Cdk2-dependent and -independent pathways in E2F-mediated S phase induction
Cdk2-dependent and -independent pathways in E2F-mediated S phase induction
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DOI：
10.1074/jbc.275.9.6337
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发表时间：
2000-03-03
影响因子：
4.8
通讯作者：
Kato, JY
中科院分区：
文献类型：
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作者：
Arata, Y;Fujita, M;Kato, JY
转录因子E2F在高等真核细胞周期的G(1)期向S期转变中起着重要作用。虽然已经发现了许多E2F诱导基因，但从E2F到S期进入的生化级联反应仍有待研究。本研究在重金属诱导的金属硫蛋白启动子的控制下，制备了稳定转染的小鼠NIH3T3细胞，表达外源的人E2F-1，并分析了E2F-1介导的染色体DNA复制起始的分子机制。血清剥夺在G(0)/G(1)中阻滞的细胞中异位表达E2F-1，使其进入G(1)期并进入S期。在G(1)进展过程中，小鼠cyclin E被诱导表达，而cyclin D1被诱导表达，随后激活Cdk2。使用Cdk抑制蛋白p27、p18和p19的实验证明，E2F-1介导的S期进入需要Cdk2的活性，而不是Cdk4的活性。小染色体维持蛋白（MCM） 4和7是dna复制起始复合体（RC）的组成部分，在细胞周期中组成性地表达，尽管MCM基因是众所周知的e2f -1诱导基因。然而，这两种蛋白与染色质的紧密关联依赖于异位E2F-1的表达。相反，另一种RC成分Cdc45蛋白被证明是E2Fs的转录靶点，在E2F-1的作用下被诱导表达并随后结合到染色质上。利用化学cdk特异性抑制剂丁内酯I进行的实验表明，Cdk2活性仅对Cdc45蛋白的染色质结合是必需的，而对Cdc45蛋白的表达或MCM4和-7的染色质结合则不需要。这些结果表明，至少有两条独立的途径在E2F下游起作用，以启动S期；一个取决于Cdk2的活性，另一个则不是。
The transcription factor E2F plays an important role in G(1) to S phase transition in the higher eukaryotic cell cycle. Although a number of E2F-inducible genes have been identified, the biochemical cascades from E2F to the S phase entry remain to be investigated. In this study, we generated stably transfected mouse NIH3T3 cells that express exogenous human E2F-1 under the control of a heavy metal-inducible metallothionein promoter and analyzed the molecular mechanism of the E2F-1-mediated initiation of chromosomal DNA replication. Ectopic E2F-1 expression in cells arrested in G(0)/G(1) by serum deprivation enabled them to progress through G(1) and to enter S phase. During the G(1) progression, mouse cyclin E, but little of cyclin D1, was induced to express, which subsequently activated Cdk2. Experiments using the Cdk inhibitory proteins p27, p18, and p19 proved that the activity of Cdk2, but not of Cdk4, was required for S phase entry mediated by E2F-1. Minichromosome maintenance proteins (MCM) 4 and 7, the components of the DNA-replication initiation complex (RC), were constitutively expressed during the cell cycle, although the MCM genes are well known E2F-1-inducible genes. However, tight association of these two proteins with chromatin depended upon ectopic E2F-1 expression. In contrast, the Cdc45 protein, another RC component, which turned out to be a transcriptional target of E2Fs, was induced to express and subsequently bound to chromatin in response to E2F-1. Experiments utilizing a chemical Cdk-specific inhibitor, butyrolactone I, revealed that Cdk2 activity was required only for chromatin binding of the Cdc45 proteins, and not for the expression of Cdc45 or chromatin binding of MCM4 and -7, These results indicate that at least two separate pathways function downstream of E2F to initiate S phase; one depends upon the activity of Cdk2 and the other does not.