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.

14-3-3γ prevents centrosome duplication by inhibiting NPM1 function

“…In contrast, the expression of a phospho-mimetic mutant, T199D, leads to centrosome amplification (24). During Sphase, Polo-Like Kinase 4 (Plk4) recruitment at the site of procentriole biogenesis initiates centriole duplication (25,26). Overexpression of Plk4 results in centrosome amplification and its depletion results in a loss of centrioles (27,28).…”

Autoamplification and competition drive symmetry breaking: Initiation of centriole duplication by the PLK4-STIL network

“…Given that both ring and spot exist in the model for hours, such a rapid transition from ring to spot might explain why intermediate states between the ring and the spot are difficult to visualize in experiments imaging endogenous PLK4 (Ohta et al, 2014;Ohta et al, 2018). Nevertheless, temporally tracking the PLK4 ring to spot transition in Drosophila has revealed intermediate states with multiple PLK4 maxima, which could correspond to the model contender sites (Dzhindzhev et al, 2017). Possibly, they had been also observed in experiments with PLK4 overexpression as a "halo" surrounding mother centrioles (Kleylein-Sohn et al, 2007).…”

“…This phosphorylation is important for the retention of STIL at the centriole (Ohta et al, 2018). Phosphorylation of the C-terminal STAN motif of STIL is required for the interaction between STIL and SAS6 (Dzhindzhev et al, 2014;Kratz et al, 2015;Moyer et al, 2015;Ohta et al, 2014). This binding is necessary for either the in-situ assembly or anchoring of the elsewhere preassembled SAS6 cartwheel (Fong et al, 2014).…”

Differential turnover of Nup188 controls its levels at centrosomes and role in centriole duplication

“…To double centrosome number, each of the centrioles must first be duplicated in S-phase by a nascent "pro" centriole that assembles perpendicularly from the parent centriole outer wall (Nigg and Holland, 2018). This is achieved by a highly coordinated series of yet to be fully defined molecular events triggered by the recruitment of polo-like-kinase 4 (Plk4) (Bettencourt-Dias et al, 2005;Habedanck et al, 2005;O'Connell et al, 2001) by Cep152 and Cep192 (Cizmecioglu et al, 2010;Hatch et al, 2010;Kim et al, 2013;Park et al, 2014;Sonnen et al, 2013) and its phosphorylation of a network of proteins including STIL, CPAP and Sas6 (Dammermann et al, 2004;Delattre et al, 2004;Dzhindzhev et al, 2017;Kemp et al, 2004;Kirkham et al, 2003;Kratz et al, 2015;Leidel et al, 2005;Leidel and Gonczy, 2003;Moyer et al, 2015;Moyer and Holland, 2019;Ohta et al, 2014;Pelletier et al, 2004;Stevens et al, 2010;Tang et al, 2011). Sas6 helps form a supramolecular cartwheel-shaped structure that serves as a scaffold for microtubule nucleation (Gonczy, 2012).…”