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 cyc lin B/cdc2. In humans, at least two cdc25 isoforms have roles in contr olling the G(2)/M transition. Here we show, using bacterially expresse d recombinant proteins, that two cdc25B splice variants, cdc25B2 and c dc25B3, are capable of activating cyclin A/cdk2 and cyclin B/cdc2, but that mitotic hyperphosphorylation of these proteins increases their a ctivity toward only cyclin B1/cdc2, Cdc25C has only very low activity in its unphosphorylated form, add following hyperphosphorylation it wi ll efficiently catalyze the activation of only cyclin B/cdc2, This was reflected by the in vivo activity of the immunoprecipitated cdc25B an d cdc25C from interphase and mitotic HeLa cells. The increased activit y of the hyperphosphorylated cdc25s toward cyclin B1/cdc2 was in large part due to increased binding of this substrate. The substrate specif icity, activities; and timing of the hyperphosphorylation of cdc25B an d cdc25C during G(2) and M suggest that these two mitotic cdc25 isofor ms are activated by different kinases and perform different functions during progression through G(2) into mitosis.