The spindle checkpoint delays anaphase onset in cells with mitotic spindle defects. Here, we show that Chk1, a component of the DNA damage and replication checkpoints, protects vertebrate cells against spontaneous chromosome missegregation and is required to sustain anaphase delay when spindle function is disrupted by taxol, but not when microtubules are completely depolymerized by nocodazole. Spindle checkpoint failure in Chk1-deficient cells correlates with decreased Aurora-B kinase activity and impaired phosphorylation and kinetochore localization of BubR1. Furthermore, Chk1 phosphorylates Aurora-B and enhances its catalytic activity in vitro. We propose that Chk1 augments spindle checkpoint signaling and is required for optimal regulation of Aurora-B and BubR1 when kinetochores produce a weakened signal. In addition, Chk1-deficient cells exhibit increased resistance to taxol. These results suggest a mechanism through which Chk1 could protect against tumorigenesis through its role in spindle checkpoint signaling.

Chk1 phosphorylates Aurora B to promote its full activation during mitotic prometaphase.

Cdc14A and Cdc14B knockout cells with double-strand breaks still arrest in G2, but they fail to repair the damage.

Chk1 is phosphorylated within its C-terminal regulatory domain by the upstream ATM/ ATR kinases during checkpoint activation, however how this modulates Chk1 function is poorly understood. Here, we show that Chk1 kinase activity is rapidly stimulated in a cell cycle phase-specific manner in response to both DNA damage and replication arrest, and that the extent and duration of activation correlates closely with regulatory phosphorylation at serines (S) S317, S345, and S366. Despite their evident co-regulation, substitutions of individual Chk1 regulatory sites with alanine (A) residues have differential effects on checkpoint proficiency and kinase activation. Thus, whereas Chk1 S345 is essential for all functions tested, mutants lacking S317 or S366 retain partial proficiency for G2/ M and S/ M checkpoint arrests triggered by DNA damage or replication arrest. These phenotypes reflect defects in Chk1 kinase induction, since the mutants are either partially (317A, 366A) or completely (345A) resistant to kinase activation. Importantly, S345 phosphorylation is impaired in Chk1 S317A and S366A mutants, suggesting that modification of adjacent SQ sites promotes this key regulatory event. Finally, we provide biochemical evidence that Chk1 catalytic activity is stimulated via a de-repression mechanism.

Activated Akt suppresses checkpoint activation by cells in late G2: although they are able to detect DNA damage, the repair pathway is put on hold until mitosis is complete.