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BubR1 phosphorylates CENP-E as a switch enabling the transition from lateral association to end-on capture of spindle microtubules

Abstract

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, powered congression of those chromosomes, their segregation in anaphase, and assembly of a spindle midzone at mitotic exit. The centromere-associated kinesin motor CENP-E, whose binding partner is BubR1, has been implicated in congression of misaligned chromosomes and the transition from lateral kinetochore-microtubule association to end-on capture. Although previously proposed to be a pseudokinase, here we report the structure of the kinase domain of Drosophila melanogaster BubR1, revealing its folding into a conformation predicted to be catalytically active. BubR1 is shown to be a bona fide kinase whose phosphorylation of CENP-E switches it from a laterally attached microtubule motor to a plus-end microtubule tip tracker. Computational modeling is used to identify bubristatin as a selective BubR1 kinase antagonist that targets the αN1 helix of N-terminal extension and αC helix of the BubR1 kinase domain. Inhibition of CENP-E phosphorylation is shown to prevent proper microtubule capture at kinetochores and, surprisingly, proper assembly of the central spindle at mitotic exit. Thus, BubR1-mediated CENP-E phosphorylation produces a temporal switch that enables transition from lateral to end-on microtubule capture and organization of microtubules into stable midzone arrays.

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Data availability

The accession numbers for apo DmBubR1c and ADP-bound DmBubR1c structures reported in this paper are PDB: 6JKK and 6JKM, respectively.

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Acknowledgements

We are grateful to Prof. Yunyu Shi for support; to Dr Hongtao Yu for reagents and Drs Guo-Qiang Chen for input. We thank all the members of our laboratories for discussion and Qiaoqiao Zhang for help on several BubR1 mutant constructs. This work was initiated by the chemical biology grant PGX-2 from the ProteoGenomics Research Laboratory, and supported in part by the National Key Research and Development Program of China (2017YFA0503600, 2017YFA0504901, 2016YFA0100500), National Natural Science Foundation of China (31320103904, 31430054, 91313303, 31621002, 31301120, 91854203, 91853115, 91753000, 31671405, 31470792, 31601097, 21672201, 31600607, and B1661138004), ‘Strategic Priority Research Program’ of the Chinese Academy of Sciences (XDB19000000 and XDB08030102), MOE Innovative team (IRT_17R102).

Author information

X. Y. and R. Z. conceived the project. Y. H., L. L., X. L. & S. Y. designed and performed most structure-activity relationship experiments. X. L., P. Y. Y., Y. Z., J. Z., J. Za., M. T., Z. W., and X. D. designed and performed chemical screening and synthesis of chemical derivative. Y. H., W. W., X. G., F. Y., J. L., Xu L., K.R., and X. D. performed in vitro reconstitution experiment and data analyses. Z. Y., Zhenye Y., L. Li and D. W. C. contributed to reagents. All authors contributed to the writing or editing of the manuscript.

Correspondence to Rongguang Zhang or Xuebiao Yao.

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