Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Abstract

The conserved process of centriole duplication requires Plk4 kinase to recruit and promote interactions between Sas6 and Sas5/Ana2/STIL (respective nomenclature of worms/flies/humans). Plk4-mediated phosphorylation of Ana2/STIL in its conserved STAN motif has been shown to promote its interaction with Sas6. However, STAN motif phosphorylation is not required for recruitment of Ana2 to the centriole. Here we show that in Drosophila, Ana2 loads onto the site of procentriole formation ahead of Sas6 in a process that also requires Plk4. However, whereas Plk4 is first recruited to multiple sites around the ring of zone II at the periphery of the centriole, Ana2 is recruited to a single site in telophase before Plk4 becomes finally restricted to this same single site. When we over-ride the auto-destruction of Plk4, it remains localized to multiple sites in the outer ring of the centriole and, if catalytically active, recruits Ana2 to these sites. Thus, it is the active form of Plk4 that promotes Ana2's recruitment to the centriole. We now show that Plk4 phosphorylates Ana2 at a site other than the STAN motif, which lies in a conserved region we term the ANST (ANa2-STil) motif. Mutation of this site, S38, to a non-phosphorylatable residue prevents the procentriole loading of Ana2 and blocks centriole duplication. Thus the initiation of procentriole formation requires Plk4 to first phosphorylate a single serine residue in the ANST motif to promote Ana2's recruitment and, secondly, to phosphorylate four residues in the STAN motif enabling Ana2 to recruit Sas6. We discuss these findings in light of the multiple Plk4 phosphorylation sites on Ana2.

Keywords: Ana2; Plk4; Sas6; centriole; centrosome.

Figure 1.

Ana2 loading onto centrioles occurs ahead of Sas6. Representative images of centrioles stained to reveal D-Plp (blue), Ana2 (red) and Sas6 (green) from D.Mel-2 cells in the indicated cell cycle stages. In late anaphase/early telophase, Ana2 is loaded onto both mother centrioles and at the sites of the nascent procentrioles, with Sas6 only on the mothers. In late telophase/cytokinesis, Ana2 and Sas6 are both on mother and at pre-procentriolar sites.

Figure 2.

Ana2 recruitment to centrioles is dependent on Asl and Plk4. Representative structured illumination images of centrioles stained to reveal D-Plp (red) and Ana2 (green), following a 5-day depletion of the indicated protein by RNAi (3 days in the case of Plk4). Approximately 30 centrioles from cells in cytokinesis/G1 were examined for each condition and were scored in the categories shown as ‘loaded’ and ‘not loaded’. The number of centrioles in each category is indicated in each panel. The terms ‘loaded/not loaded’ are used here to reflect the effective presence/absence of Ana2 in the (D-Plp) ring of the centriole.

Figure 3.

Centriolar distribution of Plk4 does not define the single site of Ana2-loading in telophase. (a) Distribution of Plk4 at centrioles throughout mitotic progression. Representative structured illumination images of centrioles stained to reveal D-Plp (red) and endogenous Plk4 (green, (i)) and monochrome (ii). In mitotic progression, the mother centriole (arrow) can be distinguished from the daughter prior to anaphase because of a more complete ring of staining given by antibodies against D-Plp/Plk4. (b) Centriolar distribution of Plk4 and Ana2 during telophase. Representative structured illumination images stained to reveal endogenous D-Plp (blue), Plk4 (green) and Ana2 (red). Note that at the stage of typical Ana2-loading (early telophase, top panel), Plk4 is still present in multiple dots in the form of a broken ring at the centriole periphery. The newly loaded Ana2 co-localizes with one of the peripheral Plk4 dots, which is not necessarily the Plk4 dot showing the strongest signal.

Figure 4.

Plk4 activity is critical for Ana2-loading. (a) Depletion of the F-box protein, Slimb, stabilizes Plk4 at the centriole ring to recruit rosettes of Ana2 in interphase. Structured illumination images of interphase centrioles from cells subjected to 5 days of treatment with control dsRNA (against GST) or dsRNA directed against Slimb. Cells are stained to reveal endogenous D-Plp (blue), Plk4 (green) and Ana2 (red). (b) Overexpression of active, but not kinase-dead, Plk4 induces Ana2 loading at multiple sites. Structured illumination images of centrioles in interphase cells following 24 h of induced expression of either active non-degradable Plk4 (ND, (i)) or kinase-dead non-degradable Plk4 (NDKD, (ii)). Cells were stained to reveal endogenous D-Plp (blue), Ana2 (red) and overexpressed un-tagged Plk4 (green). Both active and inactive Plk4-ND accumulate in ectopic rings but only active Plk4 is able to induce rosettes of Ana2.

