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Abstract
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 approximately 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 Lys306 and Lys309 within the Kinase Insert II domain are critical for Cdc7 nuclear localization.
Highlights
The Cdc7-Dbf4 kinase functions as a molecular switch for the activation of individual origins of DNA replication throughout S phase [1,2,3,4,5,6]
At 12 h post-transfection, endogenous Cdc7 was detected by indirect immunostaining with an anti-Cdc7 antibody. Both Importin-␣2 and - Interact with HuCdc7—We wanted to confirm the nuclear localization of tagged Cdc7 proteins at the onset of this investigation because several tagged recombinant Cdc7 proteins were to be used for this work
To learn how human Cdc7 (huCdc7) is transported into the nucleus, we analyzed the proteins associated with Cdc7 by a “pulldown” assay using glutathione-Sepharose beads coupled with recombinant GST or GST1⁄7Cdc7 as described under “Experimental Procedures.”
Summary
The Cdc7-Dbf4 kinase functions as a molecular switch for the activation of individual origins of DNA replication (oris) throughout S phase [1,2,3,4,5,6]. To learn how huCdc7 is transported into the nucleus, we analyzed the proteins associated with Cdc7 by a “pulldown” assay using glutathione-Sepharose beads coupled with recombinant GST or GST1⁄7Cdc7 as described under “Experimental Procedures.” PAGE and subsequent Western blot analysis of the proteins bound to the beads showed that importin-␣2, -, and MCM2 proteins interacted with Cdc7, whereas PCNA, 14-3-3 and importin-␣1 did not HuCdc7 Protein Showed Much Higher Binding Affinity for Importin- than for Importin-␣2 at Low Protein Concentrations—To determine whether the classical importin-␣/- pathway mediates Cdc7 nuclear import, we investigated the interactions between Cdc7 and importin-␣2 or - using an in vitro pull-down assay.
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