cdc25C is a phosphatase, which activates the mitosispromoting factor cyclin B1/cdc2 by dephosphorylation, and thus triggers G 2 /M transition. The activity of cdc25C itself is controlled by phosphorylation of certain aminoacid residues, which among other things determines the subcellular localization of the enzyme. Here, we describe a new phosphorylation site at threonine 236 of cdc25C, which is phosphorylated by protein kinase CK2. This phosphorylation site is located near the nuclear localization signal (amino acids 239-245). We demonstrate that cdc25C interacts with importin b and the importin a/b heterodimer but not with importin a. We further found that a cdc25C phosphorylation mutant where threonine 236 was replaced by aspartic acid as well as cdc25C phosphorylated by CK2 binds importin b or the importin a/b heterodimer less efficiently than wild type or the corresponding alanine mutant. Furthermore, the cdc25C T236D shows a retarded uptake into the nucleus in a cell import assay. Inhibition of protein kinase CK2 enzyme activity in vivo resulted in an enhanced nuclear localization of cdc25C. Thus, phosphorylation of cdc25C at threonine 236 is an important signal for the retention of cdc25C in the cytoplasm.

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Abstract. The eukaryotic cell cycle is regulated by a network of different protein kinases and phosphatases which are by various mechanisms linked to the growth suppressor p53. Cell cycle regulation is quite similar from yeast to man. Although there is no endogenous p53 in yeast expression of human p53 led to growth arrest of yeast cells which can be suppressed by simultaneous overexpression of cdc25C, a phosphatase regulating entry into mitosis. Herein, we show that overexpression of cdc25C in mammalian cells is insufficient in suppressing a p53 induced growth arrest. We further show that p53 is co-immunoprecipitated with cdc25C and p53 inhibits the cdc25C phosphatase activity in a dose-dependent manner. Thus, our data show that p53 like other binding partners of cdc25C, regulates entry into mitosis by binding to cdc25C.

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