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First published online 5 May 2004
doi: 10.1242/jcs.01108

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

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

View larger version (39K): [in a new window]   Fig. 1. Aurora-A phosphorylates CDC25B on serine 353 in vitro and in vivo. (A) MS/MS spectrum of the monophosphorylated peptide, 353-SVTPPEEQQEAEEPK-367 (doubly charged precursor ion, MH22+, at m/z 889.38) displays series of b- and y-ions [according to Biemann's nomenclature (Biemann, 1990)], intense doubly charged y13 (at m/z 756,4) together with weak mono-charged b2 (at m/z 266.9) and indicating that the serine 353 residue is phosphorylated and not threonine 355. (B) CDC25B alignment with Aurora-A consensus phosphorylation site. (C) Recombinant proteins were incubated with purified recombinant Aurora-A at 37°C for 30 minutes. The samples were analysed by w estern blotting with the 35C1 monoclonal anti-Aurora-A or affinity-purified polyclonal anti-serine 353 phosphorylated epitope – SE96 or monoclonal anti-maltose binding protein (MBP). MBP-CDC25B migrated as a doublet because of the presence of a degradation product. (D) Recombinant MBP-CDC25B and MBP-CDC25B S353A mutant were phosphorylated or not by Aurora-A as in C. Western blot analysis was performed with SE96 and anti-CDC25B antibodies. (E) Western blot analysis of CDC25B affinity-purified (15 ng/lane) from human U2OS cells expressing polyHis-tagged CDC25B, with SE96 antibody and anti-CDC25B polyclonal antibody was performed in the presence of 1000x molar excess of the phosphorylated peptide (lane 2), the unphosphorylated peptide (lane 3), or after prior incubation of the sample for 60 minutes at 30°C in the presence of phosphatase.

View larger version (38K): [in a new window]   Fig. 2. Serine 353-phosphorylated CDC25B is located at the centrosome. (A) CDC25B RNA interference. Western blot with anti-CDC25B polyclonal antibody, SE96 and anti-actin of total extracts from HeLa cells transfected or not with CDC25B RNAi or control (cont.) scrambled RNAi. (B,C) HeLa cells were fixed and immunofluorescence staining was performed as described previously (Davezac et al., 2000). Cells were also stained with 4'-6 diamino-2-phenylindole (DAPI). (B) Staining with SE96 antibodies of cells in prometaphase and interphase (top panels), and anaphase and interphase (lower panels). (C) Double immunofluorescent staining with SE96 and a -tubulin monoclonal antibody of cells in interphase (top panels) and prometaphase (lower panels); SE96 (red), -tubulin (green) and DAPI (blue). (D) HeLa C1 expressing a GFP-centrin fusion protein (Piel et al., 2001) were stained with SE96 antibodies; SE96 (red), GFP-centrin (green) and DAPI (blue).

View larger version (72K): [in a new window]   Fig. 3. Timing of serine 353 phosphorylation of CDC25B at the centrosome. Double immunofluorescence staining of HeLa cells selected at different stages of mitosis with SE96 and lamin A monoclonal antibodies (mouse anti-human lamin); SE96 (red), lamin A (green) and DAPI (blue).

View larger version (55K): [in a new window]   Fig. 4. Active Aurora-A and serine 353-phosphorylated CDC25B localise at prophase and metaphase centrosomes. (A) HeLa cells were fixed and subjected to double immunofluorescence staining with SE96 polyclonal and Aurora-A monoclonal antibodies; SE96 (green), Aurora-A (red) and DAPI (blue). Photomicrographs of cells representative of each stage of mitosis are shown. Interphase (A), prophase (B), prometaphase (C), metaphase (D), anaphase A (E) and anaphase B (F). (B) HeLa cells were fixed and stained with monoclonal Aurora-A antibodies and polyclonal antibodies against threonine 288-phosphorylated Aurora-A. Selected cell cycle phases are shown.

View larger version (51K): [in a new window]   Fig. 5. Inhibition of Aurora-A expression by RNA interference shut down serine 353 phosphorylation. HeLa C1 cells expressing GFP-centrin (Piel et al., 2001) were transfected with Aurora-A RNAi or control scrambled RNAi. Immunofluorescence staining was performed with Aurora-A monoclonal antibodies (A), with SE96 anti-serine 353-phosphorylated CDC25B polyclonal antibody (B), or with a CDC25B polyclonal antibody. Cells were also stained with DAPI (blue). (D) Lysates from HeLa cells treated as above were subjected to western blot analysis with anti-Aurora-A and anti ß-tubulin as a loading control. (E) Quantification of immunofluorescence signal was performed using Metamorph software on images taken with a CoolsnapHQ camera.