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 (Plk1 as ). When cultured in the presence of these analogs, Plk1 as cells accumulate in prometaphase with defects that parallel those found in PLK1 ⌬/⌬ 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.cell division ͉ Ect2 ͉ knockout ͉ mitosis

Polo Kinase and Cytokinesis Initiation in Mammalian Cells: Harnessing the Awesome Power of Chemical Genetics

“…29 Crucially, all effects of 3-MB-PP1 on cell growth, mitotic progression, spindle and centrosome function, Ect2 and RhoA localization, and cleavage furrow ingression were unique to Plk1 as cells, excluding the possibility that they arose through off-target inhibition of endogenous enzymes (including other Plk family members) that are common to both Plk1 wt and Plk1 as cells. 19 Similar observations were recently made in early anaphase cells exposed to BI 2536. 20,21 In support of the idea that BI 2536 affects the RhoA pathway via specific inhibition of Plk1, Petronczki et al directly compared the consequence of Plk1 RNAi and BI 2536 treatment on cells forced to exit mitosis prematurely by pharmacologic inhibition of Cdk1 activity.…”

“…As a result, these techniques provide an excellent view into the late G 2 and early mitotic functions of Plk1, but because of the ensuing SAC activation and prometaphase arrest, little insight into its late mitotic functions. To investigate the latter, Burkard et al, 19 Brennan et al 20 and Petronczki et al 21 used chemical inhibitors that can rapidly inhibit Plk1 during anaphase, after the kinase has completed its essential early mitotic functions. To do this, Brennan et al and Petronczki et al both utilized a novel pharmacologic inhibitor, BI-2536, that has been developed as a potential anti-cancer therapeutic agent.…”

“…Burkard et al 19 then used 3-MB-PP1 to acutely inhibit Plk1 as in cells that had completed chromosome alignment and already silenced the SAC. Strikingly, such cells executed anaphase and exited mitosis, but failed to initiate cytokinesis.…”

Combination of Chemical Genetics and Phosphoproteomics for Kinase Signaling Analysis Enables Confident Identification of Cellular Downstream Targets

“…Cell Culture and Treatment-Telomerase-expressing human retinal pigment epithelial (hTERT-RPE) cells in which both genomic PLK1 loci were disrupted and that expressed either wild-type Plk1 (Plk1 wt ) or an analog-sensitive Plk1 mutant (C67V/L130G, referred to as Plk1 as ) as EGFP fusions were used throughout this study (14). For differential SILAC encoding, the cells were grown in a 1:1 mixture of arginineand lysine-deficient Dulbecco's modified Eagle's medium (Invitrogen) and F12 Ham's medium (Invitrogen) supplemented with L-glutamine (2 mM; PAA Laboratories), sodium pyruvate (1 mM; Invitrogen), 1% penicillin/streptomycin (PAA Laboratories), 10% dialyzed fetal bovine serum (Invitrogen), and either 175 M unlabeled L-arginine ( as (referred to as as1 and as3) and Plk1 wt cells (wt1 and wt3), whereas in two further replicate experiments with either cell line reciprocal labeling conditions were used (as2, as4, wt2, and wt4).…”

Cytokinesis in Animal Cells

“…3) (Lee et al 2004;Yuce et al 2005;Zhao and Fang 2005;Kamijo et al 2006;Su et al 2011). The interaction between ECT2 and RacGAP1 requires phosphorylation of RacGAP1 by Polo-like kinase 1 (Plk1) in anaphase/telophase and is, instead, inhibited by Cdk1-mediated phosphorylation of ECT2 in metaphase (Yuce et al 2005;Burkard et al 2007Burkard et al , 2009Petronczki et al 2007;Wolfe et al 2009). Phosphorylation of the ECT2 carboxyterminal polybasic cluster region by Cdk1 also prevents the association of this GEF with the plasma membrane before anaphase onset (Su et al 2011).…”