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

Abstract

Edited by Xiao-Fan WangThe 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" PLK4 AS , 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.

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

“…Sequence and substrate similarity is highest between PLK2 and PLK3, followed by PLK1 and then PLK4, which is the least representative of the family [45]. Consistently, a dual C-terminal hydrophobic consensus ([ pS/pT]ΦΦ) has been ascribed to efficient PLK4 substrate phosphorylation in the literature, based on specificity studies using (PLK1)-based optimised synthetic peptide arrays [47] and a variety of recombinant protein assays [16,38]. Consistently, many physiological PLK4 substrates conform to this broad hydrophobic consensus C-terminal to the site of modification.…”

“…From the limited selection of physiological PLK4 substrates identified to date [38], we did not identify any known phosphopeptides that were statistically down-regulated in response to centrinone, likely due to our experimental procedure, in which cells were not chemically synchronised in S-phase despite centrinone-induced PLK4 stabilisation. Surprisingly, we observed a greater than 4-fold increase in levels of the phosphopeptide containing pSer237 (with respect to protein level changes) of BTRC (F-box/WD repeat-containing protein 1A), which has been previously reported to be associated with the PLK4 activator STIL [83], and 1.3-fold up-regulation of pSer3467 on the E3 ubiquitin-protein ligase MYCBP2.…”

“…Chemical-genetic PLK4 strategies have previously reported decreased phosphorylation of multiple proteins, including RUNX1, PTPN12, IL6ST, TRIM3 and SCRIB after conditional PLK4 knockdown or transgenic expression and inhibition of 'analogue-sensitive' PLK4 [13,35,38,84]. Many of these proteins have been assumed to be indirect targets of PLK4, due to the fact that they are not known to localise to the centrioles (or play known roles in centrosome or cilia biology).…”

“…This process of self-destruction ensures that centriole duplication is limited to only once per cell cycle and provides an additional mechanism for temporal control of phosphorylation of downstream targets, both directly or through priming phosphorylation events [6,24,25,34]. Although additional physiological PLK4 substrates have been identified [6,14,[35][36][37][38], a detailed characterisation of cellular PLK4 substrates phosphorylated during G1/S phase, or indeed across the phases of the cell cycle, is still lacking. Consequently, the contribution of PLK4 to centrosomal and non-centrosomal biology, ciliopathies and congenital abnormalities associated with gene dosage affects, alongside PLK4 kinase-independent functions that stabilise the kinase, remain to be established [6,[39][40][41].…”

“…Justifiably, these findings have guided the research community in the search for distinct PLK1, 2, 3 and 4 substrates. Indeed, chemical-genetic screens and phosphospecific antibodies, have led to the identification of a variety of cellular PLK4 substrates, including STIL, CEP192 and CEP131, in vertebrate cells [16,38]. While these studies also uncovered many potential additional PLK4-dependent phosphorylation sites in presumed targets, interpretation of these data were guided by their potential localisation to the centrosome, and/or an involvement in centrosomal biology, usually with the explicit assumption that PLK4 phosphorylates sites in substrates conform to a ([ pS/pT]ΦΦ) consensus.…”