Phosphorylation of β-catenin Ser60 by polo-like kinase 1 drives the completion of cytokinesis

β-Catenin is a multifunctional protein and participates in numerous processes required for embryonic development, cell proliferation, and homeostasis through various molecular interactions and signaling pathways. To date, however, there is no direct evidence that β-catenin contributes to cytokinesis. Here, we identify a novel p-S60 epitope on β-catenin generated by Plk1 kinase activity, which can be found at the actomyosin contractile ring of early telophase cells and at the midbody of late telophase cells. Depletion of β-catenin leads to cytokinesis-defective phenotypes, which eventually result in apoptotic cell death. In addition, phosphorylation of β-catenin Ser60 by Plk1 is essential for the recruitment of Ect2 to the midbody, activation of RhoA, and interaction between β-catenin, Plk1, and Ect2. Time-lapse image analysis confirmed the importance of β-catenin phospho-Ser60 in furrow ingression and the completion of cytokinesis. Taken together, we propose that phosphorylation of β-catenin Ser60 by Plk1 in cooperation with Ect2 is essential for the completion of cytokinesis. These findings may provide fundamental knowledge for the research of cytokinesis failure-derived human diseases. SYNOPSIS Phosphorylation of β-catenin Ser60 by Plk1 in cooperation with Ect2 induces RhoA activation and mediates the progression of furrow ingression and subsequent cytokinesis steps. β-Catenin is a Plk1 substrate that binds to the Plk1 polo-box domain (PBD) without priming phosphorylation. Plk1 phosphorylates β-catenin S60 during the mitotic phase. β-Catenin p-S60 is essential for the midbody localization of Ect2 and activation of RhoA. β-Catenin p-S60 is important for furrow ingression and completion of cytokinesis. Graphical Abstract Phosphorylation of β-catenin Ser60 by Plk1 in cooperation with Ect2 induces RhoA activation and mediates the progression of furrow ingression and subsequent cytokinesis steps.