Volume 27, Issue 10, Pages 1477-1484.e4 (May 2017) Ska3 Phosphorylated by Cdk1 Binds Ndc80 and Recruits Ska to Kinetochores to Promote Mitotic Progression  Qian Zhang, Sushama Sivakumar, Yujue Chen, Haishan Gao, Lu Yang, Zhu Yuan, Hongtao Yu, Hong Liu  Current Biology  Volume 27, Issue 10, Pages 1477-1484.e4 (May 2017) DOI: 10.1016/j.cub.2017.03.060 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Ska3 Is Phosphorylated at T358 and T360 by CDK1 during Mitosis (A) Ska3 is phosphorylated during mitosis. Lysates of log-phase or mitotic HeLa Tet-On cells were incubated with antibody against Ska3 and protein-A beads. Immunoprecipitated proteins were treated with mock or λ phosphatase and then resolved with SDS-PAGE and blotted with the indicated antibodies. (B) Cdk1 kinase phosphorylates Ska3. Nocodazole-arrested HeLa Tet-On cells were treated with the inhibitors of various mitotic kinases for 3 hr at the indicated concentrations: CDK1 inhibitor, RO3306 (10 μM); Aurora B inhibitor, ZM447439 (25 μM); Mps1 inhibitor Reversine (1 μM); and Plk1 inhibitor, BI2356 (500 nM). MG132 (10 μM) was added to prevent mitotic exit. The cell lysates were resolved on SDS-PAGE and blotted with anti-Ska3 antibodies. (C) Schematic drawing of hSka3 with 14 potential CDK1 sites in the C terminus marked with stars. The numbered amino acid residues are shown here. (D) GFP-Ska3 T358A or T360A fails to localize at kinetochores. HeLa cells depleted of endogenous Ska3 were transfected with indicated GFP-Ska3 WT or mutants. Expression of transgene was detected using anti-GFP antibody. This experiment was repeated three times. (E) Quantification of the kinetochore intensities of GFP-Ska3 in (D) normalized to those of anti-centromere antibodies (ACA) was shown in the lower panel. Each dot represents one kinetochore. At least ten cells (ten kinetochores per cell) were quantified for each condition. Means and SDs were shown here. (F) Cell lysates in (D) were resolved on SDS-PAGE and blotted with anti-GFP and anti-tubulin antibodies. (G) Alignment of sequences flanking T358 and T360 in Ska3 proteins from human (Hs), mouse (Mm), and Xenopus (Xl). See also Figure S1. Current Biology 2017 27, 1477-1484.e4DOI: (10.1016/j.cub.2017.03.060) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Cdk1 Phosphorylation of Ska3 Is Not Required for Ska Spindle Association (A) GFP-Ska3 is phosphorylated at T358 and T360 residues. GFP-Ska3 WT, T358A, T360A, 2A, or 2D lysates were resolved on SDS-PAGE and blotted with anti-GFP, anti-pSka3, and anti-actin antibodies. (B) Lysates of log-phase (thymidine) or mitotic HeLa cells (nocodazole) were resolved on SDS-PAGE and blotted with the indicated antibodies. (C) Recombinant 6xHis-Ska3 (141–412) WT or 2A fragments were treated with Cdk1 in the presence or absence of ATP. Proteins were then resolved on SDS-PAGE and subjected to Coomassie Brilliant Blue (CBB) staining and western blot analysis with the indicated antibodies. (D) Ska3 is required for GFP-Ska1 and GFP-Ska2 localization to the kinetochore. Mitotic HeLa Tet-On cells expressing GFP-Ska1, GFP-Ska2, or GFP-Ska3 with or without Ska3 depletion were stained with DAPI and the indicated antibodies. Representative images were shown here. This experiment was repeated three times. Quantification of the intensities of GFP-Ska1, Ska2, and Ska3 was shown in Figure S2B. (E) Ska3 phosphorylation at T358 and T360 does not affect its microtubule localization. Representative images of MG132-arrested mitotic HeLa cells expressing GFP-Ska3 WT, 2A (T358A T360A), 2D (T358A T360A), T358A, and T360A were shown in here. This experiment was repeated twice. (F) Quantification of the microtubule intensities of GFP-Ska3 in (E) normalized to those of spindle microtubules. Eight cells for each condition were quantified here. Each dot represents one cell. Means and SDs were shown here. (G) Quantification of the kinetochore intensities of GFP-Ska3 in (E) normalized to those of ACA. Thirty-five kinetochores (five kinetochores per cell) for each condition were quantified here. Each dot represents one kinetochore. Means and SDs were shown here. (H) HeLa cells with the indicated treatment were incubated at 4°C before fixation and then stained