A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation

Pooja Sharma,David R Spring,Jamie E Stokes,Maxim Rossmann,Robert Mahen,Ashok R Venkitaraman,Marko Hyvönen,Amy Emery,Bryn Hardwick,Grahame J Mckenzie,David J Huggins,Dominique L Kunciw

doi:10.1038/s41598-019-50702-2

Pooja Sharma, David R Spring + Show 10 more

Open Access

https://doi.org/10.1038/s41598-019-50702-2

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Abstract

The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.

Highlights

The human polo-like kinase polo-like kinase 1 (PLK1) coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins
While green fluorescent protein (GFP)-PLK1AAD or GFP-PLK1AM overexpression restores bipolar spindle formation, it is less effective in counteracting chromosome congression defects, with only 11.5 ± 0.7% or 5.5 ± 0.7% of cells respectively showing normal chromosomal congression (Fig. 3A). These results suggest that the Tyr pocket and the phospho-substrate binding groove of the polo-box domain (PBD) are both essential for normal chromosome congression but not bipolar spindle formation, whereas PLK1 kinase activity is essential for both processes
The canonical PLK1 PBD substrate, NEDD1 (Neural precursor cell Expressed Developmentally Downregulated gene1), is a centrosomal protein primed by CDK1 phosphorylation[47] to bind the PBD during mitotic progression[48]. Consistent with this interaction, we find that GFP-PLK1 Wild-type (PLK1Wt) can be co-immunoprecipitated with NEDD1 in extracts prepared from cells synchronized in mitosis (Fig. 4B, final right-hand lane, and Supplementary Fig. S6)

Summary

Introduction

The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. The human polo-like kinase 1 (PLK1) is a key regulator of chromosome segregation during mitosis, through its essential biological functions in chromosome condensation[1], cohesin dissociation from chromosomes[2], mitotic entry[3], centrosome maturation[4], kinetochore function[5], spindle assembly[6,7], and exit from mitosis[8,9,10] These disparate functions are mediated through the phosphorylation of proteins that bind to chromatin, centrosomes or kinetochores. Our results provide a structural and functional rationale for targeting the Tyr pocket to create selective chemical inhibitors that modulate substrate recognition by the polo-like kinases

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