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. 2025 Jul 1;36(7):br18. doi: 10.1091/mbc.E24-12-0581

Binding of CEP152 to PLK4 stimulates kinase activity to promote centriole assembly

Hazal Kübra Gürkaşlar a,b, Ingrid Hofmann a,*
Editor: Fanni Gergelyc
PMCID: PMC12327949  PMID: 40372713

Abstract

Centriole duplication is regulated by polo-like kinase 4 (PLK4) and several conserved initiator proteins. The precise timing and regulation of PLK4 activation are critical for ensuring that centriole duplication occurs only once per cell cycle. Although significant progress has been made in understanding how PLK4 is activated, many aspects remain unclear. Here, we show how CEP152 contributes to the activation of PLK4. We utilize human cell lines that have been genetically engineered to rapidly degrade CEP152. Upon degradation of CEP152, localization of PLK4 at the proximal end of the centriole is disrupted. We show that binding of CEP152 N-terminal part to PLK4 increases phosphorylation and kinase activation. CEP152 controls the localization and levels of phosphorylated PLK4 at the proximal end of the centriole. CEP152 binding to PLK4 leads to phosphorylation and activation of PLK4 that might stabilize PLK4 dimer formation, thus allowing autophosphorylation. We propose that CEP152 activates PLK4 to ensure proper centriole duplication at the onset of S-phase.

  • Formation of a new procentriole starts with the assembly of a cartwheel structure that is catalyzed by the conserved PLK4 kinase. How PLK4 itself gets activated is not precisely known.

  • The authors studied the activation of PLK4 by the centrosomal protein CEP152 in human cells using U-ExM. They found that CEP152 can phosphorylate and activate PLK4 both in vitro and in vivo.

  • Their findings suggest that CEP152 is involved in PLK4 activation in S-phase by binding to the PB1–PB2 domains of PLK4. This in turn allows PLK4 dimerization and activation through phosphorylation to ensure proper centriole duplication.

INTRODUCTION

Centrioles are microtubule-based structures that are used to assemble both centrosomes and cilia/flagella. They recruit a surrounding proteinaceous pericentriolar material (PCM) to build the centrosome that forms the poles of the mitotic spindle during cell division. Centrioles duplicate once during the cell cycle, specifically at the G1–S phase transition in that a new daughter centriole forms at the proximal end of the maternal centriole (Nigg and Holland, 2018; Yamamoto and Kitagawa, 2021; Vasquez-Limeta et al., 2022). Centriole biogenesis must be tightly regulated to maintain proper centrosome number, as abnormalities can lead to chromosome missegregation and aneuploidy and have been linked to cancer (Banterle and Gonczy, 2017; Nigg and Holland, 2018). Procentriole formation starts with the assembly of a cartwheel structure that is catalyzed by the conserved polo-like kinase 4 (PLK4) (Gonczy and Hatzopoulos, 2019). Several scaffolding components CEP192/CEP152/CEP63/CEP57 ensure the presence of PLK4 at the proximal end of the maternal centriole (Cizmecioglu et al., 2010; Hatch et al., 2010; Kim et al., 2013; Sonnen et al., 2013). CEP192 and CEP152 form distinct complexes with PLK4 by binding to the conserved polo boxes (PB) 1 and 2 located at its C-terminus. Double depletion of CEP152 and CEP192 causes a lack of centriolar PLK4 and prevents centriole assembly (Kim et al., 2013; Sonnen et al., 2013; Scott et al., 2023). CEP152 associates with nascent centrioles following disengagement during the first G1 phase (Kim et al., 2013; Park et al., 2014). To initiate centriole duplication, PLK4 phosphorylates STIL at the conserved STAN motif, promoting the recruitment of SAS-6 (Dzhindzhev et al., 2014; Ohta et al., 2014; Kratz et al., 2015; Moyer et al., 2015), a critical factor for cartwheel assembly.

The regulation of PLK4 protein levels is achieved through ubiquitylation and subsequent degradation via the proteasome. Previous studies have shown that this process is partially controlled by the SCF β-TrCP/SLIMB E3 ubiquitin ligase complex (Cunha-Ferreira et al., 2009; Rogers et al., 2009) that recognizes a conserved phosphodegron generated through homodimer-dependent trans-autophosphorylation of PLK4 (Guderian et al., 2010; Holland et al., 2010; Cunha-Ferreira et al., 2013; Klebba et al., 2013). CRL4DCAF1, which ubiquitylates PLK4 by binding to the conserved PB1–PB2 domain, is a complementary way to control PLK4 abundance to prevent centriole overduplication (Grossmann et al., 2024).

PLK4 phosphorylation is independent of the presence of STIL/SAS-6 and is already detectable before STIL/SAS-6 loading into a single site around the mother centriole (Yamamoto and Kitagawa, 2019). It will be important to assess how its activity and phosphorylation state are regulated during the cell cycle. PLK4 uniquely forms homodimers through PB1 and PB2 domains (Slevin et al., 2012) that allow trans-autophosphorylation (Guderian et al., 2010; Holland et al., 2010; Cunha-Ferreira et al., 2013; Klebba et al., 2013). CEP152 is a strong binding partner of the PB1–PB2 of PLK4 and located at the centrosome before PLK4 loading. CEP152 is found at the centrosome throughout the cell cycle, with its levels fluctuating depending on the phase. In interphase, it is predominantly localized around centrioles, while during mitosis, its presence is reduced but still significant (Cizmecioglu et al., 2010; Hatch et al., 2010). CEP152 associates with newly formed centrioles following their disengagement during the first G1 phase (Kim et al., 2013; Park et al., 2014). In early G1, CEP152 localizes at the mother centriole and then repositions PLK4 to the outer edge of a newly forming CEP152 ring at the daughter centriole (Park et al., 2014). A crucial unanswered question is how PLK4 kinase activity is temporally controlled to trigger centriole assembly.