with the indicated antibodies. Representative images were shown here. This experiment was repeated twice. (I) Quantification of the intensities of microtubules in (H) normalized to those of DNA was shown here. Fifteen cells were analyzed for each condition. Each dot represents one cell. Means and SDs were shown here. See also Figure S2. Current Biology 2017 27, 1477-1484.e4DOI: (10.1016/j.cub.2017.03.060) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Ska3 Phosphorylation by Cdk1 Promotes Direct Ska3-Ndc80 Binding (A) Ndc80 loop is required for Ska3 localization to the kinetochore. Mitotic HeLa cells (5 μM nocodazole) transfected with RNAi-resistant plasmids containing Myc-Ndc80 WT or ΔLoop were treated with Ndc80 siRNAs. Representative images of chromosome spread were shown in here. The outlined regions were amplified and shown on the right panels. This experiment was repeated twice. (B) Quantification of the kinetochore intensities of Ska3 (upper) and Myc-Ndc80 (lower) in (A) normalized to that of ACA. Means and SDs were shown here. Mock, n = 15 cells; siNdc80 and vector, n = 15 cells; siNdc80 and Ndc80-FL, n = 16 cells; siNdc80 and Ndc80-ΔLoop, n = 15 cells. (C) Cell lysates from the experiment in (A) were resolved on SDS-PAGE and blotted with the indicated antibodies. (D) Ndc80 loop promotes the Ndc80-Ska3 interaction. Nocodazole-arrested HeLa Tet-On cells expressing Myc-Ndc80 WT or ΔLoop and Myc-Nuf2 were released into MG132. Lysates of these cells were incubated with anti-Myc antibodies. Immunoprecipitated proteins were resolved on SDS-PAGE and blotted with anti-Ska3 and anti-Myc antibodies. (E) Ska3 phosphorylation at T358 and T360 is required for its binding to Ndc80. Recombinant 6xHis-Ska3 (141–412) WT, 2A (T358A T360A), or 14A (mutation of all 14 CDK sites) fragments treated with CDK1-cyclin B1 in the presence or absence of ATP were incubated with glutathione Sepharose beads pre-bound with glutathione S-transferase (GST) or GST-Nuf2-Ndc80. Pelleted proteins were resolved with SDS-PAGE and subjected to CBB staining and western blot analysis with the indicated antibodies. (F) Phosphor-mimetic 2D is sufficient for the Ska3-Ndc80 binding. The same GST pull-down assay was performed as in (E) using recombinant 6xHis-Ska3 (141–412) WT or 2D (T358D T360D). (G) Recombinant 6xHis-Ska3 (141–412) fragments treated with CDK1-cyclin B1 in the presence of ATP were incubated with beads pre-bound with GST, GST-Nuf2-Ndc80, or GST-Ndc80-Bonsai. Proteins were resolved by SDS-PAGE and subjected to Coomassie Brilliant Blue staining and western blot analysis with the indicated antibodies. Pi represents phosphorylated Ska3; Un-pi is unphosphorylated Ska3. See also Figure S3. Current Biology 2017 27, 1477-1484.e4DOI: (10.1016/j.cub.2017.03.060) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Ska3 Phosphorylation at T358 and T360 Is Required for Proper Mitotic Progression (A) Image panel showing representative images of HeLa Tet-On cells expressing GFP-Ska3 WT, 2A (T358A T36A), or 2D (T358D T360D) with or without depletion of endogenous Ska3. The time taken from nuclear envelope breakdown (NEB) to anaphase onset was measured in hr:min for every cell. (B) HeLa cells expressing Ska3 2A were delayed in anaphase onset compared to WT Ska3-expressing cells. Scatterplot depicting the time taken from NEB to anaphase onset for every cell is shown. Although 75 cells were analyzed for each condition, only cells that initiated anaphase were counted here. Cells undergoing mitotic arrest followed by cohesion fatigue were recorded in (D). Means and SDs were shown here. (C) Chromosome alignment is depicted for GFP-Ska3 WT or 2A- or 2D-expressing cells in the presence or absence of endogenous Ska3 based on live imaging in (A). Fifty to ninety cells were analyzed for each condition. Means and SDs were shown here. (D) The percentage of cells arrested in Ska3-depleted WT or 2A-expressing cells is shown. (E) Model depicting the Cdk1 phosphorylation of Ska3 at T358 and T360 to promote binding to Ndc80 and recruit Ska complex to the kinetochore. See also Figure S4. Current Biology 2017 27, 1477-1484.e4DOI: (10.1016/j.cub.2017.03.060) Copyright © 2017 Elsevier Ltd Terms and Conditions