Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics_C4PR

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 induces inhibitory effects on PLK4 and can lead to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and the extent of PLK4 signalling remains poorly characterised. We report an unbiased mass spectrometry-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing human cells exposed to the small molecule inhibitor centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to centrinone, resulting in PLK4 stabilization. A drug-resistant PLK4 mutant (G95R) confirmed several on-target effects of the compound towards PLK4. Using this experimental system, we report hundreds of centrinone-regulated phosphoproteins in U2OS cells, including centrosomal and cell cycle proteins and a variety of likely non cell-cycle substrates. Surprisingly, sequence interrogation of ~300 significantly downregulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation target sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm NMYC and PTPN12 as new 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Osteosarcoma Cell Line

SUBMITTER: Philip Brownridge  

LAB HEAD: Claire E Eyers

PROVIDER: PXD018704 | Pride | 2021-06-14

REPOSITORIES: Pride