Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131)

Ryan A. Denu; Madilyn M. Sass; James M. Johnson; Gregory K. Potts; Alka Choudhary; Joshua J. Coon; Mark E. Burkard

doi:10.1074/jbc.RA118.004867

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Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131)

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Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131)

Ryan A. Denu, Madilyn M. Sass, James M. Johnson, Gregory K. Potts, Alka Choudhary, Joshua J. Coon and Mark E. Burkard

The Journal of biological chemistry, Vol.294(16), pp.6531-6549

04/19/2019

DOI: 10.1074/jbc.RA118.004867

PMCID: PMC6484138

PMID: 30804208

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Abstract

The centrosome, consisting of two centrioles surrounded by a dense network of proteins, is the microtubule-organizing center of animal cells. Polo-like kinase 4 (PLK4) is a Ser/Thr protein kinase and the master regulator of centriole duplication, but it may play additional roles in centrosome function. To identify additional proteins regulated by PLK4, we generated an RPE-1 human cell line with a genetically engineered “analog-sensitive” PLK4AS, which genetically encodes chemical sensitivity to competitive inhibition via a bulky ATP analog. We used this transgenic line in an unbiased multiplex phosphoproteomic screen. Several hits were identified and validated as direct PLK4 substrates by in vitro kinase assays. Among them, we confirmed Ser-78 in centrosomal protein 131 (CEP131, also known as AZI1) as a direct substrate of PLK4. Using immunofluorescence microscopy, we observed that although PLK4-mediated phosphorylation of Ser-78 is dispensable for CEP131 localization, ciliogenesis, and centriole duplication, it is essential for maintaining the integrity of centriolar satellites. We also found that PLK4 inhibition or use of a nonphosphorylatable CEP131 variant results in dispersed centriolar satellites. Moreover, replacement of endogenous WT CEP131 with an S78D phosphomimetic variant promoted aggregation of centriolar satellites. We conclude that PLK4 phosphorylates CEP131 at Ser-78 to maintain centriolar satellite integrity.

cell division

centriolar satellite

centriole

centriole duplication

centrosome

CEP131

chemical biology

chemical genetics

chromosome segregation

mass spectrometry (MS)

microtubule

phosphoproteomics

PLK4

Details

Title: Subtitle: Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131)
Creators: Ryan A. Denu - University of Wisconsin–Madison
Madilyn M. Sass - University of Wisconsin–Madison
James M. Johnson - University of Wisconsin–Madison
Gregory K. Potts - University of Wisconsin–Madison
Alka Choudhary - University of Wisconsin–Madison
Joshua J. Coon - University of Wisconsin–Madison
Mark E. Burkard - University of Wisconsin–Madison
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.294(16), pp.6531-6549
DOI: 10.1074/jbc.RA118.004867
PMID: 30804208
PMCID: PMC6484138
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: Elsevier Inc
Number of pages: 19
Grant note: P41 GM108538 / National Center for Quantitative Biology of Complex Systems P30 CA014520 / University of Wisconsin Carbone Cancer Center R01 GM097245; F30CA203271 / National Institutes of Health (NIH)
Language: English
Date published: 04/19/2019
Academic Unit: Internal Medicine
Record Identifier: 9984700653502771

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