Centrosome-associated Chk1 prevents premature activation of cyclin-B–Cdk1 kinase

Abstract

Entry into mitosis occurs after activation of Cdk1, resulting in chromosome condensation in the nucleus and centrosome separation, as well as increased microtubule nucleation activity in the cytoplasm. The active cyclin-B1-Cdk1 complex first appears at the centrosome, suggesting that the centrosome may facilitate the activation of mitotic regulators required for the commitment of cells to mitosis. However, the signalling pathways involved in controlling the initial activation of Cdk1 at the centrosome remain largely unknown. Here, we show that human Chk1 kinase localizes to interphase, but not mitotic, centrosomes. Chemical inhibition of Chk1 resulted in premature centrosome separation and activation of centrosome-associated Cdk1. Forced immobilization of kinase-inactive Chk1 to centrosomes also resulted in premature Cdk1 activation. Conversely, under such conditions wild-type Chk1 impaired activation of centrosome-associated Cdk1, thereby resulting in DNA endoreplication and centrosome amplification. Activation of centrosomal Cdk1 in late prophase seemed to be mediated by cytoplasmic Cdc25B, whose activity is controlled by centrosome-associated Chk1. These results suggest that centrosome-associated Chk1 shields centrosomal Cdk1 from unscheduled activation by cytoplasmic Cdc25B, thereby contributing to proper timing of the initial steps of cell division, including mitotic spindle formation.

Cdk-mediated phosphorylation of Chk1 is required for efficient activation and full checkpoint proficiency in response to DNA damage

“…During the G2/M transition, Cdk1 phosphorylates Chk1 at Ser286 and Ser301 to promote Chk1 transport from the nucleus to the cytoplasm during prophase and trigger a positive feedback loop that contributes to Cdk1 activation in the nucleus (Kramer et al, 2004;Enomoto et al, 2009). Subsequent experiments revealed that modification of these sites could also be induced in response to DNA damage or DNA synthesis inhibition, presumably in interphase cells (Ikegami et al, 2008).…”

“…Mitotic hyper-phosphorylation of Chk1 seems to promote translocation from the nucleus to the cytoplasm in prophase, thus releasing an inhibitory effect of Chk1 on Cdk1 to promote rapid mitotic entry (Enomoto et al, 2009). It might also modulate Chk1 association with the centrosome, where Cdk1 activation begins (Kramer et al, 2004). Conversely, Chk1 S286 and S301 phosphorylation can also be induced under stress conditions, such as hydroxyurea or UV, when entry to mitosis is blocked by checkpoint activation.…”

Cyclin A/cdk2 coordinates centrosomal and nuclear mitotic events

“…Our study also reveals that cyclin A/cdk2 coordinates the activation of the centrosomal and major soluble pool of cyclin B/cdk1. Activation of cyclin B/cdk1 is coordinated such that it is first detected at the centrosome during prophase, preceding activation of the remaining cytoplasmic and translocated nuclear pools, by no more than 20-30 min (De Souza et al, 2000;Kramer et al, 2004). Inhibition of G 2 phase cyclin A/cdk2 by either depletion of cyclin A or inhibition of cdk2 resulted in the loss of this coordination of the activation of the centrosomal and soluble pools of cyclin B/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.…”