Fig. 3: Phosphorylation of PLK1-T210 blocks PLK1 dimerization.

A The T210 phospho-mimicking mutation blocks PLK1 dimerization. Lysates of HEK293T cells co-expressing V5-tagged full-length PLK1 (wild-type) and Myc-tagged full-length PLK1 mutants (K82M, S137A/D, T210A/E) were subjected to IP using a Myc-specific antibody followed by western blotting with V5-and Myc-antibodies. B, C Bis(Sulfosuccinimidyl) suberate (BS3) (20:1 Crosslinker-Protein) was added to lysates of HEK293T cells expressing Myc/V5- tagged-PLK1-WT and PLK-T210E. The crosslinking was performed for 30 min followed by quenching of unreacted BS3. Afterward, IPs using anti-V5 and anti-Myc antibodies were carried out. The products of the crosslinking were analyzed by SDS-PAGE and western blotting with PLK1- and Bora-specific antibodies. D Size exclusion chromatography of endogenous PLK1. PLK1–3xMyc cells were synchronized to the G1/S boundary using thymidine and released for 3 h or synchronized into the G2-phase using the CDK1 inhibitor RO3306. The protein extracts were loaded onto a Superose 3.2/300 size exclusion column and fractionated at a flow rate of 50 µl/min. The factions were resolved using an SDS-PAGE and probed with the anti-PLK1-pT210 antibody. E HEK293T cells were co-transfected with C-terminal CFP/YFP-fused PLK1 and -PLK1-T210E constructs alone or in combination. The FRET efficiency for the indicated constructs and combinations from three independent experiments was determined. A two-tailed unpaired t-test was performed (***p ≤ 0.001). F (Left) Confocal images of HEK293T. Cells grown on coverslips, co-transfected with YFP and mTurquoise PLK1-WT or PLK1-T210E were analyzed for FRET using the FRET co-localization analyzer ImageJ plugin. FRET-images give the calculated amount of FRET for each pixel in the merged images. The ImageJ plugin color codes the relative FRET efficiency, which is indicated by the displayed color bar. Scale bars: 10 μm. (right) Quantifications of mTurquoise/YFP emission ratios of cells expressing PLK1-WT or PLK1-T210E. Error bars indicate the standard deviation based on the emission of 10 individual cells. A two-tailed unpaired t-test was performed. (**p ≤ 0.01). G The phosphorylation of T210 by Aur-A abolishes PLK1 dimerization. (Left) Mitotic HeLa cells expressing PLK1–3xMyc enriched by Nocodazole (Noc) treatment and a shake-off were released in 10 μM MG132 or 10 μM MG132/1 μM MLN for 2 h. Lysates were resolved by SDS‐PAGE and immunoblotted for PLK1, PLK1-pT210, Cyclin B1, Aur‐A, and Aurora-pT288. (Middle) Lysates of HeLa-PLK1–3xMyc cells were subjected to IP using anti-Myc and immunoblotted for PLK1 and PLK1-pT210. (Right) The cell cycle distribution of synchronized HeLa cells represented as a bar graph.