Molecular basis of outer kinetochoreassembly on CENP-T
- Max Planck Institute of Molecular Physiology, Germany
- Chemical Genomics Centre of the Max Planck Society, Germany
- Vienna Biocenter, Austria
- Vienna Biocenter, Germany
Abstract
Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm.
Article and author information
Author details
Pim J Huis in 't Veld
Funding
European Research Council (AdG 669686 RECEPIANCE)
- Andrea Musacchio
Deutsche Forschungsgemeinschaft (CRC1093)
- Andrea Musacchio
European Molecular Biology Organization (ALTF 262-2009)
- Sadasivam Jeganathan
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2016, Huis in 't Veld et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Molecular basis of outer kinetochoreassembly on CENP-T
eLife 5:e21007.
https://doi.org/10.7554/eLife.21007
Further reading
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- Biochemistry and Chemical Biology
In healthy cells, cyclin D1 is expressed during the G1 phase of the cell cycle, where it activates CDK4 and CDK6. Its dysregulation is a well-established oncogenic driver in numerous human cancers. The cancer-related function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G-Two and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unrecognized substrate of cyclin D1–CDK4/6 in tumor cells overexpressing cyclin D1 during G1 and subsequent phases. The phosphorylation of GTSE1 mediated by cyclin D1–CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 across all cell cycle phases. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1’s role in cell cycle control and oncogenesis beyond RB phosphorylation.
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- Biochemistry and Chemical Biology
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