Asterless is a scaffold for the onset of centriole assembly.
ABSTRACT Centrioles are found in the centrosome core and, as basal bodies, at the base of cilia and flagella. Centriole assembly and duplication is controlled by Polo-like-kinase 4 (Plk4): these processes fail if Plk4 is downregulated and are promoted by Plk4 overexpression. Here we show that the centriolar protein Asterless (Asl; human orthologue CEP152) provides a conserved molecular platform, the amino terminus of which interacts with the cryptic Polo box of Plk4 whereas the carboxy terminus interacts with the centriolar protein Sas-4 (CPAP in humans). Drosophila Asl and human CEP152 are required for the centrosomal loading of Plk4 in Drosophila and CPAP in human cells, respectively. Depletion of Asl or CEP152 caused failure of centrosome duplication; their overexpression led to de novo centriole formation in Drosophila eggs, duplication of free centrosomes in Drosophila embryos, and centrosome amplification in cultured Drosophila and human cells. Overexpression of a Plk4-binding-deficient mutant of Asl prevented centriole duplication in cultured cells and embryos. However, this mutant protein was able to promote microtubule organizing centre (MTOC) formation in both embryos and oocytes. Such MTOCs had pericentriolar material and the centriolar protein Sas-4, but no centrioles at their core. Formation of such acentriolar MTOCs could be phenocopied by overexpression of Sas-4 in oocytes or embryos. Our findings identify independent functions for Asl as a scaffold for Plk4 and Sas-4 that facilitates self-assembly and duplication of the centriole and organization of pericentriolar material.
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ABSTRACT: Duplication of centrioles, namely the formation of a procentriole next to the parental centriole, is regulated by the polo-like kinase Plk4. Only a few other proteins, including STIL (SCL/TAL1 interrupting locus, SIL) and Sas-6, are required for the early step of centriole biogenesis. Following Plk4 activation, STIL and Sas-6 accumulate at the cartwheel structure at the initial stage of the centriole assembly process. Here, we show that STIL interacts with Plk4 in vivo. A STIL fragment harboring both the coiled-coil domain and the STAN motif shows the strongest binding affinity to Plk4. Furthermore, we find that STIL is phosphorylated by Plk4. We identified Plk4-specific phosphorylation sites within the C-terminal domain of STIL and show that phosphorylation of STIL by Plk4 is required to trigger centriole duplication. © 2015. Published by The Company of Biologists Ltd.
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ABSTRACT: Polo-like kinase 4 (Plk4) is a master regulator of centriole duplication, and its hyperactivity induces centriole amplification. Homodimeric Plk4 has been shown to be ubiquitinated as a result of autophosphorylation, thus promoting its own degradation and preventing centriole amplification. Unlike other Plks, Plk4 contains three rather than two Polo box domains, and the function of its third Polo box (PB3) is unclear. Here, we performed a functional analysis of Plk4's structural domains. Like other Plks, Plk4 possesses a previously unidentified autoinhibitory mechanism mediated by a linker (L1) near the kinase domain. Thus, autoinhibition is a conserved feature of Plks. In the case of Plk4, autoinhibition is relieved after homodimerization and is accomplished by PB3 and by autophosphorylation of L1. In contrast, autophosphorylation of the second linker promotes separation of the Plk4 homodimer. Therefore, autoinhibition delays the multiple consequences of activation until Plk4 dimerizes. These findings reveal a complex mechanism of Plk4 regulation and activation which govern the process of centriole duplication.Proceedings of the National Academy of Sciences 02/2015; DOI:10.1073/pnas.1417967112 · 9.81 Impact Factor
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ABSTRACT: Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4's tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl-Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.The Journal of Cell Biology 02/2015; 208(16):401-414. DOI:10.1083/jcb.201410105 · 9.69 Impact Factor