Figure S4.
PLK1- and CDK1-dependent phosphorylation is essential for branching MT nucleation in vitro and important for the spindle localization of augmin and γ-TuRC in cells. (A) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing MZT1-GFP (prey) together with NEDD1-GFP-Bio, GCP2/GCP3-GFP-Bio/γ-tubulin, or GCP4/GCP5/GCP6-GFP-Bio/γ-tubulin (bait), analyzed by Western blotting with GFP antibody. (B) Purified GFP-NEDD1, both WT and S411A mutant, were phosphorylated in vitro using [32P]ATP and active CDK1/cyclin B (see Materials and methods for details). Kinase assay reaction products were separated by SDS-PAGE (lower panel) and visualized by autoradiography (upper panel). The CDK1/cyclin B kinase alone was used as the positive control. The S411A mutation reduced the level of NEDD1 phosphorylation by 66%. (C) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing augmin-GFP-Bio (bait) together with γ-TuRCΔNEDD1-GFP, WT, or S411A mutant of γ-TuRC-GFP (prey), analyzed by Western blotting with indicated antibodies. (D and E) Coomassie blue–stained gels with augmin-SNAP (D) and γ-TuRCStN-GFP (E) that were purified from nocodazole-arrested mitotic HEK293T cells in the presence of either coexpressed untagged CDK1/cyclin B and HA-PLK1, or the combined inhibitors (augminCDK1/PLK1, augminRO/BI, γ-TuRCStN/CDK1/PLK1, and γ-TuRCStN/RO/BI). (F) TIRF microscopy images showing robust branching MT nucleation in the presence of the pair of 10 nM augminCDK1/PLK1-SNAP (blue) and 10 nM γ-TuRCStN/CDK1/PLK1-GFP (green), but not other pairs. Scale bar, 2 μm. (G–J) Immunofluorescence staining and quantification of HAUS8 intensity along the spindle (G) as well as NEDD1 intensity at the centrosome or along the spindle (H) in HeLa cells treated with or without BI2536 (300 nM) for 2 h and with STLC for 4 h. The values were normalized to the intensity of HeLa cells treated only with STLC. n = 50 cells. (K–N) Immunofluorescence staining and quantification of HAUS8 intensity along the spindle (K) as well as NEDD1 intensity at the centrosome or along the spindle (L) in HeLa cells treated with DMSO (control) or RO3306 (10 μM) for 30 min. The values were normalized to the intensity of DMSO-treated HeLa cells. n = 50 cells. Scale bars, 2 μm. Data represent mean ± SD. ***, P < 0.001, two-tailed t test.

PLK1- and CDK1-dependent phosphorylation is essential for branching MT nucleation in vitro and important for the spindle localization of augmin and γ-TuRC in cells. (A) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing MZT1-GFP (prey) together with NEDD1-GFP-Bio, GCP2/GCP3-GFP-Bio/γ-tubulin, or GCP4/GCP5/GCP6-GFP-Bio/γ-tubulin (bait), analyzed by Western blotting with GFP antibody. (B) Purified GFP-NEDD1, both WT and S411A mutant, were phosphorylated in vitro using [32P]ATP and active CDK1/cyclin B (see Materials and methods for details). Kinase assay reaction products were separated by SDS-PAGE (lower panel) and visualized by autoradiography (upper panel). The CDK1/cyclin B kinase alone was used as the positive control. The S411A mutation reduced the level of NEDD1 phosphorylation by 66%. (C) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing augmin-GFP-Bio (bait) together with γ-TuRCΔNEDD1-GFP, WT, or S411A mutant of γ-TuRC-GFP (prey), analyzed by Western blotting with indicated antibodies. (D and E) Coomassie blue–stained gels with augmin-SNAP (D) and γ-TuRCStN-GFP (E) that were purified from nocodazole-arrested mitotic HEK293T cells in the presence of either coexpressed untagged CDK1/cyclin B and HA-PLK1, or the combined inhibitors (augminCDK1/PLK1, augminRO/BI, γ-TuRCStN/CDK1/PLK1, and γ-TuRCStN/RO/BI). (F) TIRF microscopy images showing robust branching MT nucleation in the presence of the pair of 10 nM augminCDK1/PLK1-SNAP (blue) and 10 nM γ-TuRCStN/CDK1/PLK1-GFP (green), but not other pairs. Scale bar, 2 μm. (G–J) Immunofluorescence staining and quantification of HAUS8 intensity along the spindle (G) as well as NEDD1 intensity at the centrosome or along the spindle (H) in HeLa cells treated with or without BI2536 (300 nM) for 2 h and with STLC for 4 h. The values were normalized to the intensity of HeLa cells treated only with STLC. n = 50 cells. (K–N) Immunofluorescence staining and quantification of HAUS8 intensity along the spindle (K) as well as NEDD1 intensity at the centrosome or along the spindle (L) in HeLa cells treated with DMSO (control) or RO3306 (10 μM) for 30 min. The values were normalized to the intensity of DMSO-treated HeLa cells. n = 50 cells. Scale bars, 2 μm. Data represent mean ± SD. ***, P < 0.001, two-tailed t test.

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