Deficiency in Chromosome Congression by the Inhibition of Plk1 Polo Box Domain-dependent Recognition*

Polo-like kinase 1 (Plk1) is one of the key regulators of mitotic cell division. In addition to an N-terminal protein kinase catalytic domain, Plk1 possesses a phosphopeptide binding domain named polo box domain (PBD) at its C terminus. PBD is postulated to be essential for Plk1 localization and substrate targeting. Here, we developed a high-throughput screening system to identify inhibitors of PBD-dependent binding and screened a chemical library. We isolated a benzotropolone-containing natural compound derived from nutgalls (purpurogallin (PPG)) that inhibited PBD-dependent binding in vitro and in vivo. PPG not only delayed the onset of mitosis but also prolonged the progression of mitosis in HeLa cells. Although apparently normal bipolar spindles were formed even in the presence of PPG, the perturbation of chromosome alignment at metaphase plates activated the spindle assembly checkpoint pathway. These results demonstrate the predominant role of PBD-dependent binding on smooth chromosome congression at metaphase.

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

[2]  Kyung S. Lee,et al.  A Spindle Checkpoint Arrest and a Cytokinesis Failure by the Dominant-negative Polo-box Domain of Plk1 in U-2 OS Cells* 210 , 2002, The Journal of Biological Chemistry.

[3]  Hongtao Yu,et al.  Phosphorylation- and polo-box-dependent binding of Plk1 to Bub1 is required for the kinetochore localization of Plk1. , 2006, Molecular biology of the cell.

[4]  John Calvin Reed,et al.  Discovery, characterization, and structure-activity relationships studies of proapoptotic polyphenols targeting B-cell lymphocyte/leukemia-2 proteins. , 2003, Journal of medicinal chemistry.

[5]  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.

[6]  I. Sumara,et al.  The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase. , 2002, Molecular cell.

[7]  J. Peters,et al.  The Small-Molecule Inhibitor BI 2536 Reveals Novel Insights into Mitotic Roles of Polo-like Kinase 1 , 2007, Current Biology.

[8]  Xiaoqi Liu,et al.  A role for Plk1 phosphorylation of NudC in cytokinesis. , 2003, Developmental cell.

[9]  Takeharu Nagai,et al.  Shift anticipated in DNA microarray market , 2002, Nature Biotechnology.

[10]  R. Zinkowski,et al.  CENP‐E, a novel human centromere‐associated protein required for progression from metaphase to anaphase. , 1991, The EMBO journal.

[11]  Marina Bibikova,et al.  Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference. , 2005, Molecular biology of the cell.

[12]  E. Nigg,et al.  Tension-sensitive Plk1 phosphorylation on BubR1 regulates the stability of kinetochore microtubule interactions. , 2007, Genes & development.

[13]  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.

[14]  Hiroyuki Osada,et al.  M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFbeta-TrCP. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[15]  T. Kanda,et al.  Histone–GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells , 1998, Current Biology.

[16]  Erich A Nigg,et al.  Different Plk1 functions show distinct dependencies on Polo-Box domain-mediated targeting. , 2005, Molecular biology of the cell.

[17]  B. Brinkley,et al.  NudC Is Required for Plk1 Targeting to the Kinetochore and Chromosome Congression , 2006, Current Biology.

[18]  Erich A. Nigg,et al.  Polo-like kinases and the orchestration of cell division , 2004, Nature Reviews Molecular Cell Biology.

[19]  G. Fang,et al.  Cdk1 phosphorylation of BubR1 controls spindle checkpoint arrest and Plk1-mediated formation of the 3F3/2 epitope , 2007, The Journal of cell biology.

[20]  T. Hirano,et al.  Cohesin release is required for sister chromatid resolution, but not for condensin-mediated compaction, at the onset of mitosis. , 2002, Genes & development.

[21]  E. Nishida,et al.  Polo-like Kinase 1 Facilitates Chromosome Alignment during Prometaphase through BubR1* , 2007, Journal of Biological Chemistry.

[22]  R. Erikson,et al.  The serum-inducible protein kinase Snk is a G1 phase polo-like kinase that is inhibited by the calcium- and integrin-binding protein CIB. , 2003, Molecular cancer research : MCR.

[23]  R. Wolthuis,et al.  Polo-like Kinase-1 Is Required for Bipolar Spindle Formation but Is Dispensable for Anaphase Promoting Complex/Cdc20 Activation and Initiation of Cytokinesis* , 2004, Journal of Biological Chemistry.

[24]  B. Schaar,et al.  CENP-E Function at Kinetochores Is Essential for Chromosome Alignment , 1997, The Journal of cell biology.

[25]  Kiranmai Gumireddy,et al.  ON01910, a non-ATP-competitive small molecule inhibitor of Plk1, is a potent anticancer agent. , 2005, Cancer cell.

[26]  N Watanabe,et al.  Regulation of the human WEE1Hu CDK tyrosine 15‐kinase during the cell cycle. , 1995, The EMBO journal.

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

[28]  H. Osada,et al.  Mutations in the Plk gene lead to instability of Plk protein in human tumour cell lines , 2000, Nature Cell Biology.

[29]  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.

[30]  T. Kapoor,et al.  The human mitotic checkpoint protein BubR1 regulates chromosome–spindle attachments , 2005, Nature Cell Biology.

[31]  Y. Sakagami,et al.  Biological activity of purpurogallin. , 1997, Bioscience, biotechnology, and biochemistry.

[32]  Koichi Furukawa,et al.  Complex formation of Plk1 and INCENP required for metaphase–anaphase transition , 2006, Nature Cell Biology.

[33]  F. Bushman,et al.  Human Immunodeficiency Virus Type 1 cDNA Integration: New Aromatic Hydroxylated Inhibitors and Studies of the Inhibition Mechanism , 1998, Antimicrobial Agents and Chemotherapy.

[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]  T. Kapoor,et al.  Probing cell-division phenotype space and Polo-like kinase function using small molecules , 2006, Nature chemical biology.

[36]  E. Nigg,et al.  Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis. , 2007, Molecular biology of the cell.

[37]  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.

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

[39]  E. Nishida,et al.  Plk1 promotes nuclear translocation of human Cdc25C during prophase , 2002, EMBO reports.

[40]  C. Rieder.  The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells , 2004, Chromosoma.

[41]  J. Dennis,et al.  Late mitotic failure in mice lacking Sak, a polo-like kinase , 2001, Current Biology.

[42]  T. Veenstra,et al.  Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation. , 2006, Molecular cell.

[43]  K. Strebhardt,et al.  Inhibition of polo-like kinase 1 by blocking polo-box domain-dependent protein-protein interactions. , 2008, Chemistry & biology.

[44]  M. Abou-Karam,et al.  Inhibition of oncogene product enzyme activity as an approach to cancer chemoprevention. Tyrosine‐specific protein kinase inhibition by purpurogallin from Quercus sp. Nutgall , 1999, Phytotherapy research : PTR.

[45]  Bruce M. Spiegelman,et al.  Uncoupling of Obesity from Insulin Resistance Through a Targeted Mutation in aP2, the Adipocyte Fatty Acid Binding Protein , 1996, Science.

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

[47]  J. McIntosh,et al.  Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E. , 2002, Developmental cell.

[48]  T. Kapoor,et al.  Chromosomes Can Congress to the Metaphase Plate Before Biorientation , 2006, Science.

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

[50]  Paul Taylor,et al.  Inhibitors of Polo-like kinase reveal roles in spindle-pole maintenance , 2006, Nature chemical biology.