PXD018704

PXD018704 is an original dataset announced via ProteomeXchange.

Title	Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics_C4PR
Description	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.
HostingRepository	PRIDE
AnnounceDate	2024-10-07
AnnouncementXML	Submission_2024-10-07_10:39:52.539.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD018704
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Philip Brownridge
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	Label:13C(6)15N(2); Label:13C(6)15N(4); Oxidation; phosphorylated residue; Carbamidomethyl
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2020-04-21 01:56:44	ID requested
⏵ 1	2024-10-07 10:39:53	announced

Byrne DP, Clarke CJ, Brownridge PJ, Kalyuzhnyy A, Perkins S, Campbell A, Mason D, Jones AR, Eyers PA, Eyers CE, Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics. Biochem J, 477(13):2451-2475(2020) [pubmed]

10.1042/bcj20200309;

10.6019/PXD018704;

submitter keyword: Mass Spectrometry, centrinone

phosphoproteomics, Inhibitor,phosphorylation

substrate, PLK4

Claire E Eyers
contact affiliation	Centre for Proteome Research University of Liverpool Liverpool L69 7ZB
contact email	ceyers@liverpool.ac.uk
lab head
Philip Brownridge
contact affiliation	University of Liverpool
contact email	philipjb@liv.ac.uk
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/06/PXD018704

PRIDE project URI

• PRIDE
  1. PXD018704
     1. Label: PRIDE project
     2. Name: Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics_C4PR