CHK1 phosphorylates CDC25B during the cell cycle in the absence of DNA damage.
CDC25B is one of the three human phosphatases that activate the CDK-cyclin complexes, thereby triggering cell-cycle progression and division. Commitment to early mitotic events depends on the activation of a centrosomal pool of CDK1-cyclin-B1, and CDC25B is thought to be involved in initiating this centrosomal CDK1-cyclin-B1 activity. Centrosome-associated checkpoint kinase 1 (CHK1) has been proposed to contribute to the proper timing of a normal cell division cycle by inhibiting the activation of the centrosomal pool of CDK1. Here, we show that CDC25B is phosphorylated by CHK1 in vitro on multiple residues, including S230 and S563. We demonstrate these phosphorylations occur in vivo and that they are dependent on CHK1 activity. S230 CHK1-mediated phosphorylation is detected in cell extracts during S phase and G2 phase in the absence of DNA damage. We show that the S230-phosphorylated form of CDC25B is located at the centrosome from early S phase until mitosis. Furthermore, mutation of S230 to alanine increases the mitotic-inducing activity of CDC25B. Our results support a model in which, under normal cell cycle conditions and in the absence of DNA damage, CHK1 constitutively phosphorylates CDC25B during interphase and thus prevents the premature initiation of mitosis by negatively regulating the activity of CDC25B at the centrosome.
Schmitt
Estelle
E
LBCMCP-CNRS UMR5088, IFR109, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France.
Boutros
Rose
R
Froment
Carine
C
Monsarrat
Bernard
B
Ducommun
Bernard
B
Dozier
Christine
C
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D034622:RNA Interference; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D020687:cdc25 Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D012694:Serine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D020687:cdc25 Phosphatases