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

Abstract

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.

Differential mitotic degradation of the CDC25B phosphatase variants

“…CDC25B phosphatase activity appears to be highly regulated by phosphorylation mechanisms. A number of regulatory kinases that activate or inhibit CDC25B have been identified (Baldin et al, 1997a(Baldin et al, , 2003Bulavin et al, 2002;Theis-Febvre et al, 2003;Dutertre et al, 2004;Cazales et al 2005;Manke et al, 2005;Mirey et al, 2005;Lemaire et al, 2006;Schmitt et al, 2006) (our unpublished data). These phosphorylation events modulate the catalytic activity, the interactions with regulatory partners such as 14-3-3 proteins, the localization and probably also the stability of CDC25B.…”

Promoter hijack reveals pericentrin functions in mitosis and the DNA damage response

“…25 It has been reported that centrosomal Chk1 regulates this activation through its inhibition of the Cdk1-activating Cdc25B phosphatase. [26][27][28] In this model, centrosomal Chk1 localization is regulated by pericentrin, with pericentrin being controlled by Mcph1/BRIT1 (hereafter Mcph1), a centrosomal and nuclear protein that also plays a part in the DNA damage response. 29 However, an alternative model has also been advanced, in which a nuclear Chk1 signal restrains Cdk1 activation, with a different kinase activating the centrosomal Cdk1.…”

Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

“…One important aspect of centrosome biology is the control of cyclin B1/ CDK1(CDC2) activity for entry into mitosis [55]. Following DNA damage, premature initiation of mitosis is prevented by inhibiting centrosome-associated cyclin B1/ CDK1(CDC2) through CDC25 phosphatase inactivation [56,57]. In HeLa cells treated by VP16, both CDK1(CDC2) and phospho-Bcl-xL(ser49) accumulate in centrosomes during the G2 checkpoint (Supplemental Fig.…”