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Usp16 regulates kinetochore localization of Plk1 to promote proper chromosome alignment in mitosis.

Zhuo X, Guo X, Zhang X, Jing G, Wang Y, Chen Q, Jiang Q, Liu J, Zhang C - J. Cell Biol. (2015)

Bottom Line: Usp16 deubiquitinates Plk1, resulting in an enhanced interaction with kinetochore-localized proteins such as BubR1, and thereby retains Plk1 on the kinetochores to promote proper chromosome alignment in early mitosis.Down-regulation of Usp16 causes increased ubiquitination and decreased kinetochore localization of Plk1.Thus, our data unveil a unique mechanism by which Usp16 promotes the localization and maintenance of Plk1 on the kinetochores for proper chromosome alignment.

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Affiliation: Ministry of Education Key Laboratory of Cell Proliferation and Differentiation and State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China.

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Usp16 regulates the kinetochore localization of Plk1, leading to proper alignment and timely separation of chromosomes. (A) Immunostaining of Plk1 in prometaphase HeLa cells treated with negative control siRNA or siRNA targeting KLHL22, CUL3, Usp16, or both Usp16 and KLHL22. Crest was used as a centromere marker. (B) The fluorescence intensity ratios of Plk1 and Crest shown in A, determined from three independent experiments with n = 100–150. (C) HeLa cells were transfected with control siRNA or siRNA targeting Usp16, and their lysates were blotted (left three lanes). Plk1 was immunoprecipitated with anti-Plk1 antibody or IgG in a mock IP. BubR1 coimmunoprecipitated with Plk1 was blotted (right three lanes). (D) Immunostaining of Plk1 and BubR1 in HeLa cells treated with either control siRNA or siRNA targeting Usp16. (E) Immunostaining of α-tubulin (α-Tub) and Usp16 in HeLa cells treated with control siRNA or siRNA targeting Usp16. The arrow points to misaligned chromosomes. (F) Percentage of cells with chromosome misalignment shown in E, determined from three independent experiments with n = 200–250. (G) Time-lapse microscopy of RFP-H2B–expressing HeLa cells treated with siRNA targeting Usp16 or control siRNA. Arrows point to misaligned or improperly separated chromosomes. (H) Time from nuclear envelope breakdown (NEBD) to metaphase in HeLa cells shown in G, determined from three independent experiments with n = 20–25. Error bars indicate the SEM. ***, P < 0.001. NC, negative control. Bars: (A, D, E, and G) 10 µm; (A, magnified images) 1 µm.
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fig2: Usp16 regulates the kinetochore localization of Plk1, leading to proper alignment and timely separation of chromosomes. (A) Immunostaining of Plk1 in prometaphase HeLa cells treated with negative control siRNA or siRNA targeting KLHL22, CUL3, Usp16, or both Usp16 and KLHL22. Crest was used as a centromere marker. (B) The fluorescence intensity ratios of Plk1 and Crest shown in A, determined from three independent experiments with n = 100–150. (C) HeLa cells were transfected with control siRNA or siRNA targeting Usp16, and their lysates were blotted (left three lanes). Plk1 was immunoprecipitated with anti-Plk1 antibody or IgG in a mock IP. BubR1 coimmunoprecipitated with Plk1 was blotted (right three lanes). (D) Immunostaining of Plk1 and BubR1 in HeLa cells treated with either control siRNA or siRNA targeting Usp16. (E) Immunostaining of α-tubulin (α-Tub) and Usp16 in HeLa cells treated with control siRNA or siRNA targeting Usp16. The arrow points to misaligned chromosomes. (F) Percentage of cells with chromosome misalignment shown in E, determined from three independent experiments with n = 200–250. (G) Time-lapse microscopy of RFP-H2B–expressing HeLa cells treated with siRNA targeting Usp16 or control siRNA. Arrows point to misaligned or improperly separated chromosomes. (H) Time from nuclear envelope breakdown (NEBD) to metaphase in HeLa cells shown in G, determined from three independent experiments with n = 20–25. Error bars indicate the SEM. ***, P < 0.001. NC, negative control. Bars: (A, D, E, and G) 10 µm; (A, magnified images) 1 µm.