In this study, we analyzed whether PLK4 could be directly activated by binding to CEP152. We genetically engineered human cells for fast and controllable degradation of CEP152 to better understand its dynamics and the consequences of its insufficiency on PLK4 localization and function. We find that CEP152 degradation does not affect total PLK4 levels at the centrosome but perturbs PLK4 localization at the proximal end of the mother centriole, resulting in a scattered localization of PLK4 at the centriole. We show that CEP152 colocalizes with active PLK4 at the proximal end of the mother centriole. Furthermore, we unravel the requirement of CEP152 in phosphorylation and activation of PLK4.

RESULTS AND DISCUSSION

Removal of CEP152 by auxin-dependent fast degradation

To gain more insight into the function of CEP152 in the localization and activation of PLK4, we removed CEP152 from cells. Depletion using shRNA or siRNA is not well controllable and might affect centriolar structure that is the case when targeting proteins involved in centriole duplication. In addition, rapid siRNA depletion during specific cell-cycle phases is difficult. To circumvent this problem, we generated two cell lines, HeLaCEP152-AID (Figure 1) and U2OSCEP152-AID (Supplemental Figure S1), in which CEP152 is rapidly degraded by an auxin-inducible degradation system (AID). This method can conditionally induce the degradation of CEP152 by the proteasome (Figure 1A) simply by adding indole-3-acetic acid (IAA) to the cell culture medium (Nishimura et al., 2009; Nishimura and Fukagawa, 2017). All endogenous CEP152 alleles were inactivated using CRISPR/Cas9 and CEP152-AID was randomly integrated into the genome (Figure 1B). Only one mutant allele was sequenced in the blasticidin-resistant clones used in this study (Figure 1B; Supplemental Figure S1A) and the absence of endogenous CEP152 was confirmed by Western blotting (WB) (Figure 1C). CEP152-AID was degraded within 2 h after IAA addition (Figure 1D) and restored within 1 h of IAA washout (Figure 1E). Similarly, immunofluorescence (IF) signal showed a reduction of the CEP152 signal at the centrosome in response to IAA-treatment (Figure 1F; Supplemental Figure S1E). A similar effect of CEP152-AID degradation was observed in U2OS cells (Supplemental Figure S1, B–D). After 2 h of IAA depletion, we used ultrastructure expansion microscopy (U-ExM) (Gambarotto et al., 2019; Gambarotto et al., 2021) and stained centrioles with acetylated tubulin. We find that centriole length and diameter were not altered suggesting that its function was not impaired (Figure 1G; Supplemental Figure S1F). On the contrary, prolonged degradation of CEP152 for 24 to 72 h leads to a time-dependent impairment of centriole structure integrity resulting in short and broken centrioles (Figure 1H). Short-term degradation of CEP152 (2 h) did not impair centriole number (Figure 1I) compared with long-term treatment (72 h) implying that no functional defects accumulated. Together, we find that HeLaCEP152-AID and U2OSCEP152-AID are an ideal model to deplete CEP152 rapidly from centrosomal populations and study the effects of acute CEP152 deficiency on centriole assembly and function in cell lines.

FIGURE 1:

FIGURE 1:

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Fast degradation of CEP152 fused to AID. (A) Scheme of transfections and selection strategy to generate HeLa and U2OS cell lines expressing CEP152 fused to an AID. (B) Sequencing result confirming the inactivation of CEP152 alleles in HeLa CEP152-AID cells by a deletion mutation. *, point mutation. (C) HeLa CEP152-AID cells were treated with or without IAA for 2 h. Cells were lysed and analyzed by WB. (D) HeLa CEP152-AID cells treated with or without IAA for indicated timepoints. Cells were lysed and analyzed by WB. (E) HeLa CEP152-AID cells were treated with or without IAA for 2 h. IAA was washed-out for indicated timepoints and cells were lysed for Western blot analysis. The ratio of CEP152/α-tubulin is indicated below the blot. (F) IF staining of HeLa CEP152-AID cells after fixation depicts fast degradation of CEP152 upon 2 h IAA treatment. Scale bar, 1 μm. Values were normalized to the nontreated control and individual values were presented in the graph with ± SD. n = 2, ** < 0.01. (G) HeLa CEP152-AID cells were treated with IAA for 2 h and U-ExM protocol was applied. Gels were stained with acetylated tubulin and the datapoints were obtained by measuring centriole dimensions directly from U-ExM images, using line scans along the length and across the diameter of individual centrioles. To obtain biologically meaningful values, all measurements were normalized by the expansion factor, allowing us to accurately report the real, unexpanded sizes of centrioles. Scale bar, 1 μm and biological scale, 0.25 μm. Graphs represent the quantification of the length and diameter of each centriole, n = 2, ns > 0.05. (H) Representative U-ExM images of HeLa CEP152-AID cells treated with IAA for the indicated times. Centriole schemes depicting approximate centriole orientation are included (left). Quantification graph shows the accumulation of defects on centriole structure in HeLa CEP152-AID cells treated with IAA for indicated timepoints with +SD. n = 2. * = 0.05, *** < 0.001, **** < 0.0001, scale bar, 1 μm and biological scale, 0.25 μm. (right). (I) HeLa CEP152-AID cells treated with IAA for 2 and 72 h, fixed and stained with the indicated antibodies. Centriole number is counted by using centrin signal and results are shown in the graph with ± SD. ns > 0.05, ** < 0.01, **** < 0.0001. Scale bar, 1 μm.

