Figure 3. STIL is phosphorylated by Plk4 in vitro and in vivo.

(a) Schematic of STIL full length and the summary of Plk4-phosphorylated serine/threonine residues of STIL identified by MS (top, indicated in red) and phospho-specific antibodies (top, indicated in blue; Supplementary Fig. 4a). STIL N3 and C fragments were phosphorylated by Plk4 in vitro (bottom). Recombinant STIL fragments N1 (a.a. 1–360), N2 (a.a. 361–600), N3 (a.a. 601–1,000) and C (a.a. 1,018–1,287) were bacterially purified and were incubated with Plk4ΔP-FLAG proteins purified from HEK293T cells for in vitro kinase assay. The incorporation of [γ-³²P] ATP to the substrates was visualized by autoradiography, and the loaded proteins were monitored by Simply Blue Safestaining. CC (coiled-coil): a.a. 721–746; STAN: a.a. 1,061–1,147. Asterisk indicates a mobility shift of STIL C by hyper-phosphorylation. (b) In vitro kinase assay with STIL C fragment and Plk4ΔP-FLAG wild-type (WT) or kinase-dead (KD) as in a. After the kinase reaction, the resulting materials were analysed by western blotting using STIL or FLAG antibodies. Asterisk indicates a mobility shift of STIL C by hyper-phosphorylation. (c) Plk4 phosphorylates STIL at Serine 1061 in vitro. In vitro kinase assay was done as in a with the recombinant STIL C WT or S1061A proteins. The total reaction mixture was analysed by SDS–PAGE, followed by Simply Blue Safestaining or western blotting using antibodies against phospho-S1061. Note that the peptide containing S1061 was too large (53 a.a.) to be detected by MS. We therefore generated a phopho-specific antibody against S1061 to verify the existence of phosphorylation at this site. (d) Phosphatase assay of endogenous STIL in U2OS cells. Cells expressing Plk4ΔP-FLAG WT or KD were just lysed (first two lanes) or further treated with λ-phosphatase (±) (last two lanes). The resulting materials were analysed by western blot using antibodies against STIL or tubulin. The amount of expressed Plk4ΔP-FLAG WT and KD was collected by immunoprecipitate (IP) using FLAG beads, followed by western blot using FLAG antibodies. (e) U2OS cells expressing HA-STIL N3C WT or 7A+5A mutant and Plk4ΔPEST-FLAG WT or KD were lysed and analysed by western blot as in d.