Mentions: As ubiquitination promotes the removal of Plk1 from the kinetochores (Beck and Peter, 2013; Beck et al., 2013), we suspected that down-regulation of Usp16 would result in increased Plk1 ubiquitination and promote the removal of Plk1 from the kinetochores. Indeed, after Usp16 knockdown, we observed a dramatic decrease of Plk1 staining on the kinetochores in prometaphase cells (Fig. 2, A and B). On the other hand, when the CUL3-based ubiquitin ligase was suppressed by either KLHL22 or CUL3 siRNA, we observed a significant increase of Plk1 staining on the kinetochores (Fig. 2, A and B), which is consistent with previously reported results (Beck and Peter, 2013; Beck et al., 2013). Furthermore, to investigate whether Usp16 counteracts CUL3-based ubiquitination, we simultaneously knocked down both Usp16 and KLHL22 and found that the staining of Plk1 on the kinetochore remained largely stable (Fig. 2, A and B), suggesting that the dynamic equilibrium of ubiquitination and deubiquitination regulates the kinetochore localization of Plk1. To eliminate the possibility that these changes of Plk1 accumulation on the kinetochores were caused by the change of total Plk1 amount, we compared the level of total cellular Plk1 protein in cells before and after Usp16 knockdown but did not detect any difference (Fig. S1, A and B). This result suggests that Usp16 regulates the kinetochore localization but not the stability of Plk1 in early mitotic cells. To further clarify that the localization of Plk1 on kinetochores was regulated by ubiquitination/deubiquitination, we studied the interaction between Plk1 and the kinetochore-localized protein BubR1. We found that, whereas the kinetochore localization of BubR1 was unchanged in both the control and Usp16 knockdown HeLa cells, knockdown of Usp16 in the cells reduced the amount of Plk1 localized on the kinetochores (Fig. 2 D) and the binding between Plk1 and BubR1 (Fig. 2 C). On the other hand, when Usp16 was overexpressed, the Plk1–BubR1 interaction was enhanced (Fig. S1, C and D). These results strongly support the hypothesis that Usp16-mediated deubiquitination promotes kinetochore localization of Plk1 by enhancing its binding to yet-to-be-identified kinetochore-localized proteins, possibly including Usp16 and BubR1; on the other hand, ubiquitination reduces the binding of Plk1 with kinetochore-localized proteins and removes Plk1 from the kinetochores.


Usp16 regulates kinetochore localization of Plk1 to promote proper chromosome alignment in mitosis.

Zhuo X, Guo X, Zhang X, Jing G, Wang Y, Chen Q, Jiang Q, Liu J, Zhang C - J. Cell Biol. (2015)

