A novel landscape of nuclear human CDK2 substrates revealed by in situ phosphorylation
A novel landscape of nuclear human CDK2 substrates revealed by in situ phosphorylation
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DOI：
10.1126/sciadv.aaz9899
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发表时间：
2020-04-01
期刊：
影响因子：
13.6
通讯作者：
Clurman, Bruce E.
中科院分区：
文献类型：
--
作者：
Chi, Yong;Carter, John H.;Clurman, Bruce E.
细胞周期蛋白依赖性激酶2（CDK 2）控制细胞分裂，是致癌信号的中心。我们使用“原位”方法来鉴定从表达CDK 2的细胞中分离的细胞核内的CDK 2底物，所述细胞经工程改造以使用腺苷5 '-三磷酸类似物。我们确定了117个候选底物，其中40%是已知的CDK底物。先前未知的候选物被验证为CDK 2底物，包括LSD 1、DOT 1 L和Rad 54。许多染色质相关蛋白的鉴定可能已经通过标记条件来促进，所述标记条件通过内源性细胞周期蛋白保留核结构和生理性CDK 2调节。候选底物包括调节组蛋白修饰、染色质、转录和RNA/DNA代谢的蛋白质。这些蛋白质中的许多也共存于多蛋白质复合物中，包括表观遗传调节剂，这可能提供细胞分裂和CDK 2介导的其他细胞过程之间的新联系。因此，原位磷酸化显示候选底物具有高验证率，并应容易适用于其他核激酶。
Cyclin-dependent kinase 2 (CDK2) controls cell division and is central to oncogenic signaling. We used an "in situ" approach to identify CDK2 substrates within nuclei isolated from cells expressing CDK2 engineered to use adenosine 5'-triphosphate analogs. We identified 117 candidate substrates, similar to 40% of which are known CDK substrates. Previously unknown candidates were validated to be CDK2 substrates, including LSD1, DOT1L, and Rad54. The identification of many chromatin-associated proteins may have been facilitated by labeling conditions that preserved nuclear architecture and physiologic CDK2 regulation by endogenous cyclins. Candidate substrates include proteins that regulate histone modifications, chromatin, transcription, and RNA/DNA metabolism. Many of these proteins also coexist in multi-protein complexes, including epigenetic regulators, that may provide new links between cell division and other cellular processes mediated by CDK2. In situ phosphorylation thus revealed candidate substrates with a high validation rate and should be readily applicable to other nuclear kinases.