Acute removal of CEP152 disrupts the localization of PLK4 at the centrosome

To understand how the loss of CEP152 would affect PLK4 protein levels at the centrosome, we treated HeLaCEP152-AID cells with IAA for 2 h to induce rapid degradation of CEP152. Acute removal of CEP152 did not alter PLK4 protein levels at the centrosome (Figure 2A). Around 90% of PLK4 signal was detected the proximal end at 90% of the procentrioles whereas a scattered PLK4 localization at the centriole was observed at ∼10% of the procentrioles (Figure 2B). Upon fast degradation of CEP152, around 20% of the proximal PLK4 signal was lost and the scattered localization of PLK4 at the procentriole increased to 35%. This and previous data (Scott et al., 2023) suggest that the presence of CEP152 ensures proper localization of PLK4 at the proximal end of the procentriole. CEP63 and CEP152 form a heterotetrameric α-helical bundle, which is essential for their cooperative function in centrosomes (Kim et al., 2019). Binding of CEP63 to CEP152 forms a cylindrical platform that influences the level of PLK4 recruitment to the centrosome (Wei et al., 2020). We studied the effect on PLK4 loading in the absence of both CEP152 and CEP63 in cells where CEP63 was depleted in HeLaCEP152-AID cells that were additionally treated with IAA for 2 h to degrade CEP152. Centriolar structure was not impaired upon short-term degradation of CEP152 and siRNA depletion of CEP63 (Supplemental Figure S2A). As shown in Figure 2C, degradation of CEP152 did not alter PLK4 levels at the centrosome, while depletion of CEP63 slightly reduced the signal of PLK4 at the centrosome. Simultaneous depletion of CEP63 and CEP152 degradation in HeLaCEP152-AID cells, however, lead to a 3-fold reduction of the centrosomal PLK4 signal. Similar results were obtained upon depletion of CEP63 in U2OSCEP152-AID cells treated with IAA for rapid CEP152 degradation (Supplemental Figure S2B). We find that PLK4 exists in a complex with CEP63 after coimmunoprecipitation. The binding between PLK4 and CEP63 was lost in the absence of CEP152, suggesting that the interaction between PLK4 and CEP63 is not direct and mediated through binding of both PLK4 and CEP63 to CEP152, respectively (Figure 2D). Together, our data show that CEP63 alone might not be able to recruit PLK4 to the centrosome but in cooperation with CEP152 that directly binds PB1 and PB2 of PLK4 (Kim et al., 2019).

FIGURE 2:

FIGURE 2:

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Fast degradation of CEP152 disrupts PLK4 localization at the centrosome. (A) HeLa CEP152-AID cells synchronized in S-phase with Aphidicolin were treated with or without IAA for 2 h and cells were then fixed for IF analysis. Scale bar, 1 μm. PLK4 signal was normalized to the nontreated control and values were shown in the graph with ± SD. ns > 0.05. (B) S-phase synchronized HeLa CEP152-AID cells were treated with IAA for 0 or 2 h, fixed and stained with the indicated antibodies for U-ExM analysis. Arrows indicate PLK4 foci. Quantification graph of U-ExM images shows the percentage of the cells with either proximal or scattered PLK4 distribution at the centrosomes after rapid degradation of CEP152. n = 2, Scale bar, 1 μm and biological scale, 0.2 μm. (C) HeLa CEP152-AID cells transfected with control (siGL2) and CEP63 siRNAs for 48 h were treated or not with IAA for 2 h and synchronized in S-phase. Samples were stained with indicated antibodies. Images show the change in PLK4 levels at the centrosome. Scale bar, 1 μm. PLK4 signal was normalized to the nontreated control and values were shown in the graph with ± SD n = 3. * = 0.05, ** < 0.01. (D) HeLa CEP152-AID cells were transfected with siRNAs against GL2 (control) and CEP152. Cells were then transfected with empty Flag vector or Flag PLK4 for 48 h and synchronized in S-phase. Following the synchronization cells were treated with or without IAA for 2 h and cells were harvested 72 h after the first siRNA transfections for immunoprecipitation and Western blot analysis using the indicated antibodies. n = 3, * = 0.05.

CEP152 promotes PLK4 autophosphorylation and activation

Our data suggest that CEP152 is required for proximal end localization of PLK4, where the new centriole is built (Figure 2B). We therefore set out to investigate what function CEP152 might play in the regulation of the activity and phosphorylation state of PLK4. PLK4 negatively regulates its own stability through multiple trans-autophosphorylation at a degron motif in the L1 region, an unstructured linker following the PLK4 kinase domain (Holland et al., 2010; Lopes et al., 2015; Scott et al., 2023). We realized, however, that a full depletion of CEP152 is difficult to achieve upon degradation of CEP152 in HeLaCEP152-AID cells, as IAA-treatment did not lead to a complete removal of CEP152. To study a possible role of CEP152 in the activation of PLK4, we need to assure that CEP152 is not detectable on centrosomes in HeLa cells. To achieve this, we used HeLaCEP152-AID cells to degrade CEP152 and treated them additionally with CEP152 siRNA for 48 h to further reduce CEP152 levels (Figure 3A). We only analyzed those cells where the CEP152 signal was not present at centrosomes. The structure of the centrioles was not impaired following the treatment (Supplemental Figure S3A). To monitor PLK4 autophosphorylation, we used an anti-PLK4 antibody raised against phosphorylated serine 305, p-S305 PLK4 (Park et al., 2014). S305 is located within the multiphosphodegron of the PLK4 L1 domain (Sillibourne et al., 2010; Park et al., 2014). This antibody monitors autophosphorylation of PLK4, which is a consequence of kinase activation (Sillibourne et al., 2010). p-S305 PLK4 signals start to be detected and simultaneously increase before the loading of STIL/SAS-6 (Yamamoto and Kitagawa, 2019). Although total PLK4 levels at the centrosome were not impaired in the absence of CEP152 in HeLaCEP152-AID cells, the p-S305 signal was markedly decreased suggesting that CEP152 might regulate PLK4 phosphorylation and activity (Figure 3B). Similar results were also obtained in U2OSCEP152-AID cells (Supplemental Figure S3B). The p-S305 PLK4 signal clearly decreased at the daughter centriole in the absence of CEP152, suggesting that p-S305 PLK4 is removed first from the daughter centriole upon degradation of CEP152 (Supplemental Figure S3C).

