Abstract
SummaryCentrioles 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 [1]. 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 [2, 3, 4]. 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.
Highlights
In C. elegans, ZYG-1 is targeted to centrioles by SPD-2 [5, 6] whereas in Drosophila, Asterless has this function [7]
Several substrates of Polo-like kinase 4 (Plk4)/ZYG-1 have been identified to date that include SAS-6 [13], Cep152 [14], and a component of g-TuRC, Gcp6 [15], but it is not clear how phosphorylation of these proteins might affect centriole duplication
Chose to identify centriole proteins that could be phosphorylated by Plk4 in vitro
Summary
Plk4 could strongly phosphorylate Ana2 but not Sas6 (Figures 1A, S1D, and S1E), suggesting the possibility that Ana2 might be the Plk4 substrate that triggers centriole duplication. The C-terminal part of Ana2 containing the STAN motif was necessary and sufficient for this phospho-dependent interaction with Sas6 (Figure 2C), leading us to test the consequences of mutations at its four Plk4 sites. We found that the Ana2-4A mutant was unable to interact with Sas6 even after incubation with active Plk4 (Figure 2B), indicating that phosphorylation on these sites is required.
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