Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics.
PLK4
inhibitor
phosphoproteomics
protein kinase
signalling
substrate specificity
Journal
The Biochemical journal
ISSN: 1470-8728
Titre abrégé: Biochem J
Pays: England
ID NLM: 2984726R
Informations de publication
Date de publication:
17 07 2020
17 07 2020
Historique:
received:
21
04
2020
revised:
27
05
2020
accepted:
05
06
2020
pubmed:
6
6
2020
medline:
16
12
2020
entrez:
6
6
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32501498
pii: 225183
doi: 10.1042/BCJ20200309
pmc: PMC7338032
doi:
Substances chimiques
Leupeptins
0
Pyrimidines
0
Sulfones
0
centrinone
0
PLK4 protein, human
EC 2.7.1.-
Protein Serine-Threonine Kinases
EC 2.7.11.1
benzyloxycarbonylleucyl-leucyl-leucine aldehyde
RF1P63GW3K
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2451-2475Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/R000182/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/M012557/1
Pays : United Kingdom
Organisme : Cancer Research UK
ID : C1443/A22095
Pays : United Kingdom
Informations de copyright
© 2020 The Author(s).
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