Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells Journal Article

Authors: Burkard, M. E.; Randall, C. L.; Larochelle, S.; Zhang, C.; Shokat, K. M.; Fisher, R. P.; Jallepalli, P. V.
Article Title: Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells
Abstract: Polo-like kinases (Plks) play crucial roles in mitosis and cell division. Whereas lower eukaryotes typically contain a single Plk, mammalian cells express several closely related but functionally distinct Plks. We describe here a chemical genetic system in which a single Plk family member, Plk1, can be inactivated with high selectivity and temporal resolution by using an allele-specific, small-molecule inhibitor, as well as the application of this system to dissect Plk1's role in cytokinesis. To do this, we disrupted both copies of the PLK1 locus in human cells through homologous recombination and then reconstituted Plk1 activity by using either the wild-type kinase (Plk1 wt) or a mutant version whose catalytic pocket has been enlarged to accommodate bulky purine analogs (Plk1as). When cultured in the presence of these analogs, Plk1as cells accumulate in prometaphase with defects that parallel those found in PLK1Δ/Δ cells. In addition, acute treatment of Plk1as cells during anaphase prevents recruitment of both Plk1 itself and the Rho guanine nucleotide exchange factor (RhoGEF) Ect2 to the central spindle, abolishes RhoA GTPase localization to the equatorial cortex, and suppresses cleavage furrow formation and cell division. Our studies define and illuminate a late mitotic function of Plk1 that, although difficult or impossible to detect in Plk1-depleted cells, is readily revealed with chemical genetics. © 2007 by The National Academy of Sciences of the USA.
Keywords: controlled study; protein expression; unclassified drug; oncoprotein; human cell; genetics; proto-oncogene proteins; dose response; mitosis; cell cycle protein; metabolism; mammalia; cell cycle proteins; allele; homologous recombination; cell division; cell line; alleles; dose-response relationship, drug; enzyme activity; protein serine threonine kinase; physiology; genetic recombination; eukaryota; polo like kinase 1; polo-like kinase 1; protein-serine-threonine kinases; recombination, genetic; enzyme inactivation; rhoa guanine nucleotide binding protein; ect2 protein, human; anaphase; chemical genetics; mitosis spindle; mitotic spindle apparatus; purine derivative; catalysis; genetic techniques; genetic procedures; enzyme localization; rho guanine nucleotide binding protein; cytokinesis; knockout; centrosome; rho factor; guanine nucleotide exchange factor; rhoa gtp-binding protein; ect2
Journal Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 104
Issue: 11
ISSN: 0027-8424
Publisher: National Academy of Sciences  
Date Published: 2007-03-13
Start Page: 4383
End Page: 4388
Language: English
DOI: 10.1073/pnas.0701140104
PUBMED: 17360533
PROVIDER: scopus
PMCID: PMC1838611
Notes: --- - "Cited By (since 1996): 80" - "Export Date: 17 November 2011" - "CODEN: PNASA" - "Source: Scopus"
Citation Impact
MSK Authors
  1. Robert P Fisher
    28 Fisher