期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 281, 期 17, 页码 12041-12049出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M512630200
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We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin-beta binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin-alpha and -beta bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin-beta is similar to 10 times higher than for importin-alpha at low protein concentrations of an equimolar ratio. Immunodepletion of importin-beta, but not importin-alpha, abrogates Cdc7 nuclear import, and the addition of importin-beta to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-beta, but not anti-importin-alpha antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin-alpha to Cdc7. Further studies by site-directed mutagenesis suggest that Lys(306) and Lys(309) within the Kinase Insert II domain are critical for Cdc7 nuclear localization.
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