FIGURE 3:

FIGURE 3:

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PLK4 phosphorylation at S305 is dependent on CEP152 binding. (A) Experimental work-flow to yield nondetectable CEP152 signals at centrosomes in HeLa CEP152-AID cells. (B) HeLa CEP152-AID cells were transfected with control or CEP152 siRNAs for 48 h and treated with ± IAA for 2 h. Samples were fixed as in A and stained. Scale bar, 1 μm. p-S305 PLK4 and PLK4 signals were quantified in cells with no detectable CEP152 and normalized to the control after background signal subtraction. Graphs show individual values. n = 2, ns > 0.05, * = 0.05, ** < 0.01. (C) Schematic representation of GFP-tagged full-length CEP152, CEP152 1-512aa (N-terminus), 508-1710aa (C-terminus), and CEP152D43/44A constructs and their ability to bind PLK4 and to localize at the centrosome. (D) HeLa CEP152-AID cells were transfected with pEGFP-CEP152 constructs or pEGFP for 36 h. Before the fixation, cells were treated with or without IAA for 2 h. Cells were fixed after preextraction for IF analysis. Scale bar, 1 μm. n = 3, ns > 0.05, * = 0.05, *** < 0.001. (E) HEK293T cells were cotransfected with Flag-PLK4 and different pEGFP-CEP152 constructs shown in C for 36 h and then harvested for coimmunoprecipitation and Western blot analysis. Graph shows the ratio of p-S305 PLK4/Flag-PLK4 with ± SD. n = 3, * = 0.05, ** < 0.01. (F) GST-CEP152 (1-512aa) or GST alone was incubated with Zz-PLK4-His in the presence and the absence of 0.5 mM ATP for in vitro kinase assay, and then analyzed by WB. For Western blot detection, different membranes were incubated with the antibodies against p-S305 PLK4, GST, and PLK4, respectively. Activation of PLK4 was monitored with the ratio of p-S305 PLK4/PLK4 indicated under the blot. The graph shows the results of three independent experiments.

PLK4 and CEP152 interact both in vitro and in vivo in cells (Cizmecioglu et al., 2010; Hatch et al., 2010). To examine whether CEP152 interaction with PLK4 would increase PLK4 autophosphorylation at S305, we used a binding mutant or truncated versions of CEP152 that either interact with PLK4 without localizing to the centrosome or vice versa localized at the centrosome without interacting with PLK4 (Cizmecioglu et al., 2010) (Figure 3C). We found that the expression of full-length CEP152 significantly increased p-S305 PLK4 levels at the centrosome (Figure 3D). On the contrary, a CEP152 (D43A and D44A) double mutant that exhibited a significantly compromised ability to bind to the full-length PLK4 (Kim et al., 2013) did not cause activation of PLK4. Binding of an N-terminal CEP152 fragment was sufficient to induce phosphorylation of PLK4. Activation of PLK4 by CEP152 was not dependent on CEP152 localization to the centrosome as a C-terminal CEP152 fragment that localizes to the centrosome did not lead to phosphorylation of PLK4 at S305 (Figure 3D). As PLK4 expression levels were too low, immunoprecipitation of PLK4 was performed to enrich PLK4 protein levels. We show that expression of the N-terminal part of CEP152 (1-512) and full-length CEP152 expression clearly leads to an increase of pS305-PLK4 (Figure 3E). To show that CEP152 promotes PLK4 autophosphorylation directly in vitro, we performed in vitro kinase assays with recombinant Zz-PLK4 and the recombinant N-terminal fragment of CEP152, CEP152 1-512, that contains the PLK4-binding site (Cizmecioglu et al., 2010). We observed a low activity of recombinant PLK4 that is most likely caused by dimerization and trans-autophosphorylation (Cunha-Ferreira et al., 2013). Interestingly, PLK4 kinase activity markedly increased upon binding of PLK4 to the N-terminal CEP152 fragment in the presence of ATP (Figure 3F) as monitored by a clear increase in the p-S305 PLK4 signal. It is conceivable, that the low activity after PLK4 dimerization is not sufficient to initiate centriole duplication and that further binding of the PLK4 dimer to CEP152 is required to reach a threshold activity of PLK4 in order to start this process.

