14-3-3 Family Members Act Coordinately to Regulate Mitotic Progression

The mitosis promoting phosphatase, cdc25C, is a target of both the DNA replication and DNA damage checkpoint pathways. These pathways regulate cdc25C function, in part, by promoting the association of cdc25C with 14-3-3 proteins, which results in the retention of cdc25C in the cytoplasm. To determine which 14-3-3 proteins were required to regulate cdc25C function, we tested the ability of various 14-3-3 family members to form a complex with and negatively regulate cdc25C in human cells. Two 14-3-3 family members, 14-3-3epsilon and 14-3-3gamma specifically formed a complex with cdc25C but not with the 14-3-3 binding defective cdc25C mutant, S216A. In addition, 14-3-3epsilon and 14-3-3gamma inhibited the ability of cdc25C, but not the S216A mutant, to induce premature chromatin condensation (PCC) in U-2OS cells. These results suggested that the reduction in PCC by 14-3-3epsilon and 14-3-3gamma was due to inhibition of cdc25C function. In contrast, 14-3-3sigma was unable to form a complex with cdc25C, but was able to inhibit the ability of both wild type cdc25C and S216A to induce PCC. This suggests that 14-3-3sigma regulates entry into mitosis independently of cdc25C and 14-3-3epsilon and 14-3-3gamma. Thus, specific members of the 14-3-3 family of proteins may act coordinately to maintain the DNA replication checkpoint by regulating the activity of different cell cycle proteins.