Year-end Sale % OFF 
Abstract
Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore-microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore-microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80-microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1-80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80-microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore-microtubule attachment in the maintenance of genomic stability during mitosis.
Highlights
Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore–microtubule attachments
Our previous study showed that acetylation of Aurora B kinase by TIP60 protects an activation of Aurora B from dephosphorylation by the PP2A phosphatase to ensure error-free chromosome segregation during cell division [29]
Because HEC1 forms a tetrameric NDC80C together with NUF2, SPC24, and SPC25 to function in kinetochore–microtubule attachment in mitosis [4, 6, 8, 32], we tested whether TIP60 interacts with NDC80C
Summary
Our previous study showed that acetylation of Aurora B kinase by TIP60 protects an activation of Aurora B from dephosphorylation by the PP2A phosphatase to ensure error-free chromosome segregation during cell division [29]. Because HEC1 exhibits critical importance in kinetochore– microtubule binding and spindle assembly checkpoint signaling [33, 34, 37], we sought to examine whether HEC1 acetylation modulates the kinetochore–microtubule attachment in mitosis To this end, HeLa cells were treated with HEC1 siRNA to suppress endogenous HEC1 protein, followed by expressing siRNA-resistant exogenous GFP-HEC1WT, GFP-HEC1RR, or GFP-HEC1QQ. When HeLa cells were blocked in interphase or prometaphase and the activity of endogenous TIP60 was inhibited by its specific inhibitor NU9056 or shTIP60, the binding of endogenous HEC1 to recombinant GST-NUF2(1–169)-His was attenuated in a pulldown experiment (Fig. S5, C and D). Given the changing of binding affinity of TIP60 and Sirt to HEC1 in different phases illustrated above (Fig. 6D), we sought to analyze how the HEC1 acetylation level and amount of HEC1– NUF2 complex changed. With the high binding affinity of TIP60 and significantly reduced binding affinity of Sirt, the acetylation level of HEC1 was significantly elevated and elicited the chemical equilibrium’s shift toward the stabilization of HEC1–NUF2 complex and promoted highly stable attachment to microtubules
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
