Deficiency in Chromosome Congression by the Inhibition of Plk1 Polo Box Domain-dependent Recognition

Nobumoto Watanabe,Tomomi Sekine,Masatoshi Takagi,Jun-Ichi Iwasaki,Naoko Imamoto,Hisashi Kawasaki,Hiroyuki Osada

doi:10.1074/jbc.m805308200

Nobumoto Watanabe, Tomomi Sekine + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m805308200

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Abstract

Polo-like kinase 1 (Plk1) is one of the key regulators of mitotic cell division. In addition to an N-terminal protein kinase catalytic domain, Plk1 possesses a phosphopeptide binding domain named polo box domain (PBD) at its C terminus. PBD is postulated to be essential for Plk1 localization and substrate targeting. Here, we developed a high-throughput screening system to identify inhibitors of PBD-dependent binding and screened a chemical library. We isolated a benzotropolone-containing natural compound derived from nutgalls (purpurogallin (PPG)) that inhibited PBD-dependent binding in vitro and in vivo. PPG not only delayed the onset of mitosis but also prolonged the progression of mitosis in HeLa cells. Although apparently normal bipolar spindles were formed even in the presence of PPG, the perturbation of chromosome alignment at metaphase plates activated the spindle assembly checkpoint pathway. These results demonstrate the predominant role of PBD-dependent binding on smooth chromosome congression at metaphase.

Highlights

Where it is involved in the regulation of the spindle assembly checkpoint pathway and proper chromosome segregation
The bacterial lysates expressing GST-monomeric Venus (mVenus)-polo box domain (PBD) were mixed with the test compounds and put into each well; unbound PBD was washed away, and bound PBD was quantitated by spectrofluorometry
When small molecules that compete with the phosphopeptides for PBD binding are present in the assay, PBD will fluoresce less

Summary

Introduction

Where it is involved in the regulation of the spindle assembly checkpoint pathway and proper chromosome segregation. Using PPG, we have shown that PBD-dependent binding is predominantly required for stabilization of the mitotic spindle and smooth mitotic chromosome congression. Because Plk2 is expressed only in the G1 phase of the cell cycle [39], the effects of PPG on the progression of M-phase as demonstrated below are considered to be caused by the inhibition of Plk1 PBD-dependent binding.

Results

Conclusion