Plk4-dependent phosphorylation of STIL is required for centriole duplication

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.

Keywords: Centriole duplication; Centrosome; Phosphorylation; Plk4; STIL.

Fig. 1.. Identification of STIL as a Plk4 interacting protein.

(A) Bacterially purified Zz-Plk4 (N-terminal Zz-tag and C-terminal His-tag) was incubated with G1/S or mitotically arrested (double thymidine block and release) HeLa Kyoto cell lysates. Cell lysates alone served as control. Plk4 was immunoprecipitated via its Zz-tag and eluted with its interaction partners. Coimmunoprecipitating proteins were detected by staining with Colloidal Coomassie (left panel) and analyzed by mass spectrometry. Western blotting (right panel) using anti-His antibodies was performed to detect Zz-Plk4 by its His-tag in elution fractions. Cyclin B abundance was used to determine the cell cycle stages of the lysates and anti γ tubulin detection served as a loading control. (B) Mass spectrometry analysis of Zz-Plk4 pull down identified known Plk4 interaction partners, substrates or regulators and STIL as a novel Plk4 interaction partner.

Fig. 2.. STIL interacts with Plk4 in vivo.

(A) Coimmunoprecipitation of Flag-STIL and Myc-Plk4. Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitations using anti-Flag or anti-Myc antibodies. Input and IP samples were analyzed by western blotting with antibodies against Flag-tag or Plk4 and α-tubulin as loading control. (B) Coimmunoprecipitation of endogenous STIL and Plk4. Endogenous Plk4 and STIL were immunoprecipitated from HeLa lysates by anti-Plk4 and anti-STIL antibodies. IP with non-specific IgGs served as a control. Coimmunoprecipitated proteins were detected by western blotting using anti-Plk4 and anti-STIL antibodies.

Fig. 3.. The polo-boxes of Plk4 are not sufficient to mediate STIL binding.

(A) Scheme of Flag-Plk4 fragments. (B) After overexpression of Flag-Plk4-fragments (A) and GFP-STIL in HEK293T cells, cell lysates were subjected to immunoprecipitations using anti-Flag antibodies. Coprecipitation of GFP-STIL with Flag-Plk4 fragments was detected by western blotting using anti-GFP and anti-Flag antibodies.

Fig. 4.. The C-terminus of STIL interacts with Plk4.

(A) Scheme of Flag-STIL fragments. (B) After overexpression of Flag-STIL-fragments (A) and Myc-Plk4 in HEK293T cells, cell lysates were subjected to immunoprecipitations using anti-Flag antibodies. Coprecipitation of Myc-Plk4 with Flag-STIL fragments was detected by western blotting using anti-Myc and anti-Flag antibodies.

Fig. 5.. Colocalization of STIL and Plk4 at the onset of S phase.

(A–C) U2OS cells were arrested in S-phase with 1.6 µg/ml aphidicolin for 17 h, released for 5 h and then rearrested in prometaphase by addition of 100 ng/ml nocodazole for additional 16 h. The cells were released and harvested at the indicated time points. (A) For indirect immunofluorescence analysis, cells were fixed and stained with antibodies against Plk4, pericentrin, STIL and γ-tubulin. Scale bars: 10 µm (merge), 2 µm (magnifications). (B) Centrosomal localization of Plk4 and STIL was quantified for the indicated time points by counting colocalization with the centrosomal marker proteins γ-tubulin and pericentrin. For each time point on average 60 cells were counted. (C) Synchronization of the cells was confirmed by western blot analysis using antibodies against Plk1, Plk4, Cyclin E, Cyclin B1, STIL, and α-tubulin.

Fig. 6.. Phosphorylation of STIL by Plk4.

(A) Full-length Flag-STIL expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ-³²P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Indicated Flag-STIL fragments were expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies. Immunoprecipitation fractions were incubated with bacterially expressed Zz-Plk4 in the presence of [γ-³²P]-ATP, followed by SDS-PAGE and autoradiography. In vitro kinase assay with Flag-STIL fragments or Plk4 alone is shown as control. The asterisk indicates phosphorylated Flag-STIL 781-1287. 10% of each precipitation fraction was analyzed by western blotting using anti-Plk4 and anti-Flag antibodies. (C) Plk4 phosphorylation sites in the STIL protein identified by mass spectrometry analysis of bacterially purified GST-STIL 1-619 and 619-1287 phosphorylated in vitro by Zz-Plk4. Alignment of the identified sites in human, mouse, Xenopus and zebrafish STIL and Drosophila Ana2 is shown.

Fig. 7.. Phosphorylation of STIL by Plk4 triggers centriole duplication.

(A) Flag-STIL full-length or 5A mutant (S871A/S873A/S874A/S1116A/T1250A) expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ-³²P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Co immunoprecipitation of Flag-STIL wt/5A and Myc-Plk4. Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation using anti-Flag antibodies. Input and IP samples were analyzed by western blotting with antibodies against Flag- and Myc-tag. The asterisk marks an unspecific band recognized by the anti-Myc antibody. The dividing lane indicates grouping of images from different parts of the same gel, as an intervening lane was removed for presentation purposes. (C) U2OS cells transiently expressing Flag EV, Flag-STIL wt or Flag-STIL 5A were analyzed by indirect immunofluorescence using staining with anti-CP110 and mouse anti-Flag antibodies 72 h after transfection. The number of transfected cells with more than four centrioles was determined based on CP110 staining. Values in the graph are mean percentages±s.d. from three independent experiments, 50 transfected cells were analyzed in each experiment (***P<0.001, two-tailed t-test). Representative images are shown for control, Flag-STIL wt and 5A-transfected cells. Scale bars: 10 µm (merge), 2 µm (magnifications). Western blotting using antibodies against Flag and α-tubulin was performed to visualize expression of STIL constructs as indicated.