Mitonc- and g2 checkpoint-control

Checkpoints maintain the order and fidelity of the eukaryotic cell cycle by preventing cell cycle progression at inappropriate times. The DNA replication and DNA damage checkpoints block mitosis in the presence of unreplicated and damaged DNA, respectively. We have identified a pathway involving 14-3-3 proteins, that prevents Cdc25C from functionally interacting with Cdc2 when DNA damage is detected. We identified a regulatory phosphorylation site on Cdc25C that mediates the binding of 14-3-3p to Cdc25C. Cdc25C is phosphorylated on serine 216 and bound to 14-3-3p during interphase but not during mitosis. We created a mutant of Cdc25C containing alanine for serine at position 216 Cdc25(S216A). This Cdc25C mutant is not phosphorylated on serine 216 in vivo and does not bind to 14-3-3p. Cells expressing Cdc25C(S216A) advance into mitosis in the presence of damaged DNA indicating that the damage checkpoint is compromised in cells expressing a form of Cdc25C that cannot bind to 14-3-3p. The DNA damage checkpoint kinase Chklp, phosphorylates Cdc25C on serine 216, the 14-3-3p binding site. These findings suggest a model whereby unreplicated DNA activates Chkl to maintain Cdc25C in its serine 216phosphorylated form. Serine 216 phosphorylation maintains Cdc25C in a 14-3-3p bound form thereby preventing Cdc25C from activating Cdc2 and as a consequence the onset of mitosis is blocked.