1. Academic Validation
  2. Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131)

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

  • J Biol Chem. 2019 Apr 19;294(16):6531-6549. doi: 10.1074/jbc.RA118.004867.
Ryan A Denu 1 2 3 Madilyn M Sass 2 3 James M Johnson 2 3 Gregory K Potts 4 5 6 Alka Choudhary 2 3 Joshua J Coon 4 5 6 Mark E Burkard 7 3
Affiliations

Affiliations

  • 1 From the Medical Scientist Training Program.
  • 2 the Division of Hematology/Oncology, Department of Medicine.
  • 3 the University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, Wisconsin 53705.
  • 4 the Department of Chemistry.
  • 5 the Department of Biomolecular Chemistry.
  • 6 the Genome Center, and.
  • 7 the Division of Hematology/Oncology, Department of Medicine, meburkard@medicine.wisc.edu.
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.

Keywords

CEP131; PLK4; cell division; centriolar satellite; centriole; centriole duplication; centrosome; chemical biology; chemical genetics; chromosome segregation; mass spectrometry (MS); microtubule; phosphoproteomics.

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