CEP152 binding to PLK4 allows PLK4 dimerization and activation

Monomeric PLK4 is autoinhibited. Once PLK4 dimerizes, this autoinhibition is relieved, allowing for increased catalytic activity thus facilitating further autophosphorylation. PB1 and PB2 domains provide an intramolecular homodimerization platform to regulate PLK4 trans-autophosphorylation (Klebba et al., 2015) and also interact directly with CEP152 (Cizmecioglu et al., 2010; Hatch et al., 2010) and CEP192 (Kim et al., 2013). As both dimerization of PLK4 and CEP152 binding involve PB1 and PB2, we checked whether CEP152 might affect PLK4 dimerization in human cells. If this is correct, dimerization of PLK4 should be reduced in the absence of CEP152. We find that upon depletion of CEP152, homodimerization of PLK4 was reduced that might imply that binding of CEP152 to PLK4 stabilizes dimerization and leads to activation of PLK4 (Figure 4A) but does not affect PLK4 protein levels at the centrosome (Figure 2A). In fact, we find that depletion of CEP152 leads to a slight decrease in p-S305 PLK4 levels, suggesting that dimerization is necessary for activation of PLK4 through phosphorylation of S305 (Figure 4A). However, we cannot rule out that CEP152 binding to PLK4 involves a mechanism that is independent on PLK4 dimerization. CEP152 competes with another scaffold protein, CEP192, for binding to PB1 and PB2 of PLK4 (Kim et al., 2013). We asked whether expression of CEP192 would affect PLK4 activation by competing with CEP152 for PLK4 binding thus impairing PLK4 activation. We therefore expressed increasing amounts of CEP192 and checked for changes in binding between CEP152 and PLK4. As expected, we find that increasing amounts of CEP192 lead to a stepwise decrease in CEP152 binding to PLK4, suggesting that CEP192 might antagonize dimerization. Moreover, this decrease in CEP152 binding led to a decrease in p-S305 PLK4 levels (Figure 4B) as dimerization and activation of PLK4 is impaired. Together, these results suggest that binding of CEP152 to PLK4 stabilizes PLK4 dimerization leading to autophosphorylation. Klebba et al. (Klebba et al., 2015) reported that Asterless, the orthologue of CEP152 in Drosophila melanogaster has two PLK4-binding sites and that the N-terminal PLK4-binding site also affects PLK4 homodimerization. Our studies now confirm that binding of the N-terminal domain of CEP152 to PLK4 leads to enhanced PLK4 kinase activity in human cells.

FIGURE 4:

FIGURE 4:

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CEP152 regulates dimerization of PLK4. (A) HEK293T cells were transfected with siRNAs against GL2 (control) or CEP152. Cells were then cotransfected with empty Flag vector and Myc-PLK4 or Flag-PLK4 and Myc-PLK4 for 48 h. Cells were harvested 72 h after the first siRNA transfections for immunoprecipitation analysis and immunostaining. Graph on the left shows the quantification. n = 3, * = 0.05. (B) HEK293T cells were cotransfected with Flag-PLK4 and pEGFP or increasing amounts of pEGFP-CEP192 for 36 h followed by western blot detection. n = 3, * = 0.05, ** < 0.01.

CEP152 promotes PLK4 activation at the proximal end of the procentriole

From our data, it is conceivable that CEP152 regulates the activity and phosphorylation state of PLK4 at the site of procentriole formation. To analyze this in more detail, we first checked by U-ExM the colocalization of p-S305 PLK4 with CEP152 at the centriole in cells that were synchronized in S-phase. We used U2OS cells for this set of experiments, as the p-S305 PLK4 antibody did not work in U-ExM in HeLa cells presumably due to lower p-S305 PLK4 levels present in this cell line. CEP152 localizes as a continuous ring that encircles the base of the parental centriole but lacks a clear 9-fold organization (Scott et al., 2023; Sullenberger et al., 2023). p-S305 PLK4 is primarily localized at the proximal end of the parental centriole where the new centriole is formed and where it colocalizes with CEP152 (Figure 5A). Recently, it was shown that inactive PLK4 binds up to nine sites at the proximal end of parental centriole in HeLa cells (Scott et al., 2023).

FIGURE 5:

FIGURE 5:

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CEP152 regulates proximal end localization and levels of p-S305 PLK4 at the centriole. (A) U2OS WT cells were synchronized in early S-phase with 5 μg/ml aphidicolin for 16 h and active PLK4 was enriched by treatment with 0.1 μM centrinone for 16 h and washed-out for 3 h. Fixed samples were used for U-ExM analysis. (B) Experimental workflow of S-phase synchronization, centrinone-washout and IAA treatments of U2OS CEP152-AID cells. (C) U2OS CEP152-AID cells were treated as indicated in A and fixed samples were used for U-ExM analysis. Gels were stained with the indicated antibodies in ± IAA-treated U2OS CEP152-AID cells. The number of p-S305 PLK4 foci was counted and the percentage of centrioles was shown in the graph. * shows p-S305 PLK4. (D) Representative U-ExM images of different p-S305 localizations in U2OS CEP152-AID cells after the protocol shown in B. Arrowheads and arrows mark distal and proximal localization of p-S305 PLK4, respectively. Graph shows the percentage of the centrioles with different p-S305 PLK4 localizations in ± IAA-treated U2OS CEP152-AID cells. (E) Representative U-ExM images of SAS-6 localization in ± IAA-treated U2OS CEP152-AID cells. Graph shows the percentage of centrioles with SAS-6 recruitment in ± IAA treatments. Scale bars, 1 μm for 4-fold expansion images.

To examine whether CEP152 is involved in activation of PLK4 at the onset of S-phase, we used a protocol where U2OSCEP152-AID cells were arrested in S-phase and then treated with centrinone to inhibit PLK4 kinase activity in the presence and absence of CEP152 (Figure 5B). This would allow us to examine a change in the number of foci following release from kinase inhibition. Using this setup, we checked for the effect on PLK4 S305 phosphorylation upon fast degradation of CEP152 followed by costaining for total PLK4 and p-S305 PLK4 to exactly monitor the localization of active PLK4. Specifically, in control cells (in the presence of CEP152), we observed that 29% of centrioles displayed four distinct p-S305 PLK4 foci, consistent with normal PLK4 activation and localization at nascent procentriole sites. In contrast, in CEP152-depleted cells, 31% of centrioles showed no p-S305 PLK4 foci while in 26% only a single focus is present. These results demonstrate a clear reduction in the number of activated PLK4 sites upon rapid degradation of CEP152. After centrinone-washout the p-S305 PLK4 signal was visible at 4 PLK4 sites. Upon rapid degradation of CEP152, the number of p-S305 PLK4 foci clearly decreased to 0 and 1 foci at parental centrioles, suggesting that PLK4 is inactive in the absence of CEP152 at the proximal end of the procentriole (Figure 5C).

Next, we compared the localization of p-S305 PLK4 in U2OS CEP152-AID cells with and without IAA treatment. Interestingly, the localization of p-S305 PLK4 was not restricted to the proximal end of centrioles. Although 45% of centrioles exhibited only proximal-end localization of p-S305 PLK4, around 35% showed both proximal and distal-end localization. Thus, the majority of cells (∼85%) displayed proximal localization of p-S305 PLK4 (Figure 5D). Strikingly, this number dropped to 30% in CEP152-degraded U2OSCEP152-AID cells. Nearly 50% of the cells showed no p-S305 PLK4 localization. However, the number of cells with distal-end-only localization of p-S305 PLK4 increased to 25% (Figure 5D). Mislocalization of PLK4 to the distal centriole was also observed by in response to siRNA-depletion (Scott et al., 2023). The distinct localization patterns of p-S305 PLK4 observed in this study raise important questions about the spatial regulation of PLK4 activity and its functional consequences. CEP152 is required for Plk4 loading at the proximal end of the mother centriole that triggers procentriole initiation (Sullenberger et al., 2023). Our study shows that CEP152 binding not only leads to activation of PLK4 but also ensures that active PLK4 is localized the proximal end. In the absence of CEP152, active PLK4 loses its proximal localization and either localizes at the distal end of the centriole or inactivated presumably through dephosphorylation on S305.

Finally, we analyzed SAS-6 loading, which is essential for cartwheel formation and proper centriole duplication, in response to degradation of CEP152 in S-phase–arrested cells. We noticed that SAS-6 localization was clearly reduced in the absence of CEP152 (Figure 5E). These results suggest that CEP152 is also crucial for the recruitment of SAS-6 to ensure that phosphorylation of STIL by PLK4 triggers the loading of SAS-6 onto both mother and daughter centrioles after they disengage at the end of mitosis.

Based on our findings, we propose that CEP152 is involved in the activation of PLK4 in S-phase. PLK4 is slightly activated through dimerization in the absence of CEP152 (Figure 3F) but the level of kinase activity might not be enough to trigger centriole duplication in cells. Following repositioning, we propose that binding of CEP152 to PLK4 increases PLK4 kinase activity of the dimer at the proximal end of the centriole as this localization is lost in the absence of CEP152 (Figure 5D). Similar data have been gained from the CEP152 ortholog asterless (Boese et al., 2018). Apart from CEP152, STIL binding to PLK4 also stimulates its kinase activity by promoting self-phosphorylation at T170 located in the activation loop (T-loop) leading to increased activity. In cycling cells and in contrast to PLK4 phosphorylation by CEP152, STIL-induced PLK4 phosphorylation leads to subsequent destruction of PLK4 (Moyer et al., 2015) while PLK4 protein levels remain unaltered in response to CEP152 binding. Our data provide new evidence on how PLK4 is regulated over time. We anticipate that CEP152-dependent activation of PLK4 occurs during early G1 to trigger STIL recruitment to centrioles by PLK4. Phosphorylation of STIL by active PLK4 then facilitates STIL/SAS-6 interaction and centriolar loading of SAS-6. The conserved TIM domain in STIL stimulates autophosphorylation and degradation of PLK4 (Ohta et al., 2018). However, it is possible that phosphorylation of PLK4 at S305 has no effect on STIL phosphorylation and centriole assembly. It would be interesting to show how exactly CEP152 binding leads to activation of PLK4. One possibility is that CEP152 stabilizes dimerization of PLK4 by binding to PB1 and PB2. Alternatively, CEP152 might trigger a conformational change that allows more efficient kinase activation. Further investigation will shed light on the temporal activation of PLK4 to expand our understanding of centriole duplication.

MATERIALS AND METHODS

Cell culture, generation of stable cell line, transfection of plasmids, and siRNAs

HeLa (ATCC CCL-2), U2OS (ATCC HTB-96), HEK293T (ACC 635; DSMZ, Braunschweig, Germany), HeLa CEP152-AID, and U2OS CEP152-AID cells were grown in high-glucose DMEM (Life Technologies) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin–streptomycin (Sigma-Aldrich) at 37°C and 5% CO2.

HeLa CEP152-AID and U2OS CEP152-AID cells stably expressing AID-fused CEP152 were generated according to the protocol established by (Vasquez-Limeta et al., 2022). Briefly, wild-type HeLa and U2OS cells were cotransfected with pAID 1.1C-T2A-Bsr–containing gRNA-resistant CEP152 to express AID-fused CEP152 and pX458 Cas9-containing CEP152 gRNA to knockout endogenous CEP152, and they were selected with 2 and 5 μg/ml Blasticidin S HCl (Santa Cruz Biotechnology). Following the antibiotic selection, ∼60 colonies were picked and were screened for CEP152-AID expression and CEP152 knockout by WB first. The colonies with CEP152-AID expression and endogenous CEP152 KO in WB were used for genomic-DNA purification. A ∼700 bp region of CEP152 that covers gRNA-targeting sequences was amplified and cloned into a pJET blunt end cloning backbone. pJET colonies containing PCR amplified CEP152 gDNA pieces were sequenced. The degradation of AID-fused CEP152 was induced by the addition of 500 μM IAA for 2 h (Nishimura et al., 2009; Nishimura and Fukagawa, 2017). Transfections of HeLa and U2OS cells were performed with Lipofectamine 2000 and Lipofectamine RNAiMax (Life Technologies), using 2 μg of plasmid and 50 nM siRNA, respectively, to transfect 6-well plates according to the manufacturer's recommendations. HEK293T cells were used to perform immunoprecipitation experiments and transfections were performed with Polyethylenimine (Polysciences) at a final concentration of 5 μg/ml using 30 μg plasmid DNA per 15 cm2 dish (2 × 107 cells).

To analyze PLK4 phosphorylation, cells were synchronized at early S-phase by using 5 μg/ml Aphidicolin (Biomol) and PLK4 was inhibited by adding 0.1 μM centrinone for 16 h. For the activation analysis of PLK4, synchronized cells were washed with PBS five times and released into fresh ± IAA-containing medium for 3 h. For siRNA experiments, CEP63 and CEP152 siRNAs were transfected into U2OS CEP152-AID or HeLa CEP152-AID cells for 72 and 48 h, respectively, and treated with ± IAA during the last 2 to 3 h. Cells were fixed and processed for IF or expansion microscopy (U-ExM) analysis.

Antibodies

Mouse anti-FLAG M2 (1:5000 in WB; F3165), mouse anti–α-tubulin (1:1000 WB; T5168), mouse anti–acetylated-tubulin (1:1000 U-ExM; 6-11B-1), and mouse anti–γ-tubulin (1:2000 IF; GTU-88) were obtained from Sigma. Mouse anti-centrin-2 (1:500 IF; 20H5) from Merck, mouse anti-CEP152 (1:1000 WB and 1:2000 IF; P1285) from Invitrogen, mouse anti-CEP192 (1:500 WB; A302-324A), rabbit anti-STIL (1:1000 IF; A302-442A) from Bethyl, mouse anti-MYC (1:1000 WB; 9E10), rabbit anti-GST (1:1000 WB; Z-5) mouse anti-ninein (1:1000 IF; F5), mouse anti-SAS-6 (1:500 IF, 1:100 U-ExM) from Santa Cruz Biotechnology, and rabbit anti-pericentrin (1:2000 IF; ab4448) from Abcam were used in different techniques. Mouse anti-centrobin (1:10,000 IF), rabbit anti–α-tubulin (1:500 U-ExM), and rabbit anti–β-tubulin (1:500 U-ExM) were obtained from Proteintech and Guinea Pig anti–α-tubulin (1:1500 U-ExM) and guinea pig anti–β-tubulin (1:1500 U-ExM) from ABCD antibodies. Rabbit anti-CEP152 (1:5000), rabbit anti-PLK4 (1:1000 IF, 1:500 U-ExM) and mouse anti-PLK4 (1:500 WB, 1:500 IF and 1:200 U-ExM) were used as in (Cizmecioglu et al., 2010). Rabbit anti-p-S305 PLK4 (Park et al., 2014) was a gift from Kyung Lee, NIH. Secondary antibodies for IF and U-ExM were goat anti-guinea pig coupled to Alexa Flour 594, goat anti-mouse IgG, IgG1, IgG2a or IgG2b, and goat anti-rabbit IgG coupled to Alexa Fluor 488, Alexa Fluor 594, and Alexa Flour 647 (1:500–1:1000; Molecular Probes). Secondary antibodies for WB were peroxidase-conjugated donkey anti-rabbit (1:2000; Jackson Laboratories) and goat anti-mouse (1:2000; Novus).

siRNAs and gRNAs

siRNAs used in this study were directed against the following sequences: firefly luciferase (GL2, control), 5′-AACGUACGCGGAAUACUUCGA-3′; CEP152 Oligo, 5′-GGAUCCAACUGGAAAUCUATT-3′; CEP63 Oligo, 5′-GGCUCUGGCUGAACAAUCATT-3′. Target sequence of specific gRNA used against CEP152 in stable cell line generation was 5′-CTGAGCAATTGGAGATGAGC-3′. All siRNAs and gRNAs were purchased from Eurofins Genomics, Ebersberg, Germany, at high-performance liquid chromatography purity.

Plasmid constructions and molecular cloning

Full-length human CEP152 with gRNA resistance (5′-CAGAACAGTTAGAAATGAGT-3′) in pAID 1.1C-T2A-Bsr (Addgene) and CEP152D43/44A in pEGFP-C1 was synthesized by GenScript. pEGFP-C1 (Clonetech), pEGFP-C1-CEP152 WT, pEGFP-C1-CEP152 (1-512aa), pEGFP-C1-CEP152 (508-1710aa), pCMV-3Tag1A-Flag (Agilent Technologies), pCMV-3Tag1A-Flag-PLK4, pCMV-3Tag1A-Flag-PLK4K41R, and pCMV-Myc-PLK4 were provided by the laboratory (Cizmecioglu et al., 2010). pEGFP-C1-CEP192 was provided by Kyung Lee (NIH) (Kim et al., 2013). CEP152 gRNA sense and antisense were dimerized and cloned into the pSpCas9(BB)-2A-GFP (pX458, Addgene) plasmid by using the provided protocol established by (Ran et al., 2013) available on the Addgene webpage. pGEX-4T1-CEP152 (1-152) was also provided by the laboratory for recombinant protein expression. Clones were analyzed by sequencing.

Recombinant protein expression

Human GST-CEP152(1-512aa) was expressed in Escherichia coli BL21-Rosetta and purified under denaturing conditions by single-step affinity chromatography using glutathione-agarose according to the instructions of the manufacturer. CEP152 (1-512aa) was dialyzed against PBS, pH 7.0, and concentrated with Vivaspin MWCO 12 to 15 kDa.

In vitro kinase assay

For in vitro, kinase assay with recombinant Zz-PLK4-His, GST-CEP152 was purified as described above. A total of 1 μg of Zz-PLK4-His protein was incubated with 2 μg of GST-CEP152 or an equivalent molar amount of GST in kinase buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 10 μM MnCl2, and 1 mM DTT) with or without ATP (0.5 mM) for 20 min at 30°C. Reactions were stopped by adding sample buffer and heating at 95°C for 5 min. Samples were analyzed by WB. For the analysis of PLK4 phosphorylation, phospho-specific p-S305 PLK4 antibody was used (Park et al., 2014).

