Abstract
Upon prolonged arrest in mitosis, cells undergo adaptation and exit mitosis without cell division. These tetraploid cells are arrested in the subsequent G1 phase in a spindle checkpoint and p53-dependent manner. p53 has long been known to be activated by spindle poison such as nocodazole and taxol, although the underlying mechanism is still unclear. In this study, evidence was presented which demonstrated that stabilization and activation of p53 by spindle disruption required the spindle checkpoint kinase TTK/hMps1. Down regulation of TTK/hMPS1 diminishes p53 response after spindle damage. In vitro, TTK/hMPS1 phosphorylates p53 on Ser15, Thr 18, and Ser20. Ablation of TTK/hMPS1 in vivo reduces spindle damage-induced p53 Thr18 phosphorylation and p53 response. Conversely, overexpression of TTK/hMPS1 enhances Thr18 phosphoylation and stabilizes p53 by disrupting the interaction with MDM2 and by abrogating MDM2-mediated p53 ubiquinylation. In addition, TTK/hMPS1 coimmunoprecipitates with p53 after spindle damage. Upon prolonged treatment with spindle poisons, down regulation of TTK/hMPS1 leads to polyploidy, a phenomenon closely mimicked by p53 ablation. TTK/hMPS1-mediated Thr18 phosphorylation enhances p53-dependent activation of not only p21 but also Lats2, two mediators of the post-mitotic checkpoint. Furthermore, a phospho-mimicking substitution at Thr18 (T18D) is more capable than the phospho-deficient mutant (T18A) in rescuing the tetraploid checkpoint defect of the p53-depleted cells. Our data indicate that by phosphorylating p53 Thr18, TTK/hMPS1 works with p53 to prevent polyploidy after spindle damage.