Hyperphosphorylation of the N-terminal domain of Cdc25 regulates activity toward cyclin B1/Cdc2 but not cyclin A/Cdk2

Cdc25 regulates entry into mitosis by regulating the activation of cyclin B/cdc2. In humans, at least two cdc25 isoforms have roles in controlling the G(2)/M transition. Here we show, using bacterially expressed recombinant proteins, that two cdc25B splice variants, cdc25B2 and cdc25B3, are capable of activating cyclin A/cdk2 and cyclin B/cdc2, but that mitotic hyperphosphorylation of these proteins increases their activity toward only cyclin B1/cdc2, Cdc25C has only very low activity in its unphosphorylated form, add following hyperphosphorylation it will efficiently catalyze the activation of only cyclin B/cdc2, This was reflected by the in vivo activity of the immunoprecipitated cdc25B and cdc25C from interphase and mitotic HeLa cells. The increased activity of the hyperphosphorylated cdc25s toward cyclin B1/cdc2 was in large part due to increased binding of this substrate. The substrate specificity, activities; and timing of the hyperphosphorylation of cdc25B and cdc25C during G(2) and M suggest that these two mitotic cdc25 isoforms are activated by different kinases and perform different functions during progression through G(2) into mitosis.