Plk3 phosphorylates topoisomerase IIα at Thr1342, a site that is not recognized by Plk1

Abstract

The Plk (polo-like kinase) family is involved in cell-cycle machinery. Despite the possible overlapping involvement of Plk1 and Plk3 in cell-cycle distribution, the precise role of each Plk might be different. To investigate mechanisms that may differentiate their physiological roles, we compared the substrate specificities of Plk1 and Plk3 using synthetic peptides. Among these substrate peptides, topoisomerase IIα EKT1342DDE-containing synthetic peptide was strongly phosphorylated by Plk3 but not by Plk1. By modulating the topoisomerase IIα peptide, we identified residues at positions +1, +2 and +4 as determinants of differential substrate recognition between Plk1 and Plk3. Acidic residues at positions +2 and +4 appear to be a positive determinant for Plk3 but not Plk1. Variation at position +1 appears to be tolerated by Plk3, while a hydrophobic residue at +1 is critical for Plk1 activity. The direct phosphorylation of Thr1342 of topoisomerase IIα by Plk3 was demonstrated with an in vitro kinase assay, and overexpression of Plk3 induced the phosphorylation of Thr1342 in cellular topoisomerase IIα. Furthermore, the physical interaction between Plk3 and topoisomerase IIα was also demonstrated in cells in addition to phosphorylation. These data suggest that topoisomerase IIα is a novel physiological substrate for Plk3 and that Plk1 and Plk3 play different roles in cell-cycle regulation.