Inhibitors of Polo-like kinase reveal roles in spindle-pole maintenance

Abstract

Polo-like kinases (Plks) have several functions in mitotic progression and are upregulated in many tumor types. Small-molecule Plk inhibitors would be valuable as tools for studying Plk biology and for developing antitumor agents. Guided by homology modeling of the Plk1 kinase domain, we have discovered a chemical series that shows potent and selective Plk1 inhibition. The effects of one such optimized benzthiazole N-oxide, cyclapolin 1 (1), on purified centrosomes indicate that Plks are required to generate MPM2 epitopes, recruit γ-tubulin and enable nucleation of microtubules. The compound can also promote loss of centrosome integrity and microtubule nucleating ability apparently through increased accessibility of protein phosphatases. We show that treatment of living S2 cells with cyclapolin 1 leads to collapsed spindles, in contrast to the metaphase-arrested bipolar spindles observed after RNAi. This different response to protein depletion and protein inhibition may have significance in the development of antitumor agents.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Chemical structures of Plk1 inhibitors.
Figure 2: Mitotic abnormalities induced by cyclapolin 1 in HeLa cells.
Figure 3: Cyclapolin 1 induces monopolar spindles in S2 cells.
Figure 4: Time-lapse series of control and 1-treated S2 cells.
Figure 5: Cyclapolin 1 inhibits the nucleation of microtubules by centrosomes in vitro.
Figure 6: Effects of cyclapolin 1 on the restoration of the microtubule nucleating activity of salt-stripped centrosomes by cytoplasmic extracts.

References

  1. 1

    Sunkel, C.E. & Glover, D.M. Polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. J. Cell Sci. 89, 25–38 (1988).

    PubMed  Google Scholar 

  2. 2

    Llamazares, S. et al. Polo encodes a protein kinase homolog required for mitosis in Drosophila. Genes Dev. 5, 2153–2165 (1991).

    CAS  Article  Google Scholar 

  3. 3

    McInnes, C., Mezna, M. & Fischer, P.M. Progress in the discovery of polo-like kinase inhibitors. Curr. Top. Med. Chem. 5, 181–197 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Glover, D.M., Hagan, I.M. & Tavares, A.A. Polo-like kinases: a team that plays throughout mitosis. Genes Dev. 12, 3777–3787 (1998).

    CAS  Article  Google Scholar 

  5. 5

    Elia, A.E., Cantley, L.C. & Yaffe, M.B. Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science 299, 1228–1231 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Leung, G.C. et al. The Sak polo-box comprises a structural domain sufficient for mitotic subcellular localization. Nat. Struct. Biol. 9, 719–724 (2002).

    CAS  Article  Google Scholar 

  7. 7

    Elia, A.E. et al. The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell 115, 83–95 (2003).

    CAS  Article  Google Scholar 

  8. 8

    Jang, Y.J. et al. Polo-box motif targets a centrosome regulator, RanGTPase. Biochem. Biophys. Res. Commun. 325, 257–264 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Kumagai, A. & Dunphy, W.G. Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. Science 273, 1377–1380 (1996).

    CAS  Article  Google Scholar 

  10. 10

    Watanabe, N. et al. Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways. Proc. Natl. Acad. Sci. USA 102, 11663–11668 (2005).

    CAS  Article  Google Scholar 

  11. 11

    van Vugt, M.A. & Medema, R.H. Getting in and out of mitosis with Polo-like kinase-1. Oncogene 24, 2844–2859 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Glover, D.M. Polo kinase and progression through M phase in Drosophila: a perspective from the spindle poles. Oncogene 24, 230–237 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Hansen, D.V., Loktev, A.V., Ban, K.H. & Jackson, P.K. Plk1 regulates activation of the anaphase promoting complex by phosphorylating and triggering SCFbetaTrCP-dependent destruction of the APC Inhibitor Emi1. Mol. Biol. Cell 15, 5623–5634 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Moshe, Y., Boulaire, J., Pagano, M. & Hershko, A. Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome. Proc. Natl. Acad. Sci. USA 101, 7937–7942 (2004).

    CAS  Article  Google Scholar 

  15. 15

    Rauh, N.R., Schmidt, A., Bormann, J., Nigg, E.A. & Mayer, T.U. Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation. Nature 437, 1048–1052 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Liu, J. & Maller, J.L. Calcium elevation at fertilization coordinates phosphorylation of XErp1/Emi2 by Plx1 and CaMK II to release metaphase arrest by cytostatic factor. Curr. Biol. 15, 1458–1468 (2005).

