Physical and functional interactions between mitotic kinases during polyploidization and megakaryocytic differentiation

Human Polo-like kinase 3 (Plk3), a protein serine/threonine kinase, is involved in the regulation of cell cycle progression at multiple stages. Our previous studies revealed that Plk3 is closely associated with centrosomes and plays an important role in the regulation of microtubule dynamics. Here we describe the physical interaction of Plk3 with Aurora A and BubR1 kinases, and the significance of this interaction during terminal differentiation and polyploidization of megakaryocytes. Specifically, double immunofluorescence staining confirms that Plk3 and Aurora A colocalize to centrosomes or spindle poles during essentially all phases of the cell cycle and that BubR1 also exhibits spindle pole localization during metaphase. Pull-down assays show that Plk3 physically interacts with Aurora A as well as BubR1. Upon treatment with phorbol 12-myristate 13-acetate (PMA), human erythroleukemic cells (K562) underwent megakaryocytic differentiation characterized by polyploidization and expression of mature megakaryocyte surface markers such as CD41. Plk3 protein levels were seen to be increased during PMA-induced megakaryocytic differentiation of K562 cells, correlating well with the ploidy level in these cells. Similarly, Aurora A and its phosphorylated form also increased after PMA treatment. In contrast, BubR1 levels were markedly reduced. Taken together, our study suggests that Plk3 and Aurora A kinases may lie in the same regulatory pathway and that Plk3 and Aurora A as well as BubR1 may play an important role in polyploidization and megakaryocytic differentiation.