Abstract 843 Poster Session I, Saturday, 5/1 (poster 285)

G2 arrest caused by DNA damage is an important mechanism by which the fidelity of the human genome is preserved. In lower eukaryotes, G2 progression and mitotic entry requires the coordinated expression of many genes, however, mechanisms controlling their transcription remain largely unknown. We recently cloned human Cdc5 (hCdc5), a novel transcription factor that plays a positive regulatory role in G2 and mitosis ( Bernstein et al. (1997) J Biol Chem 272:5833; Bernstein et al. (1998) J Biol Chem 273:4666). Upon serum stimulation, hCdc5 is phosphorylated and translocates to the nucleus in dividing cells. Overexpression of hCdc5 in mammalian cells causes growth acceleration, while a dominant negative hCdc5 mutant delays entry into mitosis. While hCdc5 has a phase-specific effect on the cell cycle, it is uniformly expressed in dividing cells, suggesting that its activity in G2 is regulated through some post-translational mechanisms. We now demonstrate that hCdc5 is phosphorylated through a mitogen-activated pathway. We have expressed various domains of the protein as His6-fusions in E. coli, and have demonstrated that the nuclear localization, transactivation, and carboxyl domains of the protein are phosphorylated in vitro. Through the use of inhibitors, we have further demonstrated that the transactivation domain specifically is phosphorylated by protein kinase C. These studies provide the first evidence that hCdc5 is phosphorylated through a mitogen-activated signaling pathway, and lay the groundwork for investigating the role of these events in regulating hCdc5's activities during G2 and mitosis. Ultimately, the elucidation of mechanisms by which this cell cycle controller is regulated may provide new insights into how genomic fidelity is preserved.