The cell cycle-dependent regulation of Haspin, the histone H3 Thr3 kinase, is key for proper spindle assembly and nuclear envelope formation

A hallmark of mitosis is the appearance of high levels of histone phosphorylation, yet the roles of these modifications remain largely unknown. Here, we study the mitotic function and regulation of Haspin, a mitotic histone kinase. Haspin phosphorylates histone H3 at threonine 3 (H3T3ph) and, using the Xenopus egg extract system we show that the kinase activity of Haspin is important for the chromosomal targeting and activation of Aurora B, a key regulator of mitotic progression. In Xenopus egg extracts depleted of Haspin, Aurora B is no longer properly recruited to chromosomes, leading to impairment of its spindle assembly function and resulting in monoaster formation and a decrease in spindle size. Persistence of Haspin activity and H3T3ph at exit from M phase results in failure of Aurora B to dissociate from chromosomes, causing a delay in chromosome decondensation and nuclear-reformation. The temporal regulation of this mitotic histone kinase is thus critical for proper cell cycle progression.;We show that H3T3ph is restricted to M-phase through the regulation of Haspin activity by two opposing forces: an N-terminally located positive regulator consisting of phosphorylations promoted by Cyclin-dependent kinase 1 (Cdk1) and Polo-like kinase 1 (Plx1), and a C-terminal inhibitory activity that is yet to be characterized. Plx1 binds specifically in metaphase to an evolutionarily conserved polo-box binding motif on the N terminus of Haspin in a Cdk1 dependent manner. Inhibition of Plx1 or a point mutation of the Plx1-docking site abolishes Haspin phosphorylation of H3T3. Moreover, these manipulations significantly reduce the phosphorylation-dependent shift on Haspin suggesting that Plx1 positively controls Haspin via a phosphorylation-dependent mechanism. The presence of Cdk1 and Plx1 at the N terminus is needed to antagonize a Haspin inhibitory activity located immediately upstream of the kinase domain. Deletion of a six amino acid basic patch (RKKKVQ) in this region bypasses the requirement for Plx1 in the activation of Haspin and renders the kinase constitutively active.;Taken together, our data suggest that Haspin integrates signals from Cdk1 and Plk1 and transmits them through H3T3ph to Aurora B to control spindle assembly and the timing of nuclear envelope reformation.