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First published online 26 September 2006
doi: 10.1242/jcs.03200

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CHK1 phosphorylates CDC25B during the cell cycle in the absence of DNA damage

¹ LBCMCP-CNRS UMR5088, IFR109, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France
² IPBS-CNRS UMR5089, Université Paul Sabatier, 205 route de Narbonne, 31077 Toulouse, France

View larger version (27K): [in a new window]   Fig. 1. CHK1 phosphorylates CDC25B on residues S230 and S563 in vitro. (A) Alignment of corresponding CHK1 phosphorylation sites in human CDC25A and CDC25B. (B) Recombinant HA-CDC25B protein was phosphorylated (+) or not (–) by CHK1. Western blot analyses were performed with anti-CDC25B-S230-P (S230p) or anti-CDC25B-S563-P (S563p) antibodies in the presence (+) or absence (–) of an excess of competing phosphorylated peptides (pp). (C) HeLa cells were either not treated (NT) or transiently transfected with wild-type EYFP-CDC25B (WT), EYFP-CDC25B S230A or EYFP-CDC25B S563A. Cell lysates were analysed by western blotting using the anti-CDC25B-S230-P or anti-CDC25B-S563-P antibodies (S230p or S563p, respectively). The total amount of expressed fusion CDC25 protein was detected with an anti-GFP antibody, and actin was used as loading control.

View larger version (54K): [in a new window]   Fig. 2. CDC25B is phosphorylated on S230 in vivo. (A) U2OS cells conditionally expressing HA-CDC25B were synchronized by double thymidine block. At the indicated times after release, cells were harvested and subjected to western blot analyses with monoclonal anti-HA and anti-CDC25B-S230-P antibodies. Actin was used as loading control. The cell-cycle distributions were determined at each time point by flow cytometry analyses and shown as the percentage of cells in G1-, S- or G2-phase. (B) HeLa cells transiently expressing the DsRed1-tagged DNA ligase I (red) were immunostained with anti-CDC25B-S230-P (S230p, green) and anti--tubulin (blue) antibodies. (C) HeLa cells were immunostained with anti-CDC25B-S230-P (green) and anti--tubulin (red) antibodies and co-stained with DAPI. Cells were selected in interphase or at different stages of mitosis. Yellow regions in merged images indicate centrosomal colocalization of the two proteins. (D) U2OS cells expressing HA-tagged CDC25B were immunostained as in C but, in comparison to control cells, labelling with the anti-CDC25B-S230-P antibody in competition with CHK1-phosphorylated recombinant HA-CDC25B (CDC25B-p) or unphosphorylated (CDC25B) protein. (E) HeLa cells were transfected with control or CDC25B siRNA. Cells were harvested 72 hours later and immunostained with the anti-CDC25B-S230-P (red) and anti--tubulin (green) antibodies (left panel) and the percentages of cells with centrosomes stained by the anti-CDC25B-S230-P antibody were determined (right panel). Magnification, 1000x.

View larger version (54K): [in a new window]   Fig. 3. In vivo phosphorylation of CDC25B on S230 depends on CHK1. (A) U2OS cells expressing HA-CDC25B were synchronized by double thymidine block and released. Six hours after release, cells were treated or not with UCN-01 for 1 hour, and subjected to western blot analyses using anti-CDC25B-S230-P, anti-CDC25B-S563-P, anti-CDC25B-S353-P, anti-HA, anti-cdc2 and anti-cdc2-Y15-P antibodies. Actin was used as loading control. (B,C) U2OS cells expressing HA-CDC25B were transfected with control (Scr) or CHK1 siRNA, or not transfected. Forty-eight hours post-transfection, cells were subjected to (B) western blot analyses with anti-CDC25B-S230-P, anti-CDC25B-S563-P, anti-CHK1 or anti-HA antibodies, with actin as loading control or (C) immunofluorescence analyses with anti-CDC25B-S230-P (green) and anti--tubulin (red) antibodies and co-stained with DAPI. Magnification, 400x.

View larger version (10K): [in a new window]   Fig. 4. Mutation of S230 activates CDC25B. (A) Exponentially growing HeLa cells were transfected with pEYFP-C1 (control vector, VC), wild-type EYFP-CDC25B (WT) or EYFP-CDC25B S230A. The cells were fixed 8 hours or 15 hours after transfection and stained with DAPI. The number of EYFP-positive cells displaying a condensed chromatin aspect was determined. The results presented are the average of two and three independent experiments for each time point (8 hours and 15 hours, respectively). (B) Model. CHK1 negatively regulates the activity of CDC25B through multiple phosphorylation events. S230 and S563 phosphorylations, for which data are presented in this report, are indicated in black and grey, respectively. Additional phosphorylations by other kinases occuring from S-phase until entry into mitosis, result in a global phosphorylation pattern that modulates the activity of CDC25B.