In the G2 phase cell cycle checkpoint arrest, the cdc25-dependent activatio
n of cyclin B/cdc2, a critical step in regulating entry into mitosis, is bl
ocked. Studies in yeast have demonstrated that the inhibition of cdc25 func
tion involves 14-3-3 binding to cdc25, In humans, two cdc25 isoforms have r
oles in G2/M progression, cdc25B and cdc25C, both bind 14-3-3, Abrogating 1
4-3-3 binding to cdc25C attenuates the G2 checkpoint arrest, but the contri
bution of 14-3-3 binding to the regulation of cdc25B function is unknown. H
ere we demonstrate that high level over-expression of cdc25B in G2 checkpoi
nt arrested cells can activate cyclin B/cdc2 and overcome the checkpoint ar
rest. Mutation of the major 14-3-3 binding site, S323, or removal of the N-
terminal regulatory domain are strong activating mutations, increasing the
efficiency with which the mutant forms of cdc25B not only overcome the arre
st, but also initiate aberrant mitosis, We also demonstrate that 14-3-3 bin
ding to the S323 site on cdc25B blocks access of the substrate cyclin/cdks
to the catalytic site of the enzyme, thereby directly inhibiting the activi
ty of cdc25B, This provides direct mechanistic evidence that 14-3-3 binding
to cdc25B can regulate its activity, thereby controlling progression into
mitosis.