Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells.
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.
Burkard
Mark E
ME
Molecular Biology Program and Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Randall
Catherine L
CL
Larochelle
Stéphane
S
Zhang
Chao
C
Shokat
Kevan M
KM
Fisher
Robert P
RP
Jallepalli
Prasad V
PV
D000483:Alleles; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000502:physiology*; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism; D048749:Cytokinesis; D004305:Dose-Response Relationship, Drug; D005821:Genetic Techniques; D006801:Humans; D008938:Mitosis; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D011995:Recombination, Genetic; D008941:Spindle Apparatus; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*; D000097869:Polo-Like Kinase 1
D018797:Cell Cycle Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D020742:rhoA GTP-Binding Protein; D000097869:Polo-Like Kinase 1; C489850:ECT2 protein, human