Abstract

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely ‘non-canonical’ substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.

Highlights

  • Polo-like kinases (PLKs) are key cell cycle Ser/Thr kinases conserved in metazoan organisms

  • These data are consistent with a very significant decrease in Polo-like kinase 4 (PLK4) G95R binding to centrinone, but not Mg-adenosine 50-triphosphate (ATP) (Supplementary Figure S1E), which is predicted to manifest as cellular centrinone drug-resistance in the presence of physiological levels of competing ATP

  • We show that exposure of human cells to the PLK4 inhibitor centrinone generates unexpected diverse effects on the phosphoproteome

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Summary

Introduction

Polo-like kinases (PLKs) are key cell cycle Ser/Thr kinases conserved in metazoan organisms. Four kinase-domain-containing PLK family members are found in most kinomes, and each PLK polypeptide is thought to serve specific functions in cells. Polo-like kinase 4 (PLK4) is the central regulator of centriole assembly [1,2]. PLK4 activity is rate-limiting for centriole duplication, a process that requires hierarchical recruitment of many evolutionarily conserved core proteins: PLK4/SAK/ STK18/ZYG-1 itself, SAS-5/Ana2/STIL, SPD-2/DSpd-2/CEP192, Sas and Sas4/CPAP to a single assembly site on the mother centriole [3], driven by its role in phosphorylating key components of the centriolar duplication machinery, notably STIL. Overexpression of PLK4 induces centrosome amplification from pre-existing centrioles and drives de novo centriole assembly [1,2,3,4,5,6,7,8].

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