Greatwall kinase relocation from the nucleus to the cytoplasm is required at mitotic entry and is mediated by a phosphorylation-dependent mechanism targeting its central region.

The Polo kinase is a master regulator of mitosis and cytokinesis conserved from yeasts to humans. Polo is composed of an N-term kinase domain (KD) and a C-term polo-box domain (PBD), which regulates its subcellular localizations. The PBD and KD can interact and inhibit each other, and this reciprocal inhibition is relieved when Polo is phosphorylated at its activation loop. How Polo activation and localization are coupled during mitotic entry is unknown. Here we report that PBD binding to the KD masks a nuclear localization signal (NLS). Activating phosphorylation of the KD leads to exposure of the NLS and entry of Polo into the nucleus before nuclear envelope breakdown. Failures of this mechanism result in misregulation of the Cdk1-activating Cdc25 phosphatase and lead to mitotic and developmental defects in Drosophila. These results uncover spatiotemporal mechanisms linking master regulatory enzymes during mitotic entry.

Aurora B phosphorylation of the Polo kinase activation loop disrupts its binding to Map205 and central spindle microtubules, allowing it to be recruited to the site of cytokinesis.