Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 104, Issue 11, Pages 4383-4388Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0701140104
Keywords
cell division; Ect2; knockout; mitosis
Categories
Funding
- NCI NIH HHS [R01 CA 107342, R01 CA107342] Funding Source: Medline
- NIAID NIH HHS [AI 44409, R01 AI044009] Funding Source: Medline
- NIGMS NIH HHS [GM 56985, R01 GM094972, R01 GM056985] Funding Source: Medline
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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 (Pik1(as)). When cultured in the presence of these analogs, Plk1(as) cells accumulate in prometaphase with defects that parallel those found in PLK1(Delta/Delta) cells. In addition, acute treatment of Plk1(as) 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.
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