We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

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...