CDC25B phosphatase is a centrosomal substrate of the Aurora A protein kinase

Stéphanie DUTERTRE2, Martine CAZALES1, Muriel QUARANTA1, Carine FROMENT3, Valerie TRABUT1, Christine DOZIER1, Bernard MONSARRAT3, Claude PRIGENT2 and Bernard DUCOMMUN1*

1 - LBCMCP-CNRS UMR5088- IFR109 "Institut d’Exploration Fonctionnelle des Génomes"
Université Paul Sabatier, 118 route de Narbonne, 31062 TOULOUSE, FRANCE
2 -  Groupe Cycle Cellulaire - CNRS  UMR6061 - IFR97 "Génomique Fonctionnelle et Santé", Université de Rennes I, 2 avenue Léon Bernard, 35043 RENNES, FRANCE
3 - IPBS – CNRS UMR5089 205 route de Narbonne, 31077 TOULOUSE, FRANCE

* ducommun@cict.fr

INTRODUCTION
The Aurora A protein kinase, also known as the STK15 oncogene is the focus of an major interest since its overexpression has been correlated with high grade tumours(1, 2). Aurora A plays an essential role in the regulation of the events leading to the assembly of a functional mitotic apparatus and in the activation of CDK1/cyclin B1 at the centrosome (3). The CDC25B phosphatase is one of the three members of the CDC25 family in mammals that is expressed and active in late G2(4, 5). CDC25B localisation was shown to depend on NES and NLS sequences(6). CDC25B accumulation in the cytoplasm has been correlated with prophase spindle formation(5) and it was suggested that this phosphatase was responsible for the early activation of CDK1-cyclinB complexes, thus acting as a "starter" of mitosis.

RESULTS
We have shown by mass spectrometry that the CDC25B is phosphorylated in vitro by Aurora A on serine 353. Using specific antibodies raised against this phosphorylated residue, we demonstrate that this phosphorylation occurs in vivo. This phosphorylated form of CDC25B is detected in mitotic cells and is highly abundant at the centrosome from early prophase to anaphase. The centrosomal localisation of CDC25B phosphorylated on serine 353 is coincident with the activation of the Aurora A kinase, as detected with phosphoT288 specific antibodies. Furthermore, Aurora A knock-down using RNAi demonstrates that phosphorylation of CDC25B on serine 353 at the centrosome is dependent on the presence of Aurora A. At the functional level, microinjection of antibodies against S353(P) epitope in synchronised HeLa cells results in a mitotic delay and overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of the wild type CDC25B. 

CONCLUSION
In prophase CDK1-Cyclin B1 complexes are translocated to the centrosome (7) where Aurora A activity has been shown to be required for their activation and for the triggering of mitosis (3) (B). We propose, that phosphorylation of CDC25B by Aurora A is an essential step in this process  Our study provides a link between Aurora A kinase and the biochemical events that regulate the activation of CDKs. Moreover, it reemphasises the role of centrosome as the focal point of pathways participating to the onset of mitosis.

A very recent publication identifies Aurora A as a candidate low-penetrance tumour-susceptibility gene in mouse and human(8). Overexpression of CDC25B, that has also been reported in a number of tumours(9, 10), may therefore also participate in failure of the maintenance of genomic integrity and tumorogenesis. 

ACKNOWLEDGMENT
This work was supported by the C.N.R.S, l'Université Paul Sabatier, la Région Midi-Pyrénées, le pôle ARECA "Protéomique et Cancer" to BM and la Ligue Nationale Contre le Cancer to BD (Equipe labellisée 2001) and to CP (Equipe labellisée 2003). 

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