Larochelle Stéphane, Merrick Karl A, Terret Marie-Emilie, Wohlbold Lara, Barboza Nora M, Zhang Chao, Shokat Kevan M, Jallepalli Prasad V, Fisher Robert P
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Mol Cell. 2007 Mar 23;25(6):839-50. doi: 10.1016/j.molcel.2007.02.003.
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.
细胞分裂受细胞周期蛋白依赖性激酶（CDK）控制。在多细胞动物中，S期起始与Cdk2的激活同时发生，而Cdk1触发有丝分裂。Cdk1和Cdk2都需要细胞周期蛋白结合和T环磷酸化才能完全激活。多细胞动物中唯一已知的CDK激活激酶（CAK）是Cdk7，它也是转录机制的一部分。为了在体内测试对Cdk7的需求，我们在人类癌细胞中将野生型Cdk7替换为对大分子ATP类似物敏感的版本。在G1期选择性抑制Cdk7可阻止Cdk2/细胞周期蛋白复合物的激活（但不阻止其形成）并延迟S期。在G2期抑制Cdk7会阻止进入有丝分裂并破坏Cdk1/细胞周期蛋白B复合物的组装，这表明Cdk1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，通过在体细胞中结合化学遗传学和同源基因替换，我们揭示了Cdk7在细胞周期中两个不同执行点激活CDK的不同模式。
