Mutation of cyclin/cdk phosphorylation sites in HsCdc6 disrupts a late step in initiation of DNA replication in human cells
Mutation of cyclin/cdk phosphorylation sites in HsCdc6 disrupts a late step in initiation of DNA replication in human cells
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DOI：
10.1091/mbc.11.12.4117
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发表时间：
2000-12-01
影响因子：
3.3
通讯作者：
Fanning, E
中科院分区：
文献类型：
--
作者：
Herbig, U;Griffith, JW;Fanning, E
细胞周期蛋白依赖性激酶（Cdk）是促进DNA复制起始的关键酶，可能是通过磷酸化参与触发G1/S转换的关键调控蛋白。人Cdc 6（HsCdc 6）是启动DNA复制所需的蛋白质，在体外和体内被Cdk磷酸化。在这里，我们报告说，HsCdc 6与突变在潜在的Cdk磷酸化位点在体外磷酸化差的Cdk，但保留所有其他生化活性的野生型蛋白测试。将突变的HsCdc 6蛋白显微注射到人细胞中阻断了DNA复制的起始或减缓了S期进程。突变体HsCdc 6的抑制作用在G1/S转变时消失，表明Cdk对HsCdc 6的磷酸化对于人类细胞DNA复制启动的后期步骤至关重要。
Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells.