Cell cycle- and chromatin binding state-dependent phosphorylation of human MCM heterohexameric complexes: A role for cdc2 kinase

The mammalian MCM protein family, presently with six members, exists in the nuclei in two forms, chromatin-bound and unbound. The former dissociates from chromatin with progression through the S phase. Recently, we have established a procedure to isolate chromatin-bound and unbound complexes containing all six human MCM (hMCM) proteins by immunoprecipitation. In the present study, we applied this procedure to HeLa cells synchronized in each of the G₁, S, and G₂/M phases and could detect hMCM heterohexameric complexes in all three. In addition, depending on the cell cycle and the state of chromatin association, hMCM2 and 4 in the complexes were found to variously change their phosphorylation states. Concentrating attention on G₂/M phase hyperphosphorylation, we found hMCM2 and 4 in the complexes to be good substrates for cdc2/cyclin B in vitro. Furthermore, when cdc2 kinase was inactivated in temperature-sensitive mutant murine FT210 cells, the G₂/M hyperphosphorylation of the murine MCM2 and MCM4 and release of the MCMs from chromatin in the G₂ phase were severely impaired. Taken together, the data suggest that the six mammalian MCM proteins function and undergo cell cycle- dependent regulation as heterohexameric complexes and that phosphorylation of the complexes by cdc2 kinase may be one of mechanisms negatively regulating the MCM complex-chromatin association.