    CAS  Article  Google Scholar 

  17. 17

    Tung, J.J. et al. A role for the anaphase-promoting complex inhibitor Emi2/XErp1, a homolog of early mitotic inhibitor1, incytostatic factor arrest of Xenopus eggs. Proc. Natl. Acad. Sci. USA 102, 4318–4323 (2005).

    CAS  Article  Google Scholar 

  18. 18

    Hauf, S. et al. Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2. PLoS Biol. 3, e69 (2005).

    Article  Google Scholar 

  19. 19

    Clarke, A.S., Tang, T.T., Ooi, D.L. & Orr-Weaver, T.L. POLO kinase regulates the Drosophila centromere cohesion protein MEI-S332. Dev. Cell 8, 53–64 (2005).

    CAS  Article  Google Scholar 

  20. 20

    Hornig, N.C. & Uhlmann, F. Preferential cleavage of chromatin-bound cohesin after targeted phosphorylation by Polo-like kinase. EMBO J. 23, 3144–3153 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Wong, O.K. & Fang, G. Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores. J. Cell Biol. 170, 709–719 (2005).

    CAS  Article  Google Scholar 

  22. 22

    Ahonen, L.J. et al. Polo-like kinase 1 creates the tension-sensing 3F3/2 phosphoepitope and modulates the association of spindle-checkpoint proteins at kinetochores. Curr. Biol. 15, 1078–1089 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Carmena, M. et al. Drosophila polo kinase is required for cytokinesis. J. Cell Biol. 143, 659–671 (1998).

    CAS  Article  Google Scholar 

  24. 24

    Lee, K.S., Yuan, Y.L., Kuriyama, R. & Erikson, R.L. Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1. Mol. Cell. Biol. 15, 7143–7151 (1995).

    CAS  Article  Google Scholar 

  25. 25

    Neef, R. et al. Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis. J. Cell Biol. 162, 863–875 (2003).

    CAS  Article  Google Scholar 

  26. 26

    Liu, X., Zhou, T., Kuriyama, R. & Erikson, R.L. Molecular interactions of Polo-like-kinase 1 with the mitotic kinesin-like protein CHO1/MKLP-1. J. Cell Sci. 117, 3233–3246 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Lindon, C. & Pines, J. Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells. J. Cell Biol. 164, 233–241 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Spankuch-Schmitt, B., Bereiter-Hahn, J., Kaufmann, M. & Strebhardt, K. Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells. J. Natl. Cancer Inst. 94, 1863–1877 (2002).

    CAS  Article  Google Scholar 

  29. 29

    Elez, R. et al. Tumor regression by combination antisense therapy against Plk1 and Bcl-2. Oncogene 22, 69–80 (2003).

    CAS  Article  Google Scholar 

  30. 30

    Wu, S.Y. et al. Discovery of a novel family of CDK inhibitors with the program LIDAEUS: structural basis for ligand-induced disordering of the activation loop. Structure 11, 399–410 (2003).

    CAS  Article  Google Scholar 

  31. 31

    Lane, H.A. & Nigg, E.A. Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes. J. Cell Biol. 135, 1701–1713 (1996).

    CAS  Article  Google Scholar 

  32. 32

    Sumara, I. et al. Roles of polo-like kinase 1 in the assembly of functional mitotic spindles. Curr. Biol. 14, 1712–1722 (2004).

    CAS  Article  Google Scholar 

  33. 33

    Bettencourt-Dias, M. et al. Genome-wide survey of protein kinases required for cell cycle progression. Nature 432, 980–987 (2004).

    CAS  Article  Google Scholar 

  34. 34

    Donaldson, M.M., Tavares, A., Ohkura, H., Deak, P. & Glover, D.M. Metaphase arrest with centromere separation in polo mutants of Drosophila. J. Cell Biol. 153, 663–675 (2001).

    CAS  Article  Google Scholar 

  35. 35

    do Carmo Avides, M., Tavares, A. & Glover, D.M. Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes. Nat. Cell Biol. 3, 421–424 (2001).

    CAS  Article  Google Scholar 

  36. 36

    Goshima, G. & Vale, R.D. The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. J. Cell Biol. 162, 1003–1016 (2003).

    CAS  Article  Google Scholar 

  37. 37

    Laycock, J.E., Savoian, M.S. & Glover, D.M. Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo. J. Cell Sci. 119, 2354–2361 (2006).

    CAS  Article  Google Scholar 

  38. 38

    Sampaio, P., Rebollo, E., Varmark, H., Sunkel, C.E. & Gonzalez, C. Organized microtubule arrays in gamma-tubulin-depleted Drosophila spermatocytes. Curr. Biol. 11, 1788–1793 (2001).

