Usp16 regulates kinetochore localization of Plk1 to promote proper chromosome alignment in mitosis.
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.
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
License 1 - License 2
Mentions: It has been reported that Plk1 interacts with several deubiquitylases based on mass spectrometry (MS) analysis (Lowery et al., 2007). To identify any deubiquitylases that may deubiquitinate Plk1 in early mitosis, we performed reciprocal coimmunoprecipitation (coIP) assays and found that Usp16 specifically interacted with Plk1 in nocodazole-arrested prometaphase HeLa cells (Fig. 1, A and B). This interaction was verified by coIP of endogenous Plk1 and GFP-tagged Usp16 expressed in HeLa cells (Fig. 1 C). To determine whether this interaction is PBD mediated, bacteria-expressed GST-PBD or GST-PBD with H538A/K540A (2A) mutations that disrupt the protein-interacting capability of the PBD (Elia et al., 2003) was incubated with prometaphase HeLa cell lysates. Examination of the GST pull-down complexes showed that Usp16 specifically interacted with the wild-type (WT) PBD but not the PBD2A mutant (Fig. 1 D), indicating that the interaction between Plk1 and Usp16 is PBD dependent. Because the subcellular location of Plk1 undergoes dramatic changes during the cell cycle, we wanted to know the localization of Usp16 in both interphase and M phase. For this purpose, we performed immunostaining and observed that most Usp16 were cytoplasmic in interphase but were on the kinetochores from prometaphase to the end of mitosis. However, its accumulation on the kinetochores was reduced after prometaphase (Fig. 1, E–G). It was also found that Usp16 colocalized with Plk1 on kinetochores at prometaphase, and the colocalization was reduced at metaphase (Fig. 1 H).
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.