Fig. 3
Interdependence between Polo kinase activity, NLS function, and PBD function. a Mutation of NLS residues or T182D substitution abrogates the KD–PBD interaction. GST–PBD or GST-bound sepharose beads were incubated with lysates of cells transfected with different forms of Flag-KD (with the IDL) as indicated. Pulldown products were analyzed by western blots. b Mutation of NLS residues or T182D substitution abrogates the Polo-Map205 interaction. Cells were transfected as indicated and PrA-Map205 was purified. Samples were analyzed by western blotting. c Bioluminescence resonance energy transfer (BRET) reveals the impact of T182D and NLS7A mutations on the KD–PBD interaction in live cells. HEK293T cells were transfected with a fixed amount of Luc-KD (with the IDL) expression vector and increasing amounts of PBD–GFP expression vector. A third plasmid expressing a Map205 fragment (Map), which stabilizes the PBD–KD complex was co-transfected. When the KD and PBD interact, the luciferase (Luc) moiety, upon reaction with coelenterazine, transfers energy to GFP, which then fluorescences (BRET). Differences in BRET50 (the GFP/Luc ratio at which BRET is half-maximal) reflect differences in affinity. AU arbitrary units. Error bars: standard deviation of triplicate values from a representative experiment. d Mutation of NLS residues increases Polo kinase activity. Immunoprecipitated Polo-GFP (WT and mutants) were used in kinase reactions using casein as a substrate. For Polo inhibition, BI 2536 was added at 300 nM. Reactions were analyzed by autoradiography, western blots, and amido black (total protein). e Mutation of NLS-interacting PBD residues prevents the KD–PBD interaction. Experiment as in a. f Mutation of NLS-interacting PBD residues increases Polo kinase activity. Experiment as in d. g Model for coupling of Polo activation and nuclear localization. See text for details. The crystal structure of a complex between the KD (green) and the PBD (cyan) of zebrafish Polo and the inhibitory peptide from Drosophila Map205 (red) (Protein Data Bank accession no. 4J7B17) was used for structure rendering with PyMOL 1.4
