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
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Mentions: Next, we set out to investigate the function of Usp16 phosphorylation by Plk1. We initially examined the localization of Usp16 in the presence or absence of Plk1 activity. As shown in Fig. S3 A, inhibition of Plk1 with BI2536 in mitotic HeLa cells did not cause any change on the kinetochore localization of Usp16. We also expressed GFP-fused WT Plk1 and PBD2A mutant and found that disruption of the kinetochore localization of Plk1 by the 2A mutations did not affect the kinetochore localization of Usp16 in prometaphase (Fig. S3 B). Next, to find out whether the activity of Usp16 was regulated by phosphorylation, the ubiquitination status of histone H2A, a physiological substrate of Usp16 (Joo et al., 2007), was examined. We found that the level of ubiquitinated histone H2A (ubH2A) was high in interphase and low in mitotic HeLa cells (Fig. S3, C–E), suggesting that Plk1 phosphorylates and activates Usp16. To prove this, purified recombinant Xenopuslaevis Usp16 was incubated with total histones extracted from HeLa cells. The results showed that the amount of ubH2A was reduced in the presence of both Usp16 and Xenopus Plk1 compared with that in the presence of Usp16 alone and was reduced further if Usp16 was pretreated with CDK1, which supported our early result that CDK1 was a priming kinase for Plk1 (Fig. 4 A). To test this in vivo, we added BI2536 to HeLa cells and found that the level of ubH2A increased significantly, indicating that the activation of Usp16 was Plk1 dependent (Fig. 4, B and C). Moreover, the decrease of Usp16 activity as a result of the inhibition of Plk1 could be partially rescued by the expression of RNAi-resistant WT Usp16, but not the 3A mutant as judged by the ubH2A level (Fig. S3, F and G). These results strongly suggest that Plk1 phosphorylates and activates Usp16, but the exact molecular mechanism of the activation is not clear at the moment.
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.