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Decoding Polo-like kinase 1 signaling along the kinetochore–centromere axis

Abstract

Protein kinase signaling along the kinetochore–centromere axis is crucial to assure mitotic fidelity, yet the details of its spatial coordination are obscure. Here, we examined how pools of human Polo-like kinase 1 (Plk1) within this axis control signaling events to elicit mitotic functions. To do this, we restricted active Plk1 to discrete subcompartments within the kinetochore–centromere axis using chemical genetics and decoded functional and phosphoproteomic signatures of each. We observe distinct phosphoproteomic and functional roles, suggesting that Plk1 exists and functions in discrete pools along this axis. Deep within the centromere, Plk1 operates to assure proper chromosome alignment and segregation. Thus, Plk1 at the kinetochore is a conglomerate of an observable bulk pool coupled with additional functional pools below the threshold of microscopic detection or resolution. Although complex, this multiplicity of locales provides an opportunity to decouple functional and phosphoproteomic signatures for a comprehensive understanding of Plk1's kinetochore functions.

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Figure 1: Plk1 signaling at the kinetochore requires binding via its PBD.
Figure 2: High-resolution microscopy identifies discrete localization of endogenous Plk1 and kinetochore-tethered Plk1 constructs along the kinetochore–centromere axis.
Figure 3: 10-plex TMT phosphoproteomic analysis of Plk1 partitioned by locale along the kinetochore-centromere KT axis.
Figure 4: Restricting Plk1 activity along the kinetochore–centromere axis produces distinct phosphoproteomic and functional signatures.
Figure 5: Outer kinetochore tethering of Plk1 fails to restore chromosome alignment or segregation.
Figure 6: Functional and proteomic signatures and a model for Plk1 activity in the kinetochore.

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Acknowledgements

This work was supported by NIH R01 GM097245 (to M.E.B.), NIH R01 GM080148 (to J.J.C.), NIH R01 GM024364 (to E.D.S.), Cancer Center Support P30 CA014520, and http://www.Effcansah.com. The authors thank I.M. Cheeseman, S.S. Taylor, and T.J. Yen for contributing reagents, and B.A. Weaver and members of the Burkard laboratory for helpful discussions.

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Contributions

R.F.L. and M.E.B. designed the research. R.F.L., G.K.P., A.S., and J.M.J. performed experiments. R.F.L., G.K.P., A.S., J.M.J., and M.E.B. analyzed the data. M.E.B., J.J.C., and E.D.S. supervised the research. R.F.L. and M.E.B. drafted the manuscript. All authors revised and contributed to the manuscript.

Corresponding author

Correspondence to Mark E Burkard.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–12 and Supplementary Tables 1–3. (PDF 2968 kb)

Supplementary Data Set 1

Results table listing phosphopeptides encountered by mass spectrometry of Plk1 cell lines labeled with tandem mass tags. A total of 11,407 phosphopeptides were encountered from the 10 cell fractions (5 cell lines A BI- 2536). The raw 10-plex reporter ion intensities of localized phosphopeptide isoforms were log2 transformed and normalized against inhibited Plk1wt (+BI-2536) to obtain the relative phosphopeptide and protein quantitation for each cell line and condition. Terminal columns in the table indicate relative phosphopeptide expression for individual lines after BI-2536 treatment. See Online Methods section for details of analysis. (XLSX 8750 kb)

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Lera, R., Potts, G., Suzuki, A. et al. Decoding Polo-like kinase 1 signaling along the kinetochore–centromere axis. Nat Chem Biol 12, 411–418 (2016). https://doi.org/10.1038/nchembio.2060

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