cdc25C induces mitosis by activating the cdc2-cyclin B complex. The intracellular localization of cyclin B1 is regulated in a cell cycle-specific manner, and its entry into the nucleus may be required for the initiation of mitosis. To determine the cellular localization of cdc25C, monoclonal antibodies specific for cdc25C were developed and used to demonstrate that in human cells, cdc25C is retained in the cytoplasm during interphase. A deletion analysis identified a 58-amino-acid region (amino acids 201 to 258) in cdc25C that was required for the cytoplasmic localization of cdc25C. This region contained a specific binding site for 14-3-3 proteins, and mutations in cdc25C that disrupted 14-3-3 binding also disrupted the cytoplasmic localization of cdc25C during interphase. cdc25C proteins that do not contain a binding site for 14-3-3 proteins showed a pancellular localization and an increased ability to induce premature chromosome condensation. The cytoplasmic localization of cdc25C was not altered by ␥ irradiation or treatment with the nuclear export inhibitor leptomycin B. These results suggest that 14-3-3 proteins may negatively regulate cdc25C function by sequestering cdc25C in the cytoplasm.In eukaryotic cells, an active cyclin-dependent kinase complex, cdc2-cyclin B1, promotes entry into mitosis. Prior to mitosis, kinase activity is inhibited by phosphorylation of the cdc2 catalytic subunit on two residues, threonine 14 (T14) and tyrosine 15 (Y15) (reviewed in reference 46). Several kinases, including wee1 (3, 21, 48), mik1 (32), and myt1 (36, 42), phosphorylate cdc2 at residues T14 and Y15 and inhibit mitotic progression. Entry into mitosis is dependent on dephosphorylation of the T14 and Y15 residues, resulting in the formation of an active cdc2-cyclin B complex (reviewed in reference 46). Inhibition of cdc2 dephosphorylation is a target of the DNA replication and DNA damage checkpoints in both yeast and mammalian cells (reviewed in references 45 and 46

Control of proliferation and differentiation by the retinoblastoma tumor suppressor protein (pRB) and related family members depends upon their interactions with key cellular substrates. Efforts to identify such cellular targets led to the isolation of a novel protein, EID-1 (for E1A-like inhibitor of differentiation 1). Here, we show that EID-1 is a potent inhibitor of differentiation and link this activity to its ability to inhibit p300 (and the highly related molecule, CREB-binding protein, or CBP) histone acetylation activity. EID-1 is rapidly degraded by the proteasome as cells exit the cell cycle. Ubiquitination of EID-1 requires an intact C-terminal region that is also necessary for stable binding to p300 and pRB, two proteins that bind to the ubiquitin ligase MDM2. A pRB variant that can bind to EID1, but not MDM2, stabilizes EID-1 in cells. Thus, EID-1 may act at a nodal point that couples cell cycle exit to the transcriptional activation of genes required for differentiation.