Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis

We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initia tors of mitosis in human cells, To differentiate between the functions of t he two proteins, we have microinjected expression constructs encoding Cdc25 B or Cdc25C or their CFP-chimeras into synchronized tissue culture cells. T his assay allows us to express the proteins at defined points in the cell c ycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether t hey enter mitosis prematurely or at the correct time. We find that overexpr essing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mito sis, whether or not DNA replication is complete, whereas overexpressing Cdc 25C does not cause premature mitosis, Overexpressing Cdc25C together with c yclin B1 does shorten the G2 phase and can override the unreplicated DNA ch eckpoint, but much less efficiently than overexpressing Cdc25B. These resul ts suggest that Cdc25B and Cdc25C do not respond identically to the same ce ll cycle checkpoints. This difference may be related to the differential lo calization of the proteins; Cdc25C is nuclear throughout interphase, wherea s Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data sugges t that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.