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MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components

Abstract

The genomic stability of all organisms depends on the precise partition of chromosomes to daughter cells. The spindle assembly checkpoint (SAC) senses unattached kinetochores and prevents premature entry to anaphase, thus ensuring that all chromosomes attach to opposite spindle poles (bi-orientation) during mitosis1. MPS1 is an evolutionarily conserved protein kinase required for the SAC and chromosome bi-orientation2,3,4. Yet, its primary cellular substrate has remained elusive. We show that fission yeast Mph1 (MPS1 homologue) phosphorylates the kinetochore protein Spc7 (KNL1/Blinkin homologue) at the MELT repeat sequences. This phosphorylation promotes the in vitro binding to the Bub1–Bub3 complex, which is required for kinetochore-based SAC activation (Mad1–Mad2–Mad3 localization) and chromosome alignment. Accordingly, a non-phosphorylatable spc7-12A mutation abolishes kinetochore targeting of Bub1–Bub3, whereas a phospho-mimetic spc7-12E mutation forces them to localize at kinetochores throughout the entire cell cycle, even in the absence of Mph1. Thus, MPS1/Mph1 kinase locating at the unattached kinetochores initially creates a mark, which is crucial for SAC activation and chromosome bi-orientation. This mechanism seems to be conserved in human cells.

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Figure 1: Schizosaccharomyces pombe Mph1 kinase targets Bub1 to kinetochores.
Figure 2: Phosphorylation of Spc7 by Mph1 promotes Bub1 targeting to kinetochores.
Figure 3: Phosphorylation of KNL1 by MPS1 promotes BUB1 targeting to kinetochores in human cells.
Figure 4: Phosphorylation of Spc7 by Mph1 promotes direct association between the Bub1–Bub3 complex and Spc7.
Figure 5: Phosphorylation of Spc7 by Mph1 is required for SAC activation and chromosome bi-orientation.

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Acknowledgements

We thank S. Hauf for critical reading of the manuscript and for yeast strains, and all of the members of our laboratory for their valuable support and discussion. This work was supported in part by JSPS Research Fellowships and Grant-in-Aid for Research Activity Start-up (to Y.Y.), the Global COE Program and a Grant-in-Aid for Specially Promoted Research (to Y.W.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Most experiments were performed by Y.Y. except those in Fig. 3 and Supplementary Fig. S4, which were performed by C.Y. and Y.T. Experimental design and interpretation of data were conducted by all authors. Y.W. supervised the project and Y.Y. and Y.W. wrote the paper.

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Correspondence to Yoshinori Watanabe.

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Yamagishi, Y., Yang, CH., Tanno, Y. et al. MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components. Nat Cell Biol 14, 746–752 (2012). https://doi.org/10.1038/ncb2515

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