Different Plk1 Functions Show Distinct Dependencies on Polo-Box Domain-mediated Targeting

Abstract

Polo-like kinase 1 (Plk1) has multiple important functions during M-phase progression. In addition to a catalytic domain, Plk1 possesses a phosphopeptide-binding motif, the polo-box domain (PBD), which is required for proper localization. Here, we have explored the importance of correct Plk1 subcellular targeting for its mitotic functions. We either displaced endogenous Plk1 through overexpression of the PBD or introduced the catalytic domain of Plk1, lacking the PBD, into Plk1-depleted cells. Both treatments resulted in remarkably similar phenotypes, which were distinct from the Plk1 depletion phenotype. Cells depleted of Plk1 mostly arrested with monoastral spindles, because of inhibition of centrosome maturation and separation. In contrast, these functions were not impaired in cells with mislocalized Plk1. Instead, these latter cells showed a checkpoint-dependent mitotic arrest characterized by impaired chromosome congression. Thus, whereas chromosome congression requires localized Plk1 activity, other investigated Plk1 functions are less dependent on correct PBD-mediated targeting. This opens the possibility that PBD-directed drugs might be developed to selectively interfere with a subset of Plk1 functions.

Deficiency in Chromosome Congression by the Inhibition of Plk1 Polo Box Domain-dependent Recognition

“…Overexpressed PBD perturbed the subcellular localization of endogenous Plk1, implicating the requirement of the PBD for proper localization of Plk1 (23,27). There are many defects induced by Plk1 depletion (e.g.…”

“…Although the roles of Plk1 in mitotic progression as described above have been elucidated mainly by depletion of Plk1 using small interfering RNA or specific antibodies, the role of PBD-dependent binding has been examined through overexpression of the PBD (23,27). Overexpressed PBD perturbed the subcellular localization of endogenous Plk1, implicating the requirement of the PBD for proper localization of Plk1 (23,27).…”

Regulation of microtubule-based microtubule nucleation by mammalian polo-like kinase 1

“…Loss of the function of Plk1 or its homologs in diverse organisms results in various defects in centrosome maturation and bipolar spindle formation, such as diminished centrosomal MT nucleation, weakened bipolar spindles, and monopolar spindles and consequently induces spindle checkpoint-dependent mitotic arrest and apoptotic cell death. Expression of a dominant-negative polo-box domain (PBD), a phospho-Ser/Thr-binding module (21,22), also induces a spindle defect similar to that of Plk1 depletion (23,24), suggesting that PBD-dependent Plk1 function is required for this event.…”

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

“…8,9 When overexpressed in vivo, the PBD causes delocalization of the endogenous pool of Plk1 and elicits a strong checkpoint-dependent prometaphase arrest. 10,11 However, the vast majority of these cells display mature centrosomes and bipolar spindles, unlike those injected with Plk1-specific antibodies or depleted of Plk1 via RNA interference (RNAi) or genetic deletion of the PLK1 locus (see below). These results support the notion that delocalized Plk1 is sufficient to mature centrosomes and induce bipolar spindle assembly in prometaphase.…”

“…Although discussed in the context of chemical genetics, the same scientific logic underlies the rescue of RNAi-generated phenotypes by RNAi-resistant transgenes. 10 To date, chemical genetics has been practiced primarily in yeast, where gene disruption and replacement are standard techniques. Recently, however, analogous tools for gene targeting studies in human somatic cells have been developed.…”