Study of the docking-dependent PLK1 phosphorylation of the CDC25B phosphatase.

CDC25 (A, B and C) phosphatases control cell cycle progression through the timely dephosphorylation and activation of cyclin-dependent kinases (CDK). At mitosis the CDC25B phosphatase activity is dependent on its phosphorylation by multiple kinases impinging on its localisation, stability and catalytic activity. Here we report that prior phosphorylation of CDC25B by CDK1 enhances its substrate properties for PLK1 in vitro, and we also show that phosphorylated S50 serves as a docking site for PLK1. Using a sophisticated strategy based on the sequential phosphorylation of CDC25B with (16)O and (18)O ATP prior to nanoLC-MS/MS analysis we identified 13 sites phosphorylated by PLK1. This study illustrates the complexity of the phosphorylation pattern and of the subsequent regulation of CDC25B activity.

The polo-like kinase 1 regulates CDC25B-dependent mitosis entry.

Activation of cyclin-dependent kinase complexes (CDK) at key cell cycle transitions is dependent on their dephosphorylation by CDC25 dual-specificity phosphatases (CDC25A, B and C in human). The CDC25B phosphatase plays an essential role in controlling the activity of CDK1-cyclin B complexes at the entry into mitosis and together with polo-like kinase 1 (PLK1) in regulating the resumption of cell cycle progression after DNA damage-dependent checkpoint arrest in G2. In this study, we analysed the regulation of CDC25B-dependent mitosis entry by PLK1. We demonstrate that PLK1 activity is essential for the relocation of CDC25B from the cytoplasm to the nucleus. By gain and loss of function analyses, we show that PLK1 stimulates CDC25B-induced mitotic entry in both normal conditions and after DNA-damage induced G2/M arrest. Our results support a model in which the relocalisation of CDC25B to the nucleus at the G2-M transition by PLK1 regulates its mitotic inducing activity.

NanoLC-MS/MS analysis provides new insights into the phosphorylation pattern of Cdc25B in vivo: full overlap with sites of phosphorylation by Chk1 and Cdk1/cycB kinases in vitro.

NanoLC-MS/MS analysis was used to characterize the phosphorylation pattern in vivo of CDC25B3 (phosphatase splice variant 1) expressed in a human cell line and to compare it to the phosphorylation of CDC25B3 by Cdk1/cyclin B and Chk1 in vitro. Cellular CDC25B3 was purified from U2OS cells conditionally overexpressing the phosphatase. Eighteen sites were detectably phosphorylated in vivo. Nearly all existing (S/T)P sites were phosphorylated in vivo and in vitro. Eight non(S/T)P sites were phosphorylated in vivo. All these sites could be phosphorylated by kinase Chk1, which phosphorylated a total of 11 sites in vitro, with consensus sequence (R/K) X(2-3) (S/P)-non P. Nearly half of the sites identified in this study were not previously described and were not homologous to sites reported to be phosphorylated in other CDC25 species. We also show that in vivo a significant part of CDC25B molecules can be hyperphosphorylated, with up to 13 phosphates per phosphatase molecule.

CDC25B phosphorylated by pEg3 localizes to the centrosome and the spindle poles at mitosis.

The phosphatase CDC25B is one of the key regulators that control entry into mitosis through the dephosphorylation and subsequent activation of the cyclin-dependent kinases. Here we study the phosphorylation of CDC25B at mitosis by the kinase pEg3, a member of the KIN1/PAR-1/MARK family. Using mass spectrometry analysis we demonstrate that CDC25B is phosphorylated in vitro by pEg3 on serine 169, a residue that lies within the B domain. Moreover, using phosphoepitope-specific antibodies we show that serine 169 is phosphorylated in vivo, that this phosphorylated form of CDC25B accumulates during mitosis, and is localized to the centrosomes. This labelling is abrogated when pEg3 expression is repressed by RNA interference. Taken together, these results support a model in which pEg3 contributes to the control of progression through mitosis by phosphorylation of the CDC25 phosphatases.

Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition.

Aurora-A protein kinase, which is the product of an oncogene, is required for the assembly of a functional mitotic apparatus and the regulation of cell ploidy. Overexpression of Aurora-A in tumour cells has been correlated with cancer susceptibility and poor prognosis. Aurora-A activity is required for the recruitment of CDK1-cyclin B1 to the centrosome prior to its activation and the commitment of the cell to mitosis. In this report, we demonstrate that the CDC25B phosphatase, an activator of cyclin dependent kinases at mitosis, is phosphorylated both in vitro and in vivo by Aurora-A on serine 353 and that this phosphorylated form of CDC25B is located at the centrosome during mitosis. Knockdown experiments by RNAi confirm that the centrosome phosphorylation of CDC25B on S353 depends on Aurora-A kinase. Microinjection of antibodies against phosphorylated S353 results in a mitotic delay whilst overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of CDC25B. Our results demonstrate that Aurora-A phosphorylates CDC25B in vivo at the centrosome during mitosis. This phosphorylation might locally participate in the control of the onset of mitosis. These findings re-emphasise the role of the centrosome as a functional integrator of the pathways contributing to the triggering of mitosis.