Differential phosphorylation of Cdc25C phosphatase in mitosis

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Abstract

Cdc25 dual-specificity phosphatases coordinate entry into mitosis through activating dephosphorylation of the Mitosis-Promoting Factor, Cdk1-cyclin B1. Activation of Cdc25C at the G2/M transition, involves its dissociation from 14-3-3, together with its hyperphosphorylation on several sites within its regulatory N-terminal domain, mediated by cyclin-dependent kinases and Plk1. Growing evidence suggests that phosphorylation intermediates are likely to precede complete hyperphosphorylation of Cdc25C. To address whether such variants occur in mitotic cells, we raised antibodies directed against different mitotic phosphorylation sites of human Cdc25C, and characterized the phosphorylated species detectable in HeLa cells. In the present study, we provide first-time evidence for the existence of multiple species of Cdc25C in mitotic cell extracts, including full-length and splice variants with different phosphorylation patterns, thereby revealing an intricate network of Cdc25C phosphatases, likely to have distinct biological functions.

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Materials and methods

Cell culture, synchronization, and siRNA transfection. HeLa cells were cultured in DMEM supplemented with 10% FCS, at 37 °C in an atmosphere containing 5% CO2. Cell culture media, serum, and antibiotics were purchased from Invitrogen. Cells were synchronized in mitosis for 12 h with 250 μg/ml Nocodazole, and collected by mitotic shakeoff. siRNA smart-pools targeting Cdc25C and control siRNA (non-targeting) were obtained from Dharmacon. 50 nM siRNA were transfected twice at a 24 h interval with

Characterization of human Cdc25C variants in interphasic and mitotic cells

In order to characterize the different species of Cdc25C present in interphasic and mitotic HeLa cells, we probed cell extracts by Western blotting with a generic antibody directed against the C-terminus of the phosphatase (C-20). We identified several forms of Cdc25C, which we classified into three categories, according to their electrophoretic mobility (Fig. 1A). In asynchronous HeLa cell extracts, two major species of Cdc25C were detected: a doublet in the 60 kDa range (molecular weight range

Discussion

Mitotic hyperphosphorylation of Cdc25C is associated with an increase in its phosphatase activity and is readily detected through a significant shift in its electrophoretic mobility [3], [15], [16], [17], [18], [19], [20], [26]. Although it is commonly assumed that Cdc25C oscillates between an hypophosphorylated (Ser216 phosphorylated) inactive state, and an hyperphosphorylated active mitotic form, growing evidence suggests that intermediate phosphorylation variants are likely to occur [18],

Acknowledgments

This work was supported by the CNRS (Centre National de la Recherche Scientifique) and grants from the Association de Recherche contre le Cancer (ARC) and the French National Research Agency (ANR) to MCM. J.B. was supported by fellowships from the French Ministry of Research and La Ligue Nationale Contre le Cancer. We thank J.M. Donnay and J.-C. Mazur from the CRBM animal core facility for immunizations. We thank B. Ducommun, V. Baldin, P. Coopman, D. Fesquet, V. Dulic, and all members of the

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