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

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.

[1]  D. Glover.  Polo kinase and progression through M phase in Drosophila: a perspective from the spindle poles , 2005, Oncogene.

[2]  M. Pagano,et al.  Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[3]  B. Alberts,et al.  Isolation of centrosomes from Drosophila embryos. , 1999, Methods in cell biology.

[4]  P. Fischer,et al.  Progress in the discovery of polo-like kinase inhibitors. , 2005, Current topics in medicinal chemistry.

[5]  J. Maller,et al.  Calcium Elevation at Fertilization Coordinates Phosphorylation of XErp1/Emi2 by Plx1 and CaMK II to Release Metaphase Arrest by Cytostatic Factor , 2005, Current Biology.

[6]  J. Adler,et al.  A role for the anaphase-promoting complex inhibitor Emi2/XErp1, a homolog of early mitotic inhibitor 1, in cytostatic factor arrest of Xenopus eggs. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[7]  D. Glover,et al.  Polar expeditions — provisioning the centrosome for mitosis , 2003, Nature Cell Biology.

[8]  Hiroyuki Osada,et al.  Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Kumagai,et al.  Purification and Molecular Cloning of Plx1, a Cdc25-Regulatory Kinase from Xenopus Egg Extracts , 1996, Science.

[10]  J. R. Daum,et al.  Polo-like Kinase 1 Creates the Tension-Sensing 3F3/2 Phosphoepitope and Modulates the Association of Spindle-Checkpoint Proteins at Kinetochores , 2005, Current Biology.

[11]  K. Hoe,et al.  Polo-box motif targets a centrosome regulator, RanGTPase. , 2004, Biochemical and biophysical research communications.

[12]  G. Fang,et al.  Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores , 2005, The Journal of cell biology.

[13]  Michael B. Yaffe,et al.  The Molecular Basis for Phosphodependent Substrate Targeting and Regulation of Plks by the Polo-Box Domain , 2003, Cell.

[14]  J. Dennis,et al.  The Sak polo-box comprises a structural domain sufficient for mitotic subcellular localization , 2002, Nature Structural Biology.

[15]  C. Sunkel,et al.  polo encodes a protein kinase homolog required for mitosis in Drosophila. , 1991, Genes & development.

[16]  C. Sunkel,et al.  Organized microtubule arrays in γ-tubulin-depleted Drosophila spermatocytes , 2001, Current Biology.

[17]  J. Pines,et al.  Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells , 2004, The Journal of cell biology.

[18]  E. Nigg,et al.  Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis , 2003, The Journal of cell biology.

[19]  D. Glover,et al.  Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo , 2006, Journal of Cell Science.

[20]  B. Kronenberger,et al.  Tumor regression by combination antisense therapy against Plk1 and Bcl-2 , 2003, Oncogene.

[21]  Jürgen Bereiter-Hahn,et al.  Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells. , 2002, Journal of the National Cancer Institute.

[22]  Michael B Yaffe,et al.  Proteomic Screen Finds pSer/pThr-Binding Domain Localizing Plk1 to Mitotic Substrates , 2003, Science.

[23]  F. Uhlmann,et al.  Preferential cleavage of chromatin‐bound cohesin after targeted phosphorylation by Polo‐like kinase , 2004, The EMBO journal.

[24]  Xiaoqi Liu,et al.  Molecular interactions of Polo-like-kinase 1 with the mitotic kinesin-like protein CHO1/MKLP-1 , 2004, Journal of Cell Science.

[25]  P. Jackson,et al.  Plk1 regulates activation of the anaphase promoting complex by phosphorylating and triggering SCFbetaTrCP-dependent destruction of the APC Inhibitor Emi1. , 2004, Molecular biology of the cell.

[26]  D. Glover,et al.  Requirement of Hsp90 for centrosomal function reflects its regulation of Polo kinase stability , 2001, The EMBO journal.

[27]  D. M. Glover,et al.  Genome-wide survey of protein kinases required for cell cycle progression , 2004, Nature.

[28]  R. Erikson,et al.  Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1 , 1995, Molecular and cellular biology.

[29]  D. Glover,et al.  Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes , 2001, Nature Cell Biology.

[30]  D. Ooi,et al.  POLO kinase regulates the Drosophila centromere cohesion protein MEI-S332. , 2005, Developmental cell.

[31]  L. Lehmann,et al.  SAK/PLK4 Is Required for Centriole Duplication and Flagella Development , 2005, Current Biology.

[32]  Su-Ying Wu,et al.  Discovery of a novel family of CDK inhibitors with the program LIDAEUS: structural basis for ligand-induced disordering of the activation loop. , 2003, Structure.

[33]  E. Nigg,et al.  Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation , 2005, Nature.

[34]  H. Lane,et al.  Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes , 1996, The Journal of cell biology.

[35]  David M. Glover,et al.  Metaphase Arrest with Centromere Separation in polo Mutants of Drosophila , 2001, The Journal of cell biology.

[36]  D. Glover,et al.  Drosophila Polo Kinase Is Required for Cytokinesis , 1998, The Journal of cell biology.

[37]  M. V. Vugt,et al.  Getting in and out of mitosis with Polo-like kinase-1 , 2005, Oncogene.

[38]  D. Glover,et al.  Abnormal spindle protein, Asp, and the integrity of mitotic centrosomal microtubule organizing centers. , 1999, Science.

[39]  D. Glover,et al.  Polo-like kinases: a team that plays throughout mitosis. , 1998, Genes & development.

[40]  Jan Ellenberg,et al.  Roles of Polo-like Kinase 1 in the Assembly of Functional Mitotic Spindles , 2004, Current Biology.

[41]  C. Sunkel,et al.  The Drosophila POLO kinase localises to multiple compartments of the mitotic apparatus and is required for the phosphorylation of MPM2 reactive epitopes. , 1998, Journal of cell science.

[42]  C. Sunkel,et al.  The conserved mitotic kinase polo is regulated by phosphorylation and has preferred microtubule‐associated substrates in Drosophila embryo extracts. , 1996, The EMBO journal.

[43]  C. Sunkel,et al.  polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. , 1988, Journal of cell science.

[44]  G. Goshima,et al.  The roles of microtubule-based motor proteins in mitosis , 2003, The Journal of cell biology.

[45]  J. Scholey,et al.  Spindle pole organization in Drosophila S2 cells by dynein, abnormal spindle protein (Asp), and KLP10A. , 2005, Molecular biology of the cell.

[46]  D. Glover,et al.  Drosophila dd4 mutants reveal that γTuRC is required to maintain juxtaposed half spindles in spermatocytes , 2003, Journal of Cell Science.

[47]  B. Alberts,et al.  Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. , 1998 .

[48]  Karl Mechtler,et al.  Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2 , 2005, PLoS biology.