SUMOylation Promotes Nuclear Import and Stabilization of Polo-like Kinase 1 to Support Its Mitotic Function  Donghua Wen, Jianguo Wu, Lei Wang, Zheng Fu  Cell Reports  Volume 21, Issue 8, Pages 2147-2159 (November 2017) DOI: 10.1016/j.celrep.2017.10.085 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 2147-2159DOI: (10.1016/j.celrep.2017.10.085) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Ubc9 Interacts with PLK1 in a Phosphorylation-Dependent Manner (A) Reciprocal co-immunoprecipitation of endogenous PLK1 and Ubc9 from HeLa cell lysates untreated or treated with nocodazole. (B) In-vitro-translated Ubc9 was used in a pull-down assay with either GST or GST-PLK1 immobilized on agarose beads (upper panel). Loading controls are shown below (CBB, Coomassie blue staining). (C) 293T cells were transfected with plasmids encoding Myc-tagged WT Ubc9 (Ubc9-WT) or mutant Ubc9 (Ubc9-S71A or Ubc9-S71D). Cell lysates were subjected to pull-down assays using beads coated with GST-PLK1 PBD WT or a mutant (MUT) deficient in phosphopeptide binding. Beads coated with GST alone were used as a negative control. (D) Ponceau S staining was used to indicate equal loading of the assays. See also Figure S1. Cell Reports 2017 21, 2147-2159DOI: (10.1016/j.celrep.2017.10.085) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Ubc9 Positively Regulates PLK1’s Protein Stability (A) U2OS cells were transfected with increasing amounts of pCMV-Myc-Ubc9 (0, 0.5, 1, and 2 μg). 48 hr post-transfection, cells were harvested for western blot using indicated antibodies. (B) U2OS cells were infected with lentiviral shRNA constructs that target either the 3′ UTR of endogenous Ubc9 (no. 1 and no. 2) or serve as a control (shCTL). Protein levels of PLK1, Ubc9, and β-actin were examined by western blot. (C) U2OS cells with stable Ubc9 knockdown were transfected with increasing amounts of pCMV-myc-Ubc9 (0, 0.5, 1, 2, 3, and 4 μg), and lysates were subjected to western blot. LE, longer exposure; SE, shorter exposure. (D) U2OS cells were transfected with pCMV-Myc-Ubc9 or empty vector for 24 hr and then treated with either DMSO or 2 μM MG132 for 16 hr. (E) U2OS cells with stable Ubc9 knockdown were treated with DMSO or 2 μM MG132 for 16 hr. Cells were harvested for western blot. (F) Control (CTL) and Ubc9-overexpressing U2OS cells were treated with nocodazole for 16 hr, mitotic cells were collected by shake-off and replated in fresh medium in the presence or absence of 20 μg/mL cycloheximide (CHX) for the indicated times, and endogenous PLK1 protein levels were monitored by immunoblotting (left panel). (G) Control (shCTL) and Ubc9 knockdown U2OS cells were treated as described in (F), and PLK1 protein levels were monitored by immunoblotting (left panel). Quantification of endogenous PLK1 protein levels relative to β-actin expression is shown in the right panel. The data are presented as the mean ± SEM. ∗p < 0.05. Cell Reports 2017 21, 2147-2159DOI: (10.1016/j.celrep.2017.10.085) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 PLK1 Is Subject to SUMO Modification (A) U2OS cells were transfected with Myc-PLK1, Flag-Ubc9, and HA-SUMO-1 or HA-SUMO-2 for 48 hr and then harvested for IP using anti-Myc antibody. The immunoprecipitated products were subjected to western blot using the indicated antibodies. ∗ indicates non-specific protein bands. (B) In vitro SUMOylation assays were performed by incubating bacterially expressed PLK1 with the SUMO-conjugation machinery: Aos1/Uba2; Ubc9; and SUMO-1. A SUMO-1 mutant (MUT) with deletion of the 2 C-terminal glycine residues required for its conjugation was included as a negative control. The SUMOylation status of PLK1 was examined by western blot. (C) U2OS cells were transfected with the indicated constructs. After 48 hr, the cells were lysed, immunoprecipitated using anti-PLK1 antibody, and immunoblotted with the indicated antibodies. (D) 293T cells were lysed under denaturing conditions, immunoprecipitated using anti-SUMO-1 antibody, and immunoblotted with anti-PLK1 antibody. (E) U2OS cells were transfected with indicated constructs, lysed, immunoprecipitated using anti-Myc antibody, and immunoblotted with the indicated antibodies. (F) Alignment of PLK1 regions containing the identified SUMO acceptor site K492 from multiple species. See also Figure S2. Cell Reports 2017 21, 2147-2159DOI: (10.1016/j.celrep.2017.10.085) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 SUMOylation Regulates PLK1’s Nuclear Import (A) The cellular localization of endogenous PLK1 in control (shCTL) and Ubc9 knockdown (shUbc9) U2OS cells was examined by immunofluorescence (IF) staining using anti-PLK1 antibody (red). Similar results were obtained from 3 independent experiments. The scale bar represents 10 μm. (B) An additional set of samples was subjected to subcellular fractionation. The levels of endogenous PLK1 in nuclear and cytoplasmic fractions were determined by immunoblotting with anti-PLK1 antibody. The relative purity of the nuclear and cytoplasmic fractions was confirmed by sequential probing for the nuclear marker lamin A/C and the cytoplasmic marker α-tubulin. C, cytoplasm; N, nucleus; W, whole cell lysate. (C) U2OS cells were transfected with Flag-tagged PLK1 WT or a Flag-tagged mutant PLK1 (K492R, SUMO-1-K492R, or Ubc9-WT). The cellular localization of ectopically expressed WT and mutant PLK1 was examined by IF using anti-Flag antibody (green). Similar results were obtained from 3 independent experiments. The scale bar represents 10 μm. (D) An additional set of samples was subjected to subcellular fractionation. The levels of exogenous PLK1 in nuclear and cytoplasmic fractions were determined by immunoblotting with anti-Flag antibody. The relative purity of the nuclear and cytoplasmic fractions was confirmed as indicated in (B). (E) The same set of cells as in (D) were treated with 5 ng/mL leptomycin B (LMB) for 16 hr and subjected to subcellular fractionation. See also Figures S3 and S4. Cell Reports 2017 21, 2147-2159DOI: (10.1016/j.celrep.2017.10.085) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 SUMOylation of PLK1 Enhances Its Protein Stability (A and B) U2OS cells expressing Myc-tagged PLK1 WT or a Myc-tagged mutant PLK1 (K492R or SUMO-1-K492R) were treated with nocodazole for 16 hr, and mitotic cells were collected by shake-off and replated in fresh medium in the presence or absence of 20 μg/mL CHX for the indicated times. Ectopically expressed WT and mutant PLK1 protein levels were monitored by immunoblotting (A). Quantification of exogenous protein levels relative to β-actin expression is shown in (B). The data are presented as the mean ± SEM. ∗p < 0.05. (C) U2OS cells were transfected with PLK1 WT or mutant PLK1 (K492R or SUMO-1-K492R) along with ubiquitin and then synchronized at the late stage of M phase by treating with Taxol for 16 hr and subsequently released in the presence of hesperidin, an Aurora B inhibitor, for 45 min. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody and blotted with the indicated antibodies. (D) U2OS cells expressing Myc-tagged PLK1 WT, K492R, or SUMO-1-K492R were treated with Taxol for 16 hr. Half of the cells were then released in the presence of hesperidin for 45 min to enrich cells at the later stage of M phase. Cell lysates were subject to IP, followed by western blot. Cell Reports 2017 21, 2147-2159DOI: (10.1016/j.celrep.2017.10.085) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 SUMOylation of PLK1 Contributes to Normal Cell Cycle Progression and Genome Stability (A) U2OS cells stably expressing PLK1 WT or K492R were infected with lentivirus encoding shRNA targeting the 3′ UTR of endogenous PLK1. Cell cycle distribution was determined by flow cytometry. The expression of exogenous PLK1 was analyzed by western blots. (B) Endogenous PLK1 was knocked down in U2OS cells stably expressing PLK1 WT, K492R, SUMO-1-K492R, or Ubc9-WT constructs. Cells were stained with anti-pH3 antibody and PI and analyzed by flow cytometry. (C and D) The same set of cells as described in (B) were infected with lentiviral mCherry-H2B constructs and subjected to time-lapse imaging. Representative time-lapse images are shown with the acquisition time relative to the onset of mitosis indicated on each image (C). White arrows indicate misaligned chromosomes or lagging chromosomes. The scale bar represents 10 μm. The duration of nuclear envelope breakdown (NEBD) to anaphase onset in each cell line is summarized in (D). Fifty cells per cell line were monitored. The data are presented as the mean ± SEM. ∗p < 0.05. See also Figures S5 and S6. Cell Reports 2017 21, 2147-2159DOI: (10.1016/j.celrep.2017.10.085) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 Model for SUMOylation-Mediated Regulation of PLK1’s Function Cell Reports 2017 21, 2147-2159DOI: (10.1016/j.celrep.2017.10.085) Copyright © 2017 The Author(s) Terms and Conditions