Abstract

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.

Highlights

  • The Polo kinase is a master regulator of mitosis and cytokinesis conserved from yeasts to humans

  • To test the importance of polo-box domain (PBD) function for this localization pattern, we mutated residues analogous to those required for phosphobinding in human Plk[1] (W395F, H518A, K520A = PoloPBDmut) 14

  • PoloPBDmut-GFP fails to localize to kinetochores and the midbody, consistent with previous work showing that Plk[1] and orthologs require prior phosphorylation of their targets to localize at these sites[8] (Supplementary Fig. 1b)

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Summary

Introduction

The Polo kinase is a master regulator of mitosis and cytokinesis conserved from yeasts to humans. 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 Cdc[25] phosphatase and lead to mitotic and developmental defects in Drosophila. Polo relocalizes to the central spindle and remains enriched at the midbody ring in late stages of cytokinesis Polo localization at these structures requires the polo-box domain (PBD), a Cterminal protein interaction domain that defines the PLK family[10, 11]. The activities of the KD and PBD of Polo are regulated during cell division by an intramolecular mechanism governed by posttranslational modifications and protein interactions[13]. Phosphorylation of the KD at the activation loop was proposed to promote the dissociation of the KD from the PBD, the precise mechanism is unclear[15, 17, 18]

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