Figure 5.

Plk4 phosphorylates Ana2 at conserved serine 38. (a) (i) Transient co-overexpression of Ana2-FLAG with active or kinase-dead non-degradable Plk4 (Plk4^ND or Plk4^NDKD respectively) in D.Mel-2 cells. Cell extracts were subjected to western blotting to reveal the Ana2-FLAG protein. (ii) ³⁵S-Met-Ana2 protein synthesized by coupled transcription–translation in vitro and incubated with MBP-Plk4 and ATP in vitro and analysed by SDS-PAGE and autoradiography. Plk4-mediated phosphorylation in vivo or in vitro results in a shift in the electrophoretic mobility of Ana2. (b) Phosphorylation of Ana2 in which all 21 in vitro phosphorylation sites (highlighted in blue in electronic supplementary material, figure S1D) have been mutated to alanine residues. Assays are carried out using either the in vivo co-overexpression assay (i) or the in vitro phosphorylation assay (ii). None of the 21 mutations abolishes the band-shift. (c) The phosphorylation site responsible for the band shift is located within the N-terminal 140 amino acids of Ana2. Of the three Ana2 fragments, Ana2^1–140, Ana2^141–280 and Ana2^281–420, only Ana2^1–140 displays the band-shift in both the co-overexpression (i) and the in vitro phosphorylation assay (ii). (d) An S38A-Ana2 mutation, but not mutations in other candidate sites in the N-terminal 140 amino acids, abolishes the band-shift resulting from incubation with Plk4 in vitro. (e) The S38A mutation abolishes the shift in electrophoretic mobility of Ana2 following phosphorylation by Plk4 in either the in vivo co-overexpression assay (i) or in the in vitro assay (ii). (f) S38 (green) lies within a conserved region of Ana2—the ANST motif (highlighted grey). Partial alignment of Drosophila melanogaster Ana2 (top line) with STIL orthologues from Danio rerio, Xenopus laevis, Homo sapiens, Gallus gallus and Anolis carolinensis.

Figure 6.

S38 is essential for centriole duplication and Ana2 loading. (a) Control D.Mel-2 cells and cells expressing either Ana2-WT or Ana2-S38A transgenes from the constitutive Actin-5 promoter were treated with either control dsRNA or dsRNA targeting the UTRs of endogenous Ana2 (Ana2-UTR RNAi). Cells completely lacking centrosomes were scored after three rounds of treatment. Bars represent s.d. (b) Structured illumination images of centrioles stained with anti-D-Plp (red) and anti-Ana2 antibodies (in the case of untransfected D.Mel-2 cells, green) or anti-Myc (in the case of cells expressing Myc-tagged Ana2 transgenes) following three rounds of 4-day treatment with the indicated dsRNAs. Approximately 30 centrioles from interphase cells were scored for each condition and allocated to the categories ‘loaded’ and ‘not loaded’. The number of centrioles in each category is indicated in each panel.

Figure 7.

Sequential steps in the loading of Ana2 and Sas6 onto the site of the procentriole. Depiction of the spatial organization of a mother centriole (lower part) and the molecular interactions (upper part) of Plk4 (brown), Ana2 (blue) and Sas6 (green) during progression through anaphase (left), telophase (central), and late telophase-cytokinesis (right) in Drosophila cells. At the metaphase–anaphase transition, cells have a ring of Plk4 around the periphery of the centriole (Zone III) resembling a string of beads; Ana2 and Sas6 are present within the centriole core (Zone I). Plk4 will first phosphorylate Ana2 on S38 (single yellow site). In early telophase, Plk4 is lost from many, but not all, peripheral sites as a result of its proteolysis and Ana2 has begun to associate with one of the Plk4 sites, not necessarily the one with the highest levels of Plk4, but likely the one with the highest level of kinase activity (represented by the differential shading of Plk4). The Ana2 recruited to this site has been phosphorylated upon serine 38 in the ANST motif and associates either with Plk4 or another protein (X?, see Discussion). In late telophase-cytokinesis, Plk4 is lost from all but the procentriole site. Ana2 has been phosphorylated by Plk4 at four residues in the STAN motif (four yellow sites) and so can now bind to and recruit Sas6 dimers, which assemble into ninefold symmetrical rings.