14-3-3 Gamma is required to enforce both the incomplete S phase and G2 DNA damage checkpoints

Checkpoint pathways inhibit mitotic progression by inducing the phosphorylation of serine 216 in cdc25C resulting in the generation of a 14-3-3 binding site on cdc25C. Two 14-3-3 isoforms, 14-3-3epsilon and 14-3-3gamma form a complex with cdc25C and inhibit cdc25C function. To examine the contribution of 14-3-3gamma to checkpoint regulation, the expression of 14-3-3gamma was inhibited in HCT116 cells using vector based RNA interference. A transient reduction in the expression of 14-3-3gamma in HCT116 cells resulted in an override of both the incomplete S phase and the G(2) DNA damage checkpoint. A 14-3-3gamma knockdown clone also showed an override of both checkpoint pathways. These phenotypes were reversed upon expression of a shRNA resistant 14-3-3gamma cDNA. Override of the G(2) DNA damage checkpoint pathway was accompanied by a decrease in the levels of inhibitory phosphorylation on cdc25C and cdk1. However, there was no difference in the gamma-H2AX foci formation and levels of phospho-chk1 and phospho-chk2, suggesting that activation of the DNA damage checkpoint response and subsequent activation of the checkpoint kinases Chk1 and Chk2 was not perturbed. These results suggest that the override of checkpoint observed in 14-3-3gamma knockdown cells is due to failure to inhibit cdc25C function.