Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a criticial threonine residue of Cdc7 bY Cdks.
huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D. , Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.
Masai
H
H
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, CREST, Japan.
Matsui
E
E
You
Z
Z
Ishimi
Y
Y
Tamai
K
K
Arai
K
K
D000957:Antigens, Viral, Tumor / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000502:physiology; D018844:Cyclin-Dependent Kinases / Q000502:physiology*; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D013912:Threonine / Q000378:metabolism*
D000957:Antigens, Viral, Tumor; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D064111:Minichromosome Maintenance Complex Component 2