Importin-beta mediates Cdc7 nuclear import by binding to the kinase insert II domain, which can be antagonized by importin-alpha.
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.
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario P3E 2C6, Canada.
Byung Ju
BJ
Kim
Hoyun
H
Lee
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D010750:Phosphoproteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014162:Transfection; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D019913:Nuclear Localization Signals; D010750:Phosphoproteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases