Plk4 phosphorylates Ana2 to trigger Sas6 recruitment and procentriole formation

Abstract

Centrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4) counterpart in other organisms. Although Plk4's centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. Here we show that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent.

Figure 1

Plk4 Phosphorylates Ana2 at Four Essential Serine Residues in the Conserved STAN Motif (A) GST-Ana2 or GST were incubated with active (MBP-Plk4) or inactive (MBP-Plk4^KD) Plk4 in the presence of γ-[³²P]ATP and run on SDS-PAGE (Coomassie), photographed, dried, and directly subjected to autoradiography. (B) Plk4 phosphorylation sites (red bars) revealed by mass spectrometry (see Figure S1G) of which four serines are in the conserved STAN motif (blue box). Green box is predicted coiled-coil. (C) Alignment of STAN motifs in Ana2 orthologs showing the four conserved Plk4 phosphorylation sites (arrows). (D) Plk4 phosphorylates C-terminal portion (indicated in B) of wild-type Ana2 (GST-Ana2-C-WT) but not mutant with alanine substitutions at the four mapped Plk4 sites (GST-Ana2-C-4A). (E) Cell lines overexpressing either Ana2-WT or Ana2-4A from the actin5c promoter were treated with either control dsRNA (GST-RNAi) or dsRNA targeting the UTRs of endogenous Ana2 (Ana2-UTR RNAi). Cells with no centrosomes were scored after three rounds (12 days in total) of dsRNA treatment. Error bars represent SEM. (F) Micrographs from (E) showing centrosomes revealed by D-Plp and Asl costaining in the two cell lines following Ana2-UTR RNAi. Insets show D-Plp and Asl in monochrome from one pole (indicated by a white arrow). DNA is stained with DAPI (blue). Scale bar represents 10 μm.

Figure 2

Plk4 Triggers a Direct, Phosphorylation-Dependent Interaction between Ana2 and Sas6 In Vitro and In Vivo (A) In vitro screen for differential interactions of centriolar proteins with Ana2 with and without phosphorylation by Plk4. GST-Ana2 treated with active (MBP-Plk4) or inactive (MBP-Plk4^KD) Plk4 and incubated with ³⁵S-Met-labeled Sas4, Bld10, Rcd4, Ana1, or Sas6 synthesized by in vitro transcription and translation. Affinity-purified complexes were analyzed by SDS-PAGE (Coomassie) followed by autoradiography. Note: Sas6 interacts with Plk4-prephosphorylated Ana2 but not with Ana2 treated with the inactive kinase. See Figure S2A for longer exposure showing interaction with Bld10. (B) GST, GST-tagged wild-type Ana2 (GST-Ana2-WT), or the four alanine substitution mutant (GST-Ana2-4A) were treated with either MBP-Plk4 or MBP-Plk4^KD and incubated in vitro with ³⁵S-Met-labeled Sas6. The resulting complex was analyzed by SDS-PAGE (Coomassie) and autoradiography. Sas6 specifically interacts with Ana2, when the four conserved serines in the STAN motif are pre-phosphorylated by Plk4. (C) GST-Ana2, GST-Ana2-N (residues 1–280), or GST-Ana2-C (residues 281–420) incubated with either MBP-Plk4 or MBP-Plk4^KD and ³⁵S-Met-labeled Sas6 and subjected to SDS-PAGE and autoradiography. C-terminal but not N-terminal part of Ana2 binds to Sas6 in vitro. (D) FLAG-tagged wild-type Ana2 (FLAG-Ana2^WT) or the four alanine substitution mutant (FLAG-Ana2^4A) were transiently cooverexpressed with Myc-tagged Sas6 (Sas6-Myc) and either the degron mutant (Plk4^ND) or degron/kinase dead (Plk4^NDKD) forms of Plk4 in D.Mel-2 cells. Input and anti-FLAG-immunoprecipitates were subjected to SDS-PAGE and Western blotting to reveal the indicated antigens.

Figure 3

Plk4 and Ana2 Are Essential for Sas6 Loading onto Centrioles, while Ana2 Loading Is Independent of Sas6 (A) Structured illumination images of centrioles showing exogenous GFP-tagged wild-type Ana2 and its mutant form with alanine substitutions in the four serines phosphorylated by Plk4 (Ana2-WT-GFP and Ana2-4A-GFP, respectively) both associate with two distinct puncta in interphase centrioles (compare to endogenous Ana2; Figure S3). Note that endogenous Ana2 was not depleted in these experiments. (B) Categories of Ana2 localization in interphase centrioles following control (GST dsRNA) or Sas6 RNAi. (C) Categories of Sas6 localization following control (GST), Ana2, or Plk4 RNAi. Numbers of centrioles counted and imaged are shown on the right hand columns in (B) and (C). (D and E) Structured illumination images of centrioles (Sas6, green; D-Plp, red) at the indicated cell cycle stages of control dsRNA-treated cells (upper panels), Ana2 dsRNA (D, lower panel), or Plk4 dsRNA (E, lower panel)-treated cells. Scale bars represent 0.5 μm.

Figure 4

Plk4-Mediated Phosphorylation of Four Serine Residues within the STAN Motif of Ana2 Is Critical for Sas6 Recruitment to Centrioles Endogenous Ana2 was depleted using dsRNA directed against its UTRs in two cell lines, one stably expressing wild-type Ana2 (Ana2-WT) and the other, the alanine substitution mutant at the Plk4 sites in the STAN motif (Ana2-4A). Cells were immunostained to reveal the indicated proteins and analyzed by structured illumination microscopy. (A) The substituting Ana2-WT, both Ana2-WT and endogenous Sas6 colocalize at both central and the peripheral puncta in the great majority of interphase centrioles. (B) Ana2-4A loads onto mother and procentriole and Sas6 only onto the mother in the great majority of interphase centrioles. Scale bars represent 0.5 μm. Numbers of centrioles observed in each category are indicated on the right.