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J. Biol. Chem., Vol. 281, Issue 17, 12041-12049, April 28, 2006

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Importin- Mediates Cdc7 Nuclear Import by Binding to the Kinase Insert II Domain, Which Can Be Antagonized by Importin-^*

Byung Ju Kim and Hoyun Lee¹

From the Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario P3E 2C6, Canada, Tumour Biology Group, Northeastern Ontario Regional Cancer Program at the Sudbury Regional Hospital, Sudbury, Ontario P3E 5J1, Canada, and the Department of Medical Sciences, Northern Ontario School of Medicine, Sudbury, Ontario P3E 2C6, Canada

We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin- binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin- and - bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin- is 10 times higher than for importin- at low protein concentrations of an equimolar ratio. Immunodepletion of importin-, but not importin-, abrogates Cdc7 nuclear import, and the addition of importin- to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-, but not anti-importin- antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin- to Cdc7. Further studies by site-directed mutagenesis suggest that Lys³⁰⁶ and Lys³⁰⁹ within the Kinase Insert II domain are critical for Cdc7 nuclear localization.

Received for publication, November 28, 2005 , and in revised form, January 25, 2006.

^* This work was supported by Canadian Institutes of Health Research Grant MOP-57706 and the National Cancer Institute of Canada Grant 016072 (to H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed. Tel.: 705-522-6237 (ext. 2703); E-mail: hlee{at}hrsrh.on.ca.

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