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-␤ 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 ϳ10 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 Lys 306 and Lys 309 within the Kinase Insert II domain are critical for Cdc7 nuclear localization.

Identification and Characterization of Human Cdc7 Nuclear Retention and Export Sequences in the Context of Chromatin Binding

“…Therefore, it was previously suggested that kinase inserts may play regulatory functions (15). Consistent with this prediction, we reported previously that importin-␤ binds to Cdc7 at its Kinase Insert II and induces Cdc7 import into the nucleus (19).…”

“…Cells grown on a glass coverslip were transfected with plasmids using Lipofectamine PLUS TM as suggested by the supplier (Invitrogen). Plasmid Constructs-The pEGFP⅐huCdc7 recombinant plasmid was constructed by cloning a full-length cDNA encoding the entire human Cdc7 into the SmaI site of pEGFP-C1 (Clontech) (19). Various Cdc7 deletion mutants were generated by PCR-based cloning using pfu DNA polymerase (Invitrogen).…”

“…To generate tandem GFP-tagged (2ϫGFP) constructs, pEGFP⅐ Cdc7NES1, pEGFP⅐Cdc7NES2, pEGFP⅐Cdc7NES1⅐NES2, and pEGFP⅐MEKKNES were digested with AgeI and then ligated with a 790-bp fragment generated by double-digesting pEGFP-C1 with AgeI and XmaI. pGEX⅐huCdc7 recombinant plasmid was constructed as described previously (19). Various Cdc7 deletion mutants were generated by cloning mutant DNA fragments into pGEX-2T or pGEX-5X-1 (Amersham Biosciences).…”

Proliferating cell nuclear antigen in the cytoplasm interacts with components of glycolysis and cancer

“…We have recently found that PCNA contains a nuclear localization sequence (NLS) within the amino acid 101-120 segment [35], and its nuclear import appears to be mediated by importin b as PCNA binds to importin-b, but not importin-a [36]. These data suggest that cells have an active mechanism for the cytoplasmic localization of PCNA, which is yet to be elucidated.…”

Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication

“…52 For the resolution of Cdc7 phospho-isoforms, sample buffer was supplemented with 8M urea and SDS-PAGE was carried out utilizing a 10% resolving gel and at least 1 cm 3% stacking gel, run at 125 volts for 2.5 h to maximize resolution. Representative images of at least two independent experiments are shown.…”