Plk4 autophosphorylates a 24-aa region to promote its own destruction. (A) Schematic showing the localization of the 24-aa region that is required for Plk4 to promote its own destruction. The alignment of this region, which is rich in potential phosphorylation sites (S/T residues), is shown. This putative β-TrCP–binding motif is contained within this region and marked by a line. (B) GST-Plk4 kinase assays were performed using His-Plk4^KD (aa 1–416) as a substrate. GST-Plk4^WT phosphorylates itself and His-Plk4^KD, but note that phosphorylation of His-Plk4^KD is reduced by the Δ24 mutation. Coomassie-stained gel shows the purified protein, and the autoradiogram shows the incorporation of γ[³²P]ATP. Assays were performed for 60 min, and activity was measured by scintillation counting. (C) His-Plk4 (aa 1–416) kinase assay was performed using histone H1 as a substrate. Despite the fact that WT and Δ24 His-Plk4 have similar activity toward histone H1, His-Plk4^Δ24 displays reduced autophosphorylation. Coomassie-stained gel shows the purified protein, and autoradiogram shows the incorporation of γ[³²P]ATP. Assays were performed for 60 min, and activity was measured by scintillation counting. (D) Immunoblot showing the relative expression levels of WT, AA, Δ24, and KD Plk4-EYFP. (E) Graph quantifying of the proportion of cells with more than four centrioles 48 h after induction of Plk4-EYFP. (F) Graph quantifying the mean number of centrioles per cell in cells with more than four centrioles. Counting was performed 48 h after induction of Plk4-EYFP. (G) Immunofluorescence images acquired 48 h after Plk4-EYFP was induced. Insets depict an enlargement of centrioles. Red, SAS-6; green, Plk4-EYFP; blue, DNA. Bars, 10 µm. C, Coomassie. Bars represent the mean of at least three independent experiments with >280 cells per condition. Error bars represent the SEM.