们在蛋白激酶上鉴定了1000多个磷酸化位点。有趣的是，一半的激酶磷酸肽在有丝分裂中上调。我们的数据揭示了许多未知的M期诱导的磷酸化位点的激酶建立有丝分裂功能。我们还发现了涉及许多蛋白激酶的潜在磷酸化网络，这些蛋白激酶以前没有参与有丝分裂进程。这些结果为激酶的功能研究及其通过位点特异性磷酸化的调节提供了广泛的知识基础。
Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.