Cell cycle regulation of chromatin binding and nuclear localization of human Cdc7-ASK kinase complex.
BACKGROUND
During the course of DNA replication, regulation of cellular localization and chromatin binding of involved factors plays critical roles. Cdc7 kinase is required for DNA replication and its kinase activity is cell cycle-regulated by its activation subunit Dbf4/ASK. In mammals, it is not known at which time point during the cell cycle Cdc7 and Dbf4/ASK proteins are imported into nuclei and loaded on to chromatin.


RESULTS
We have constructed a series of truncation and deletion derivatives of ASK and expressed them as fusion proteins with GFP in mammalian cells. Both Dbf4-motif-M and -C conserved in Dbf4/ASK protein family are required for huCdc7 kinase activation. Two stretches of amino acid sequences, NLS1 (P346KKKRIK) and NLS2 (K201RVGSGAQKTRTGRLKK), are important for ASK nuclear localization. In stable transformants expressing GFP-fused full-length ASK under the tetracycline inducible promoter, GFP-ASK protein accumulates in nuclei at the telophase, but its binding to chromatin does not reach a maximum until late G1, whereas huCdc7 is imported into nuclei and binds to chromatin at early G1. An important substrate of Cdc7-ASK at the G1/S transition is likely to be MCM. Indeed, over-expression of both huCdc7 and ASK results in the elevated phosphorylation of endogenous MCM2 protein, as manifested by appearance of the mobility-shifted form on SDS-PAGE, but does not cause any significant effects on cell cycle progression.


CONCLUSIONS
Nuclear localization and chromatin binding of endogenous huCdc7 and GFP-ASK expressed during the post-mitotic phase are independently regulated. Although GFP-ASK is presumably imported into nuclei through its two nuclear localization signals at telophase, it may require additional signals for chromatin binding, the level of which increases at late G1 phase.
Sato
Noriko
N
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. nrksato@ims.u-tokyo.ac.jp
Sato
Megumi
M
Nakayama
Masahito
M
Saitoh
Rika
R
Arai
Ken-ichi
K
Masai
Hisao
H
D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D002467:Cell Nucleus; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000378:metabolism*; D019927:Cyclin E / Q000378:metabolism; D004261:DNA Replication; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation; D016193:G1 Phase; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D007834:Lasers; D008164:Luminescent Proteins / Q000378:metabolism; D064111:Minichromosome Maintenance Complex Component 2; D008938:Mitosis; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D016196:S Phase; D017384:Sequence Deletion
D018797:Cell Cycle Proteins; D002843:Chromatin; D019927:Cyclin E; C485678:DBF4 protein, human; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D049452:Green Fluorescent Proteins; C068619:CDC7 protein, human; C489403:Cdc7 protein, mouse; D017346:Protein Serine-Threonine Kinases; D064111:Minichromosome Maintenance Complex Component 2