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

Mark E Burkard; Catherine L Randall; Stéphane Larochelle; Chao Zhang; Kevan M Shokat; Robert P Fisher; Prasad V Jallepalli

doi:10.1073/pnas.0701140104

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

Journal article

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

Mark E Burkard, Catherine L Randall, Stéphane Larochelle, Chao Zhang, Kevan M Shokat, Robert P Fisher and Prasad V Jallepalli

Proceedings of the National Academy of Sciences - PNAS, Vol.104(11), pp.4383-4388

03/13/2007

DOI: 10.1073/pnas.0701140104

PMCID: PMC1838611

PMID: 17360533

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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 (Plk1(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.

Alleles

Catalysis

Cell Cycle Proteins - physiology

Cell Line

Centrosome - metabolism

Cytokinesis

Dose-Response Relationship, Drug

Genetic Techniques

Humans

Mitosis

Polo-Like Kinase 1

Protein Serine-Threonine Kinases - physiology

Proto-Oncogene Proteins - genetics

Proto-Oncogene Proteins - physiology

Recombination, Genetic

rhoA GTP-Binding Protein - metabolism

Spindle Apparatus

Details

Title: Subtitle: Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells
Creators: Mark E Burkard - Memorial Sloan Kettering Cancer Center
Catherine L Randall
Stéphane Larochelle
Chao Zhang
Kevan M Shokat
Robert P Fisher
Prasad V Jallepalli
Resource Type: Journal article
Publication Details: Proceedings of the National Academy of Sciences - PNAS, Vol.104(11), pp.4383-4388
Publisher: Proceedings of the National Academy of Sciences
DOI: 10.1073/pnas.0701140104
PMID: 17360533
PMCID: PMC1838611
ISSN: 0027-8424
eISSN: 1091-6490
Grant note: GM 56985 / NIGMS NIH HHS R01 AI044009 / NIAID NIH HHS AI 44409 / NIAID NIH HHS R01 GM094972 / NIGMS NIH HHS R01 CA107342 / NCI NIH HHS R01 CA 107342 / NCI NIH HHS R01 GM056985 / NIGMS NIH HHS
Language: English
Date published: 03/13/2007
Academic Unit: Internal Medicine
Record Identifier: 9984700653702771

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