CELL CYCLE-DEPENDENT 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 i n the nuclei in two forms, chromatin-bound and unbound. The former dis sociates from chromatin with progression through the S phase. Recently , we have established a procedure to isolate chromatin-bound and unbou nd complexes containing all six human MCM (hMCM) proteins by immunopre cipitation. In the present study, we applied this procedure to HeLa ce lls synchronized in each of the G(1), S, and G(2)/M phases and could d etect hMCM heterohexameric complexes in all three. In addition, depend ing on the cell cycle and the state of chromatin association, hMCM2 an d 4 in the complexes were found to variously change their phosphorylat ion states. Concentrating attention on G(2)/M phase hyperphosphorylati on, we found hMCM2 and 4 in the complexes to be good substrates for cd c2/cyclin B in vitro. Furthermore, when cdc2 kinase was inactivated in temperature-sensitive mutant murine FT210 cells, the G(2)/M hyperphos phorylation of the murine MCM2 and MCM4 and release of the MCMs from c hromatin in the G(2) 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 mechani sms negatively regulating the MCM complex-chromatin association.