Figure 2.
Figure 2. T-loop phosphorylation of Polo by Aurora B or interference with the interdomain interaction in Polo promotes its dissociation from Map205. (A) The phosphomimetic mutation T182D of Polo reduces its interaction with Map205. Cells were transfected as indicated and PrA-Map205 was purified. Purification products and whole cell extracts (WCE) were analyzed by Western blotting. (B) Overexpression of Aurora B results in hyperphosphorylation of Polo at Thr182 and abrogates its interaction with Map205. PoloWT-GFP– and PoloT182A-GFP–expressing cells were transfected with PrA-Map205 and Aurora B–Myc as indicated and treated for 1 h with okadaic acid (100 nM) to inhibit phosphatases. PrA-Map205 was purified, and Western blots were performed. (C) BI 2536 affects the localization of Polo in cytokinesis. (C, top) Immunofluorescence in cells expressing PoloWT-GFP. BI 2536 treatment (1 µM for 10 min) reduces the localization of Polo on microtubules but not at the midbody. Bars, 5 µm. (C, bottom) Line scans of the GFP fluorescence intensity along the intercellular bridge (n = 20; MT, microtubules; MB, midbody). Error bars indicate SD. (D) BI 2536 reduces the interaction between Polo-GFP and PrA-Map205. Cells were transfected as indicated and treated for 10 min with BI 2536, and PrA-Map205 was purified. Samples were analyzed by Western blotting. (E) BI 2536 disrupts the in vitro interaction between a fragment of Map205 and Polo. GST-Map205254-375 or GST-bound Sepharose beads were incubated with cell lysates and then treated with BI 2536 or ATP for 1 h as indicated. Pull-down products and the supernatant were analyzed by Western blots. (F) Binding of BI 2536 to the KD catalytic cleft is incompatible with binding of the PBD. (F, top left) Structure of the co-complex between the KD and PBD from zPlk1 and a peptide from Map205 (Protein Data Bank [PDB] accession no. 4J7B). (F, top right) Structure of human Plk1 KD bound to BI 2536 (PDB accession no. 2RKU). (F, bottom) Structural alignment of the two KD structures. BI 2536 induces a steric clash with a conserved region of the PBD comprising Pro394 involved in its intramolecular interaction with the KD. Structural alignment and rendering was performed using PyMOL 1.4 built-in commands.

T-loop phosphorylation of Polo by Aurora B or interference with the interdomain interaction in Polo promotes its dissociation from Map205. (A) The phosphomimetic mutation T182D of Polo reduces its interaction with Map205. Cells were transfected as indicated and PrA-Map205 was purified. Purification products and whole cell extracts (WCE) were analyzed by Western blotting. (B) Overexpression of Aurora B results in hyperphosphorylation of Polo at Thr182 and abrogates its interaction with Map205. PoloWT-GFP– and PoloT182A-GFP–expressing cells were transfected with PrA-Map205 and Aurora B–Myc as indicated and treated for 1 h with okadaic acid (100 nM) to inhibit phosphatases. PrA-Map205 was purified, and Western blots were performed. (C) BI 2536 affects the localization of Polo in cytokinesis. (C, top) Immunofluorescence in cells expressing PoloWT-GFP. BI 2536 treatment (1 µM for 10 min) reduces the localization of Polo on microtubules but not at the midbody. Bars, 5 µm. (C, bottom) Line scans of the GFP fluorescence intensity along the intercellular bridge (n = 20; MT, microtubules; MB, midbody). Error bars indicate SD. (D) BI 2536 reduces the interaction between Polo-GFP and PrA-Map205. Cells were transfected as indicated and treated for 10 min with BI 2536, and PrA-Map205 was purified. Samples were analyzed by Western blotting. (E) BI 2536 disrupts the in vitro interaction between a fragment of Map205 and Polo. GST-Map205254-375 or GST-bound Sepharose beads were incubated with cell lysates and then treated with BI 2536 or ATP for 1 h as indicated. Pull-down products and the supernatant were analyzed by Western blots. (F) Binding of BI 2536 to the KD catalytic cleft is incompatible with binding of the PBD. (F, top left) Structure of the co-complex between the KD and PBD from zPlk1 and a peptide from Map205 (Protein Data Bank [PDB] accession no. 4J7B). (F, top right) Structure of human Plk1 KD bound to BI 2536 (PDB accession no. 2RKU). (F, bottom) Structural alignment of the two KD structures. BI 2536 induces a steric clash with a conserved region of the PBD comprising Pro394 involved in its intramolecular interaction with the KD. Structural alignment and rendering was performed using PyMOL 1.4 built-in commands.

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