    CAS  Article  Google Scholar 

  39. 39

    Barbosa, V., Gatt, M., Rebollo, E., Gonzalez, C. & Glover, D.M. Drosophila dd4 mutants reveal that gammaTuRC is required to maintain juxtaposed half spindles in spermatocytes. J. Cell Sci. 116, 929–941 (2003).

    CAS  Article  Google Scholar 

  40. 40

    Tavares, A.A., Glover, D.M. & Sunkel, C.E. The conserved mitotic kinase polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts. EMBO J. 15, 4873–4883 (1996).

    CAS  Article  Google Scholar 

  41. 41

    Liu, X., Zhou, T., Kuriyama, R. & Erikson, R.L. Molecular interactions of Polo-like-kinase 1 with the mitotic kinesin-like protein CHO1/MKLP-1. J. Cell Sci. 117, 3233–3246 (2004).

    CAS  Article  Google Scholar 

  42. 42

    Blagden, S.P. & Glover, D.M. Polar expeditions–provisioning the centrosome for mitosis. Nat. Cell Biol. 5, 505–511 (2003).

    CAS  Article  Google Scholar 

  43. 43

    Moritz, M., Zheng, Y., Alberts, B.M. & Oegema, K. Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. J. Cell Biol. 142, 775–786 (1998).

    CAS  Article  Google Scholar 

  44. 44

    do Carmo Avides, M. & Glover, D.M. Abnormal spindle protein, Asp, and the integrity of mitotic centrosomal microtubule organizing centers. Science 283, 1733–1735 (1999).

    CAS  Article  Google Scholar 

  45. 45

    de Carcer, G., do Carmo Avides, M., Lallena, M.J., Glover, D.M. & Gonzalez, C. Requirement of Hsp90 for centrosomal function reflects its regulation of Polo kinase stability. EMBO J. 20, 2878–2884 (2001).

    CAS  Article  Google Scholar 

  46. 46

    Logarinho, E. & Sunkel, C.E. The Drosophila POLO kinase localises to multiple compartments of the mitotic apparatus and is required for the phosphorylation of MPM2 reactive epitopes. J. Cell Sci. 111, 2897–2909 (1998).

    CAS  PubMed  Google Scholar 

  47. 47

    Morales-Mulia, S. & Scholey, J.M. Spindle pole organization in Drosophila S2 cells by dynein, abnormal spindle protein (Asp), and KLP10A. Mol. Biol. Cell 16, 3176–3186 (2005).

    CAS  Article  Google Scholar 

  48. 48

    Moritz, M. & Alberts, B.M. Isolation of centrosomes from Drosophila embryos. Methods Cell Biol. 61, 1–12 (1999).

    CAS  PubMed  Google Scholar 

  49. 49

    Bettencourt-Dias, M. et al. SAK/PLK4 is required for centriole duplication and flagella development. Curr. Biol. 15, 2199–2207 (2005).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We would like to thank many colleagues at Cyclacel and the Cancer Research UK Cell Cycle Genetics Group who have contributed to this project. D.M.G. also acknowledges Cancer Research UK Programme support, a Special Cambridge Nehru Bursary and a Cancer Research UK PhD studentship to A.M. We thank M. Savoian and V. Archambault for their comments on the manuscript.

Author information

Affiliations

Authors

Contributions

A.M. carried out all the studies involving D. melanogaster S2 cells, human cells, and isolated preparations of centrosomes. C.Mc. completed the computational experiments and contributed to project management and manuscript writing. M.M. was responsible for in vitro screening and contributed intellectually. C.Me. was responsible for the synthesis of benzthiazole analogs described. C.Mi. and F.S. evaluated effects of 1 on human cell lines. L.C. carried out RNAi and immunostaining of HeLa cells. P.T. and M.W. were responsible for the development and application of LIDAEUS, and P.M.F. contributed to the medical chemistry and project directions.

Corresponding author

Correspondence to David Glover.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Mitotic phenotypes following Plk1 RNAi in HeLa cells. (PDF 102 kb)

Supplementary Fig. 2

Cyclapolin 1 does not affect phosphorylation of histone H3 on Ser10. (PDF 67 kb)

Supplementary Fig. 3

Dose-dependent loss of γ-tubulin and MPM2 phosphoepitope for cyclapolin 1-treated centrosomes. (PDF 179 kb)

Supplementary Table 1 (PDF 84 kb)

Supplementary Table 2 (PDF 228 kb)

Supplementary Table 3 (PDF 9 kb)

Supplementary Methods (PDF 84 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

McInnes, C., Mazumdar, A., Mezna, M. et al. Inhibitors of Polo-like kinase reveal roles in spindle-pole maintenance. Nat Chem Biol 2, 608–617 (2006). https://doi.org/10.1038/nchembio825

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing