Importin-β mediates Cdc7 nuclear import by binding to the Kinase Insert II domain, which can be antagonized by importin-α

Abstract

We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin-β binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin-α and -β bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin-β is ∼10 times higher than for importin-α at low protein concentrations of an equimolar ratio. Immunodepletion of importin-β, but not importin-α, abrogates Cdc7 nuclear import, and the addition of importin-β to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-β, but not anti-importin-α antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin-α 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. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.