Identification of CDK2 substrates in human cell lysates.
Identification of CDK2 substrates in human cell lysates.
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DOI：
10.1186/gb-2008-9-10-r149
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发表时间：
2008-10-13
期刊：
影响因子：
12.3
通讯作者：
Clurman BE
中科院分区：
文献类型：
--
作者：
Chi Y;Welcker M;Hizli AA;Posakony JJ;Aebersold R;Clurman BE
工程化激酶和硫代磷酸盐富集用于鉴定人细胞裂解物中的许多候选CDK 2底物。蛋白质磷酸化调节多种生物过程。然而，大量的蛋白激酶及其底物产生了极其复杂的磷酸化蛋白质组。细胞周期蛋白依赖性激酶-CDKs -包括一类调节细胞周期进程的酶，并在肿瘤发生中发挥重要作用。然而，尽管进行了大量研究，但已知的哺乳动物CDK底物数量有限。全面了解CDK功能需要鉴定其底物网络。我们描述了一种简单而有效的方法来识别复杂细胞裂解物中潜在的细胞周期蛋白A-CDK 2靶点。使用激酶工程策略结合化学富集和质谱，我们确定了180个潜在的细胞周期蛋白A-CDK 2底物和200多个磷酸化位点。这些候选人中约有10%在与细胞分裂相关的途径中发挥作用，绝大多数参与其他基本的细胞过程。我们已经验证了几个候选人作为直接细胞周期蛋白A-CDK 2底物，在我们通过质谱鉴定的相同位点上磷酸化，我们还发现一种新的底物，核糖体蛋白RL 12，在体内表现出位点特异性CDK 2依赖性磷酸化。我们使用工程化激酶和硫代磷酸富集的方法来鉴定细胞裂解物中大量的候选CDK 2底物。这些结果与其他最近的蛋白质组学研究一致，并表明CDKs通过细胞底物的大网络调节细胞分裂。这些方法是通用的，可以很容易地适用于鉴定许多其他蛋白激酶的直接底物。
Engineered kinases and thiophosphate enrichment were used to identify many candidate CDK2 substrates in human cell lysates. Protein phosphorylation regulates a multitude of biological processes. However, the large number of protein kinases and their substrates generates an enormously complex phosphoproteome. The cyclin-dependent kinases - the CDKs - comprise a class of enzymes that regulate cell cycle progression and play important roles in tumorigenesis. However, despite intense study, only a limited number of mammalian CDK substrates are known. A comprehensive understanding of CDK function requires the identification of their substrate network. We describe a simple and efficient approach to identify potential cyclin A-CDK2 targets in complex cell lysates. Using a kinase engineering strategy combined with chemical enrichment and mass spectrometry, we identified 180 potential cyclin A-CDK2 substrates and more than 200 phosphorylation sites. About 10% of these candidates function within pathways related to cell division, and the vast majority are involved in other fundamental cellular processes. We have validated several candidates as direct cyclin A-CDK2 substrates that are phosphorylated on the same sites that we identified by mass spectrometry, and we also found that one novel substrate, the ribosomal protein RL12, exhibits site-specific CDK2-dependent phosphorylation in vivo. We used methods entailing engineered kinases and thiophosphate enrichment to identify a large number of candidate CDK2 substrates in cell lysates. These results are consistent with other recent proteomic studies, and suggest that CDKs regulate cell division via large networks of cellular substrates. These methods are general and can be easily adapted to identify direct substrates of many other protein kinases.
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影响因子：
2.9
作者：
HIRIYANNA, KT;BAEDKE, D;INGEBRITSEN, TS
通讯作者：
INGEBRITSEN, TS
影响因子：
4.8
作者：
Kane, S;Sano, H;Lienhard, GE
通讯作者：
Lienhard, GE
影响因子：
4.3
作者：
Graub, Remo;Lancero, Hope;Bernstein, Harold S.
通讯作者：
Bernstein, Harold S.
DOI：
10.1083/jcb.149.5.1073
发表时间：
2000-05-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Hirota T;Morisaki T;Nishiyama Y;Marumoto T;Tada K;Hara T;Masuko N;Inagaki M;Hatakeyama K;Saya H
通讯作者：
Saya H
DOI：
10.1073/pnas.0708966105
发表时间：
2008-02-05
影响因子：
11.1
作者：
Blethrow, Justin D.;Glavy, Joseph S.;Shokat, Kevan M.
通讯作者：
Shokat, Kevan M.