Usp16 regulates the kinetochore localization of Plk1, leading to proper alignment and timely separation of chromosomes. (A) Immunostaining of Plk1 in prometaphase HeLa cells treated with negative control siRNA or siRNA targeting KLHL22, CUL3, Usp16, or both Usp16 and KLHL22. Crest was used as a centromere marker. (B) The fluorescence intensity ratios of Plk1 and Crest shown in A, determined from three independent experiments with n = 100–150. (C) HeLa cells were transfected with control siRNA or siRNA targeting Usp16, and their lysates were blotted (left three lanes). Plk1 was immunoprecipitated with anti-Plk1 antibody or IgG in a mock IP. BubR1 coimmunoprecipitated with Plk1 was blotted (right three lanes). (D) Immunostaining of Plk1 and BubR1 in HeLa cells treated with either control siRNA or siRNA targeting Usp16. (E) Immunostaining of α-tubulin (α-Tub) and Usp16 in HeLa cells treated with control siRNA or siRNA targeting Usp16. The arrow points to misaligned chromosomes. (F) Percentage of cells with chromosome misalignment shown in E, determined from three independent experiments with n = 200–250. (G) Time-lapse microscopy of RFP-H2B–expressing HeLa cells treated with siRNA targeting Usp16 or control siRNA. Arrows point to misaligned or improperly separated chromosomes. (H) Time from nuclear envelope breakdown (NEBD) to metaphase in HeLa cells shown in G, determined from three independent experiments with n = 20–25. Error bars indicate the SEM. ***, P < 0.001. NC, negative control. Bars: (A, D, E, and G) 10 µm; (A, magnified images) 1 µm.
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fig2: Usp16 regulates the kinetochore localization of Plk1, leading to proper alignment and timely separation of chromosomes. (A) Immunostaining of Plk1 in prometaphase HeLa cells treated with negative control siRNA or siRNA targeting KLHL22, CUL3, Usp16, or both Usp16 and KLHL22. Crest was used as a centromere marker. (B) The fluorescence intensity ratios of Plk1 and Crest shown in A, determined from three independent experiments with n = 100–150. (C) HeLa cells were transfected with control siRNA or siRNA targeting Usp16, and their lysates were blotted (left three lanes). Plk1 was immunoprecipitated with anti-Plk1 antibody or IgG in a mock IP. BubR1 coimmunoprecipitated with Plk1 was blotted (right three lanes). (D) Immunostaining of Plk1 and BubR1 in HeLa cells treated with either control siRNA or siRNA targeting Usp16. (E) Immunostaining of α-tubulin (α-Tub) and Usp16 in HeLa cells treated with control siRNA or siRNA targeting Usp16. The arrow points to misaligned chromosomes. (F) Percentage of cells with chromosome misalignment shown in E, determined from three independent experiments with n = 200–250. (G) Time-lapse microscopy of RFP-H2B–expressing HeLa cells treated with siRNA targeting Usp16 or control siRNA. Arrows point to misaligned or improperly separated chromosomes. (H) Time from nuclear envelope breakdown (NEBD) to metaphase in HeLa cells shown in G, determined from three independent experiments with n = 20–25. Error bars indicate the SEM. ***, P < 0.001. NC, negative control. Bars: (A, D, E, and G) 10 µm; (A, magnified images) 1 µm.
Mentions: As ubiquitination promotes the removal of Plk1 from the kinetochores (Beck and Peter, 2013; Beck et al., 2013), we suspected that down-regulation of Usp16 would result in increased Plk1 ubiquitination and promote the removal of Plk1 from the kinetochores. Indeed, after Usp16 knockdown, we observed a dramatic decrease of Plk1 staining on the kinetochores in prometaphase cells (Fig. 2, A and B). On the other hand, when the CUL3-based ubiquitin ligase was suppressed by either KLHL22 or CUL3 siRNA, we observed a significant increase of Plk1 staining on the kinetochores (Fig. 2, A and B), which is consistent with previously reported results (Beck and Peter, 2013; Beck et al., 2013). Furthermore, to investigate whether Usp16 counteracts CUL3-based ubiquitination, we simultaneously knocked down both Usp16 and KLHL22 and found that the staining of Plk1 on the kinetochore remained largely stable (Fig. 2, A and B), suggesting that the dynamic equilibrium of ubiquitination and deubiquitination regulates the kinetochore localization of Plk1. To eliminate the possibility that these changes of Plk1 accumulation on the kinetochores were caused by the change of total Plk1 amount, we compared the level of total cellular Plk1 protein in cells before and after Usp16 knockdown but did not detect any difference (Fig. S1, A and B). This result suggests that Usp16 regulates the kinetochore localization but not the stability of Plk1 in early mitotic cells. To further clarify that the localization of Plk1 on kinetochores was regulated by ubiquitination/deubiquitination, we studied the interaction between Plk1 and the kinetochore-localized protein BubR1. We found that, whereas the kinetochore localization of BubR1 was unchanged in both the control and Usp16 knockdown HeLa cells, knockdown of Usp16 in the cells reduced the amount of Plk1 localized on the kinetochores (Fig. 2 D) and the binding between Plk1 and BubR1 (Fig. 2 C). On the other hand, when Usp16 was overexpressed, the Plk1–BubR1 interaction was enhanced (Fig. S1, C and D). These results strongly support the hypothesis that Usp16-mediated deubiquitination promotes kinetochore localization of Plk1 by enhancing its binding to yet-to-be-identified kinetochore-localized proteins, possibly including Usp16 and BubR1; on the other hand, ubiquitination reduces the binding of Plk1 with kinetochore-localized proteins and removes Plk1 from the kinetochores.

Bottom Line: Usp16 deubiquitinates Plk1, resulting in an enhanced interaction with kinetochore-localized proteins such as BubR1, and thereby retains Plk1 on the kinetochores to promote proper chromosome alignment in early mitosis.Down-regulation of Usp16 causes increased ubiquitination and decreased kinetochore localization of Plk1.Thus, our data unveil a unique mechanism by which Usp16 promotes the localization and maintenance of Plk1 on the kinetochores for proper chromosome alignment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ministry of Education Key Laboratory of Cell Proliferation and Differentiation and State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China.

Show MeSH
Related in: MedlinePlus