Cell lysis, coimmunoprecipitation and Western blot analysis

Cell lysates were prepared as described previously (Hanle-Kreidler et al., 2022). Cells were harvested and washed with PBS. For Western blot analysis cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, and 50 mM NaF) and for coimmunoprecipitation experiments transfected HEK293T cells were lysed in NP40 lysis buffer (40 mM Tris, pH 7.5, 150 mM NaCl, 0.5% NP40, 5 mM EDTA, 10 mM β-glycerophosphate, 5 mM NaF, 1 mM dithiothreitol, 0.1 mM Na3VO4, and protease inhibitors). Anti-Flag M2 Affinity Gel was used to pull-down Flag-tagged proteins. Beads were washed three times with TBS (50 mM Trizma-HCl, with 150 mM NaCl, pH 7.4), once with 0.1 M glycine HCl, pH 3.5 and again three times with TBS (50 mM Trizma-HCl, with 150 mM NaCl, pH 7.4). Cell lysates and beads were incubated on a rotating wheel overnight at 4°C. Beads were washed three times with NP40 buffer and proteins were eluted with 3X flag peptide with 250 ng/μl concentration in NP40 buffer for 30 min.

Samples were run on SDS–PAGE, and proteins were transferred onto a nitrocellulose membrane by using Bio-Rad Trans-Blot turbo transfer system. All the membranes were blocked with 5% Skim milk in PBS-T. Blocked membranes were incubated with determined primary antibody solutions that were prepared in 5% Skim milk in PBS-T overnight. After primary antibodies were collected, membranes were washed three times with PBS-T and incubated with horseradish peroxidase (HRP)-conjugated mouse or rabbit secondary antibodies for 1 h at room temperature.

Expansion microscopy

The U-ExM protocol was adapted from previously described protocol by (Gambarotto et al., 2021). U2OS CEP152-AID cells were seeded on 12-mm coverslips. After the treatments were completed, preextraction was performed before MeOH fixation. Fixed samples were incubated with formaldehyde/acrylamide mixture for 4 h at 37°C. Gel polymerization, protein denaturation and first round of gel expansion were performed as described previously by (Gambarotto et al., 2021). To label the proteins with primary antibody, gels were incubated with primary antibody solution in 2% BSA in PBS for 2 d at 4°C. The labeled gels were stained with the secondary antibodies for 2 to 3 h at 37°C and the second round of expansion was performed. Expanded gels were fixed in a 35-mm glass bottom imaging chamber coated with poly-D-Lysine. Images were acquired as Z-stacks and were deconvolved by using either Zeiss or Huygens deconvolution software.

IF of fixed specimens

For indirect IF, HeLa and U2OS cells were treated as described previously in this article. Coverslips were incubated in BRB80 preextraction buffer (80 mM PIPES pH 6.8, 1 mM MgCl2, 1 mM EGTA and 0.1% TritonX-100) for 30 s, and then fixed with ice-cold MeOH for 10 min at −20°C. Cells were blocked in blocking buffer (3% BSA, 0.1% Triton X-100 and 0.2% NaN3 in PBS) for 20 min. Both primary and secondary antibodies were prepared in blocking solution. Coverslips were incubated with primary antibodies for 1 h at room temperature and washed with PBS for three times. After 1 h of secondary antibody incubation, the coverslips were washed again with PBS for three times and were mounted onto glass slides with ProLong Gold with DAPI (4′,6-diamidino-2-phenylindole) (Molecular Probes). For cell imaging in both IF and U-ExM, the Zeiss motorized inverted Observer.Z1 microscope was used, containing mercury arc burner HXP 120°C and LED module Colibri. Filter combinations as follows: DAPI (49), green fluorescent protein (GFP) (38 HE), DsRed (43 HE), and HXP120 for Far-Red, and with the detector gray-scale charge-coupled device camera AxioCam MRm system and 40x or 63 ×/1.4 Oil Pln Apo DICII objective. Zeiss Apotome confocal imaging device was used during z-stack imaging. For the signal intensity analysis, all images in the same set of experiments were acquired with the same settings (exposure time, Z-stack size, and Z-stack number) and maximum projection images were used during the analysis. Centrosomal signal intensity of PLK4, p-S305 PLK4 or CEP152 was quantified by randomly choosing cells by drawing a 3.5μm2 circle around the centrosome. The same circle was used to measure the background signal within the cells and this signal was subtracted from the measured signal from the centrosome.

Statistical analysis

GraphPad Prism, version 10 (GraphPad Software) was used to perform a statistical analysis of the data collected from at least three independent experiments. The data were represented as either individual values or the mean ± SD. To perform a statistical analysis of fold change data, all values were normalized to a control group and a logarithmic transformation was applied to obtain normally distributed data. Logarithmically transformed values were further analyzed by a one-sample, two-tailed t test against the mean of the control group, which was set to 1.0, or by a paired, two tailed t test for comparisons among the test groups to determine the significance. If data were normally distributed and not normalized to a control group, an unpaired, two-tailed t test with Welch's correction was used for the statistical analysis. The P values below 0.05 were considered statistically significant (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001).

Supplementary Material

mbc-36-br18-s001.pdf (995.9KB, pdf)

ACKNOWLEDGMENTS

We thank Kyung Lee, NIH, for reagents, Jadranka Loncarek, NIH, for sharing their detailed protocol to generate AID-cell lines, Holger Lorenz, University of Heidelberg, for help with the deconvolution software, and to Alexandra Turi da Fonte Dias for comments on the article.

Abbreviations used:

AID

auxin-inducible degradation system

DAPI

4',6-diamidino-2-phenylindole

GST

glutation S transferase

IAA

indole-3-acetic acid

PB

polo-box

PLK4

polo-like kinase 4

U-exM

ultrastructure expansion microscopy

WB

western blotting.

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E24-12-0581) on May 15 2025.

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Supplementary Materials

mbc-36-br18-s001.pdf (995.9KB, pdf)

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