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J Biol Chem, Vol. 275, Issue 9, 6337-6345, March 3, 2000

Cdk2-dependent and -independent Pathways in E2F-mediated S Phase Induction^*

Yukinobu Arata, Masatoshi Fujita^§, Kiyoshi Ohtani^¶, Sho Kijima^¶, and Jun-ya Kato

From the  Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, the ^§ Laboratories of Viral Oncology, Research Institute, Aichi Cancer Center, Nagoya 464, and the ^¶  Human Gene Sciences Center, Graduate School of Dentistry, Tokyo Medical and Dental University, Tokyo 113-8510, Japan

The transcription factor E2F plays an important role in G₁ 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 G₀/G₁ by serum deprivation enabled them to progress through G₁ and to enter S phase. During the G₁ 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-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.

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^* This work was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-743-72-5541; Fax: 81-743-72-5549; E-mail: jkata@bs.aist-nara.ac.jp.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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