Use of the Novel Plk1 Inhibitor ZK-Thiazolidinone to Elucidate Functions of Plk1 in Early and Late Stages of Mitosis

Abstract

Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis. INTRODUCTIONThe error-free segregation of chromosomes during cell division is necessary for the maintenance of correct ploidy and genomic integrity, and errors in cell division are presumed to lead to aneuploidy and cancer (Rajagopalan and Lengauer, 2004). To ensure that daughter cells receive the correct complement of chromosomes, two key events need to be coordinated. First, chromosomes must be equally segregated, a process that depends on the mitotic spindle. Second, cytokinesis, the process dividing the cell into two, must occur between the two sets of segregated chromosomes. Both of these processes require the activity of a key cell cycle regulator, the Polo-like kinase 1 (Plk1). Plks form a conserved subfamily of serine/threonine protein kinases. The first member to be identified was Polo in Drosophila melanogaster (Llamazares et al., 1991) and, subsequently, four Plk family members have been identified in mammals Barr et al., 2004).Plk1 contains an N-terminal kinase domain and a phosphopeptide-binding C-terminal regulatory polo-box domain (PBD; Leung et al., 2002;Elia et al., 2003b). In vertebrates Plk1 has been implicated in the activation of Cdk1-cyclin B upon entry into mitosis, centrosome maturation via the recruitment of the ␥-tubulin ring complex (␥-TuRC), spindle formation, sister chromatid separation by cohesin removal from the chromosome arms, promotion of anaphase onset through direct phosphorylation of the APC/C complex as well as the inhibition of the APC/C inhibitor Emi1, and finally, mitotic exit and cytokinesis (reviewed in Barr et al., 2004). Fitting with these diverse functions, Plk1 localizes to the centrosomes, spindle poles, and kinetochores in prophase and metaphase, the central spindle in anaphase, and the midbody during cytokinesis. These localizations require the function of the PBD (Jang et al., 2002;Seong et al., 2002) and priming-kinases to generate phosphorylated docking sites that are subsequently recog...

Mitotic drivers—inhibitors of the Aurora B Kinase

“…TAL is a thiazolidinone developed by Bayer Schering Pharma that inhibits Plk1 in vitro with an IC 50 of 19 nM. Again, the phenotypes observed in cell based assays are entirely consistent with Plk1 inhibition [41]. Importantly, both BI 2536 and TAL induce a penetrant monopolar spindle phenotype which in turn activates the spindle checkpoint resulting in a mitotic arrest phenotype.…”

Trachyspic acid 19-butyl ester, a new inhibitor of Plk1 polo box domain-dependent recognition from uncharacterized fungus RKGS-F2684

“…They showed the same range of activity to that of purpurogallin with IC 50 values of 102 (1), 128 (2) and 114 (purpurogallin) μM. Several Plk1 inhibitors have been reported including purpurogallin, [6][7][8] however, most of them contain an aromatic moiety in their molecules apart from the structures of 1 and 2, which have a 5,5-spiroacetal core with alkyl chain and tricarboxylic acid moiety. Also 2 has been isolated as an inhibitor of heparanase and the activity has not been investigated in detail.…”

“…5 Thus, Plk1 is considered to be an attractive target for the treatment of human cancers. [6][7][8] Several inhibitors of its catalytic activity are developed and some of them have proceeded to clinical trials. 9,10 We established a screening system for the inhibitors of PBD-dependent binding.…”

PRC1 associates with the hsp70i promoter and interacts with HSF2 during mitosis

“…Recent studies have also reported that PRC1 acts as a docking site for Plk1 in a phosphorylationdependent manner [7,21]. Given that HSF2 and PRC1 interact in the early stages of mitosis, an intriguing question for future studies would be to determine if HSF2 acts in a similar manner to bring PRC1 into proximity with an as yet undetermined binding partner.…”