Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells
Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells
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DOI：
10.1016/j.molcel.2007.02.003
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发表时间：
2007-03-23
期刊：
影响因子：
16
通讯作者：
Fisher, Robert P.
中科院分区：
文献类型：
--
作者：
Larochelle, Stephane;Merrick, Karl A.;Fisher, Robert P.
细胞分裂受细胞周期蛋白依赖性激酶（CDK）控制。在后生动物中，S期开始与Cdk 2的激活相一致，而Cdk 1触发有丝分裂。Cdk 1和Cdk 2都需要细胞周期蛋白结合和T环磷酸化才能发挥全部活性。在后生动物中唯一已知的CDK激活激酶（CAK）是Cdk 7，它也是转录机制的一部分。为了测试体内对Cdk 7的需求，我们用对人类癌细胞中的大体积ATP类似物敏感的版本替换了野生型Cdk 7。选择性抑制Cdk 7在G1期阻止Cdk 2/细胞周期蛋白复合物的激活（但不形成），并延迟S期。在G2期抑制Cdk 7阻断有丝分裂的进入并破坏Cdk 1/细胞周期蛋白B复合物的组装，表明Cdk 1激活的两个步骤-细胞周期蛋白结合和T环磷酸化-是相互依赖的。因此，通过结合化学遗传学和体细胞中的同源基因置换，我们揭示了Cdk 7在细胞周期中两个不同的执行点激活CDK的不同模式。
Cell division is controlled by cyclin-dependent kinases (CDKs). In metazoans, S phase onset coincides with activation of Cdk2, whereas Cdk1 triggers mitosis. Both Cdk1 and -2 require cyclin binding and T loop phosphorylation for full activity. The only known CDK-activating kinase (CAK) in metazoans is Cdk7, which is also part of the transcription machinery. To test the requirements for Cdk7 in vivo, we replaced wild-type Cdk7 with a version sensitive to bulky ATP analogs in human cancer cells. Selective inhibition of Cdk7 in G1 prevents activation (but not formation) of Cdk2/cyclin complexes and delays S phase. Inhibiting Cdk7 in G2 blocks entry to mitosis and disrupts Cdk1/cyclin B complex assembly, indicating that the two steps of Cdk1 activation-cyclin binding and T loop phosphorylation-are mutually dependent. Therefore, by combining chemical genetics and homologous gene replacement in somatic cells, we reveal different modes of CDK activation by Cdk7 at two distinct execution points in the cell cycle.