Journal: Journal of Molecular Cell Biology

Article Title: Methylation of PLK1 by SET7/9 ensures accurate kinetochore–microtubule dynamics

doi: 10.1093/jmcb/mjz107

Figure Lengend Snippet: PLK1 K191 is methylated during G2 phase and mitosis. ( A ) Characterization of the specificity of the PLK1-K191 me2 antibody. HEK293T cells were co-transfected with GFP-SET7/9 and FLAG-Plk1 WT or FLAG-PLK1 K191R followed by western blotting analyses of PLK1 and PLK1-K191 me2 , respectively. ( B ) HeLa cells were arrested by nocodazole or synchronized to the indicated time points by double thymidine release and probed for PLK1-K191 me2 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-K191 me2 antibody. ( D ) Representative immunofluorescence staining of PLK1 and PLK1-K191 me2 in HeLa cells at different mitotic stages. Scale bar, 10 μm. ( E ) Co-localization analysis of K191-methylated (K191 me2 ) and Thr210-phosphorylated (pT210) PLK1 at prometaphase kinetochores in HeLa cells. Scale bar, 10 μm.

Article Snippet: Anti-dimethylated K191-PLK1 rabbit antibody (PLK1-K191 me2 ) was generated by YenZym LLC.

Techniques: Methylation, Transfection, Western Blot, Immunoprecipitation, Incubation, SDS Page, Immunofluorescence, Staining

Journal: Journal of Molecular Cell Biology

Article Title: Methylation of PLK1 by SET7/9 ensures accurate kinetochore–microtubule dynamics

doi: 10.1093/jmcb/mjz107

Figure Lengend Snippet: 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-K191 me2 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-PLK1 WT and FLAG-PLK1 K191R were purified from HEK293T cells co-transfected with GFP-SET7/9 and FLAG-PLK1 WT or FLAG-PLK1 K191R . 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-K191 me2 antibody. ( D ) Cartoon representation of ADP docked onto PLK1 or dimethylated PLK1 (PLK1-K191 me2 ). Residues for ADP binding are shown as sticks. ( E ) Representative mitotic phenotypes in PLK1-depleted HeLa cells expressing GFP-PLK1 WT or GFP-PLK1 K191R shown by time-lapse microscopy and visualized with mCherry-H2B. Scale bar, 10 μm. ( F ) Quantification of chromosome misalignment of HeLa cells expressing GFP-PLK1 WT ( n = 108) or GFP-PLK1 K191R ( n = 101). Data represent mean ± SEM from three independent experiments. ( G ) Quantification of mitotic duration of PLK1-depleted HeLa cells expressing PLK1 WT , PLK1 T210D , and PLK1 K191R 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.

Article Snippet: Anti-dimethylated K191-PLK1 rabbit antibody (PLK1-K191 me2 ) was generated by YenZym LLC.

Techniques: Activity Assay, Incubation, In Vitro, Methylation, Phospho-proteomics, Purification, Concentration Assay, Transfection, Binding Assay, Expressing, Time-lapse Microscopy, Control