Figure 1(A) β-Catenin is hyperphosphorylated during the mitotic phase. HeLa CCL2 cells were synchronized at the G1/S phase using a double thymidine block and released using medium containing 200 ng/mL nocodazole (Noc.). Cells were harvested at the indicated time points and treated with (+) or without (-) λ-phosphatase (λ-PPase). The resulting cell lysates were used for immunoblot analysis. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE. Asterisk indicates the phosphorylated form of Cdc25C. (B) Treatment with the Plk1 kinase inhibitor BI 2536 converts the slower migrating β-catenin form to the faster migrating β-catenin form. Thymidine (S phase) or Nocodazole (M-phase)-arrested HeLa CCL2 cells were treated with the indicated kinase inhibitors to inhibit CK1δ/ε (IC261), CK1δ (D4476), Plk1 (BI2536), GSK3β (TWS119), CDK2/5/1/9 (Dinaciclib), Aurora kinase A/B (ZM447439), or Aurora kinase A (VX680). The resulting cell lysates were used for immunoblot analyses. Only BI2536 converted slower migrating (red arrowhead) to faster migrating β-catenin (blue arrowhead). (C) Plk1-specific inhibitors rapidly abolished the phosphorylated form of β-catenin (red arrowhead). Nocodazole-arrested HeLa CCL2 cells were treated with two different Plk1 inhibitors, BI2536 and GSK461364, for the indicated times. Asterisk indicates the phosphorylated form of Cdc25C. BI2536 or GSK461364 treatment converted slower migrating (red arrowhead) to faster migrating β-catenin (blue arrowhead). (D) Asynchronously growing HeLa CCL2 cells were silenced for either control luciferase (shGL) or Plk1 (shPlk1) using lentivirus-based shRNA infection. The resulting cells were synchronized using a double thymidine block and released using medium containing 200 ng/mL nocodazole (Noc.) Cells were harvested at the indicated time points and used for immunoblot analyses. Asterisk indicates the phosphorylated form of Cdc25C. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE. (E) β-Catenin interacts with Plk1 PBD in a phosphorylation-independent manner. Total cell lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were incubated and pull-downed with bacterially purified/bead-associated GST-Plk1 PBD wild type (WT), GST-Plk1 PBD H538A and K540M mutant (AM), or GST only. The resulting precipitates were subjected to immunoblot analyses. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE. (F) Total cell lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were immunoprecipitated with two different Plk1 antibodies. Each antibody (gifted from Kyung S. Lee, NIH/NCI) recognized either Plk1 N-terminus (N-term. Ab.) or C-terminus (C-term. Ab.). The resulting precipitates were subjected to immunoblot analysis. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE. (G) Lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were mixed with either control (phosphate-buffered saline only, +PBS), 10 μg/ml of high-affinity Plk1 PBD binding phospho-peptide (PLHSpT, +P), or 10 μg/ml of non-phospho-peptide (PLHST, +NP). The resulting cell lysates were immunoprecipitated with anti-Plk1 antibody. Precipitates were subjected to immunoblot analysis. Immunoblotting with anti-Dvl2 antibody represents a positive control of phospho-peptide (PLHSpT, +P) competition for Plk1 binding. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE. (H) β-Catenin is directly phosphorylated by Plk1. Bacterially purified full-length GST-β-catenin was incubated with either HA-Plk1 wild type (WT) or HA-Plk1 kinase defective mutant K82M (KM) purified from insect cells in the presence of [γ-32P] ATP. Incorporation of γ-32P into GST-β-catenin was confirmed by SDS-PAGE followed by autoradiography analysis.