Human Cdc7-related kinase complex - In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a critical threonine residue ofCdc7 by Cdks

huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, bi nds 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) Mel. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression sys tem. To facilitate purification of the kinase complex, glutathione S-transf erase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GS T-huCdc7 protein is inert as a kinase on its own, and phosphorylation absol utely depends on the presence of the ASK subunit, It autophosphorylates bot h 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 C dks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks fa cilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration betwe en Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc 7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corre sponding to the activating threonine of Cdk, with alanine (T376A mutant) dr amatically reduces kinase activity, indicative of kinase activation by phos phorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cd c2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.