Genetic dissection of Polo-like kinase 1's functions in human cell division

Human cell division is a highly regulated process to ensure each daughter cell contains the same genetic complement. One key regulator of this process is Polo-like kinase 1 (Plk1), member of a conserved serine/threonine kinase family. Plk1 itself is subject to regulation at several levels, including its localization and phosphorylation of its activation loop. Plk1's localization to the central midzone and evidence from other systems raised the hypothesis that Plk1 is required for the completion of cell division. Due its essential requirement for bipolar spindle formation, direct analysis of Plk1's function in late mitotic events has been difficult. We have developed a chemical genetics system to inhibit Plk1 in human cells. First, we eliminated endogenous protein by homologous inactivation of the PLK1 locus in the human retinal pigment epithelial cell line, followed by complementation with a modified PLK1 transgene that can be rapidly and specifically inhibited. When Plk1 activity was inhibited during anaphase, cells failed complete cytokinesis. Further, Plk1 controls the localized activation of the RhoA GTPase by triggering Ect2-Cyk-4 complex formation at the central spindle. The application of chemical genetics permitted the definition of Plk1's function during late mitosis. Additionally, we used the gene targeting strategies and chemical genetics to evaluate the contribution of activation loop phosphorylation to kinase activity. We created an allelic series of cell lines in which the T210 codon was mutated. Although T210D substitution in vitro causes an increase in kinase activity, the in vivo situation is more complex. When T210D is the sole form of Plk1 in the cell, bipolar spindles are formed, but cells are unable to properly align their chromosomes during prometaphase, in part due to hypophosphorylation of BubR1. This unexpected recessive phenotype repeals the idea that T210D is simply a constitutively active mutant. Full Plk1 activation is more complex than phosphorylation of T210D.