Fig 2: Methylation of PLK1 fine tunes PLK1 kinase activity at kinetochore. (A) SET7/9-depleted HeLa cells expressing Hec1-targeted PLK1 kinase sensor were imaged live after 8 h release from thymidine arrest. The first and third panels, color-coded images of the emission ratio; the second and fourth panels, mCherry-H2B (mCh-H2B) merged with YFP signal of the sensor. Note that suppression of SET7/9 resulted in mitotic arrest. Also, the emission ratio decreased drastically when anaphase began due to the localization change of Hec1. Scale bar, 10 µm. (B) Statistical analyses of the FRET/CFP emission ratio on centromeres at the indicated time points. Data represent mean ± SEM; > 150 kinetochores of each categories from five different cells. (C) Statistical analyses of the FRET/CFP emission ratio of HeLa cells expressing mCherry-PLK1WT, PLK1T210D, PLK1T210A, and PLK1K191R. Non-methylatable PLK1 (PLK1K191R) retained higher PLK1 activity similar to constitutively active PLK1T210D. Data represent mean ± SEM; > 150 kinetochores of each categories from five different cells. (D) Representative immunofluorescence images of HeLa cells depleted of SET7/9. Cells were fixed, permeabilized, and stained for anti-BubR1 and ACA antibodies, respectively. Scale bar, 10 µm. (E) Statistical analyses of BubR1-positive kinetochores in HeLa cells expressing mCherry-PLK1WT, PLK1T210D, PLK1K191R, and SET7/9 siRNA, respectively. Data represent mean ± SEM from three independent experiments. Statistical significance was tested by two-sided t-test. **P < 0.01.

Fig 3: Dimethylation of K191 on PLK1 attenuates its kinase activity. (A) GST-PLK1 was incubated with 6× His-tagged Aurora A plus Bora in the presence of 1 mM SAM for in vitro methylation assay. The methylation and phosphorylation levels of PLK1 were analyzed by PLK1-K191me2 and PLK1-pT210 antibodies, respectively. (B) Kinetics of PLK1 (purified as in A) kinase activity in the presence of Aurora A and SET7/9 with increased concentration of ATP. Data represent mean ± SEM from three independent experiments. (C) FLAG-PLK1WT and FLAG-PLK1K191R were purified from HEK293T cells co-transfected with GFP-SET7/9 and FLAG-PLK1WT or FLAG-PLK1K191R. ATP agarose beads were used as affinity matrices to absorb purified PLK1 with increased amount (0.25–1 µg). The methylation level of PLK1 was analyzed with the anti-PLK1-K191me2 antibody. (D) Cartoon representation of ADP docked onto PLK1 or dimethylated PLK1 (PLK1-K191me2). Residues for ADP binding are shown as sticks. (E) Representative mitotic phenotypes in PLK1-depleted HeLa cells expressing GFP-PLK1WT or GFP-PLK1K191R shown by time-lapse microscopy and visualized with mCherry-H2B. Scale bar, 10 µm. (F) Quantification of chromosome misalignment of HeLa cells expressing GFP-PLK1WT (n = 108) or GFP-PLK1K191R (n = 101). Data represent mean ± SEM from three independent experiments. (G) Quantification of mitotic duration of PLK1-depleted HeLa cells expressing PLK1WT, PLK1T210D, and PLK1K191R with or without MAD2 depletion. An aliquot of SET7/9 siRNA-treated cells were used as a control. NEBD, nuclear envelope breakdown. At least 103 cells per group were examined from three independent experiments. Data represent mean ± SEM. Statistical significance was tested by two-sided t-test. **P < 0.01; NS indicates non-significant, P > 0.05.

Fig 4: PLK1 K191 is methylated during G2 phase and mitosis. (A) Characterization of the specificity of the PLK1-K191me2 antibody. HEK293T cells were co-transfected with GFP-SET7/9 and FLAG-Plk1WT or FLAG-PLK1K191R followed by western blotting analyses of PLK1 and PLK1-K191me2, respectively. (B) HeLa cells were arrested by nocodazole or synchronized to the indicated time points by double thymidine release and probed for PLK1-K191me2 and other indicated proteins. (C) Immunoprecipitation of endogenous PLK1 from asynchronized or nocodazole-synchronized HeLa cells. HeLa cell extracts were incubated with an anti-PLK1 antibody. After extensive washes, immunoprecipitates were resolved by SDS–PAGE followed by western blotting analyses using indicated antibodies. The methylation level of PLK1 was detected by PLK1-K191me2 antibody. (D) Representative immunofluorescence staining of PLK1 and PLK1-K191me2 in HeLa cells at different mitotic stages. Scale bar, 10 µm. (E) Co-localization analysis of K191-methylated (K191me2) and Thr210-phosphorylated (pT210) PLK1 at prometaphase kinetochores in HeLa cells. Scale bar, 10 µm.

Fig 5: PLK1 is a cognate substrate of SET7/9 in vitro and in vivo. (A) Immunoprecipitation of endogenous PLK1 from prometaphase-synchronized HeLa cells. Clarified extracts from mitotic HeLa cells were incubated with an anti-PLK1 antibody and immunoprecipitates were resolved by SDS–PAGE followed by western blotting analyses using indicated antibodies. (B) Immunoprecipitation of FLAG-PLK1 from HEK293T cells co-transfected with GFP or GFP-SET7/9. The immunoprecipitates were analyzed by an anti-GFP western blotting. (C) Recombinant GST-SET7/9 or GST proteins were incubated with His-PLK1 for 4 h, and their interactions were assessed by Coomassie Brilliant Blue (CBB)-stained SDS–PAGE gel and western blot with an anti-His antibody blotting analysis. (D) GST-3×MBTWT and GST-3×MBTDN bound agarose beads were used as affinity matrices to absorb methylated PLK1 from HEK293T cells co-transfected with FLAG-PLK1 and GFP-SET7/9. (E) Aliquots of purified GST-PLK1 were incubated with 0.5 µg GST-SET7/9 in the presence or absence of 1 mM S-(5'-adenosyl)-Lmethionine (SAM). PLK1 methylation was detected by dimethyl lysine antibody. Methylated lysine residues in PLK1 from in vitro methylation reaction were identified using mass spectrometric analysis. (F) Diagram of PLK1 functional domains relative to newly identified lysine residues bearing methylation. (G) Characterization of K191 methylation in vitro. Aliquots of purified GST-PLK1 and GST-PLK1 mutants (K191R and K474R/K492R; 0.5 µg) were incubated with 0.5 µg of His-SET7/9 in the presence or absence of 1 mM SAM. Methylated PLK1 was detected by western blotting analyses using an anti-dimethyl lysine antibody.