Journal: The Journal of Biological Chemistry

Article Title: The 68-kDa Telomeric Repeat Binding Factor 1 (TRF1)-associated Protein (TAP68) Interacts with and Recruits TRF1 to the Spindle Pole during Mitosis *

doi: 10.1074/jbc.M113.526244

Figure Lengend Snippet: Phospho-regulation of TRF1-TAP68 interaction and centrosomal localization during mitosis. A, immunoblot analysis of TAP68 in HeLa cells synchronized at the indicated cell cycle phases. Note the mobility shift (pTAP68) from mitotic cell lysates. B, HeLa cell lysates (35 μg) from synchronized interphase and mitotic cells were treated with λ-phosphatase or the phosphatase inhibitor, okadaic acid, followed by Western blotting with an anti-TAP68 antibody. C, bacterially expressed histidine-tagged TAP68 fusion proteins, both wild-type and mutant (TAP68T221A and TAP68T457A), were phosphorylated in vitro in the presence of [32P]ATP and NEK2A kinase. Samples were separated by 6–16% gradient SDS-PAGE. The gel was dried and subsequently incubated with x-ray film. Note that in the presence of NEK2A, there was a dramatic incorporation of 32P into the wild-type TAP68 and TAP68T457A mutant, but not the TAP68T221A mutant. D, purified His-tagged TAP68-WT and TAP68-T457A were subjected to in vitro phosphorylation by recombinant PLK1 kinase. The left panel shows Coomassie Blue staining of the gel. The right panel shows the result of autoradiography. E, HeLa cell lysates from synchronized G1 and nocodazole-treated mitotic cells ectopically expressing wild-type or the indicated FLAG-TAP68 mutants were immunoblotted with an anti-FLAG and anti-α-tubulin antibodies, respectively. F, lysates from mitotic HeLa cells ectopically expressing FLAG-tagged TAP68-WT and the indicated mutants, immunoprecipitated with an anti-TRF1 antibody, and then immunoblotted with anti-TRF1 and anti-FLAG antibodies. TAP68T221A, which cannot be phosphorylated by NEK2A, was co-precipitated with TRF1 (lane 4), and its phospho-mimicking mutant TAP68T221E did not interact with TRF1 (lane 6). In contrast, the mutant with mimics Thr-457 phosphorylation by PLK1, TAP68T457E, interacted with TRF1, whereas the TAP68T457A mutant did not (lanes 10 and 8, respectively). Thus, phosphorylation of TAP68 at Thr-221 by NEK2A releases it from TRF1, whereas phosphorylation at Thr-457 by PLK1 is required for the TRF1-TAP68 interaction. G, representative images of HeLa cells ectopically expressing wild-type and mutant FLAG-TAP68. Cells were fixed and stained for TAP68 (red), TRF1 (green), and DNA (blue). Bars, 10 μm. Both wild-type FLAG-TAP68 and TRF1 are co-localized to the centrosome at metaphase (panels a and b, arrows). A merged image shows the co-localization of TRF1 and wild-type FLAG-TAP68 to the centrosome of mitotic cells (panel d). However, in HeLa cells ectopically expressing TAP68T221A, which cannot be phosphorylated by NEK2A, TAP68T221A remains associated with TRF1 at the telomere in late prometaphase cells (panel f) as evident in the merged image (panel h). In HeLa cells ectopically expressing TAP68T457A, which cannot be phosphorylated by PLK1, TAP68T457A localizes to the centrosome (panel j) with little TRF1 (panel i) as evidenced in the merged image (panel l, arrows), illustrating that PLK1-mediated phosphorylation is essential for TAP68-TRF1 association at the centrosome. Indeed, TRF1 co-localizes with TAP68 in HeLa cells ectopically expressing the phospho-mimicking mutant, TAP68T457E.

Article Snippet: To verify whether Thr-221 is a substrate for NEK2A or Thr-457 is a substrate for PLK1, 2 μg of purified His-TAP68 fusion proteins, both wild-type and mutants, were incubated with 1 unit of active Nek2A or PLK1 (Upstate Biotechnology, Lake Placid, NY) in kinase buffer containing 20 m m HEPES, pH 7.5, 10 m m MgCl 2 , 5 m m EGTA, 1 m m dithiothreitol, 10 μ m ATP, and 5 μCi of [ 32 P]ATP (PerkinElmer Life Sciences) in a total volume of 30 μl.

Techniques: Western Blot, Mobility Shift, Mutagenesis, In Vitro, SDS Page, Incubation, Purification, Phospho-proteomics, Recombinant, Staining, Autoradiography, Expressing, Immunoprecipitation

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

Journal: Oncotarget

Article Title: Disulfiram, a drug widely used to control alcoholism, suppresses self-renewal of glioblastoma and overrides resistance to temozolomide

doi:

Figure Lengend Snippet: (A) SF188 cells were exposed to DSF for 24 hrs using DMSO as a solvent control. Protein and transcript levels of PLK1 were assessed using immunoblotting or qRT-PCR. (B-C) PLK1 inhibition with siRNA inhibits SF188 cell growth and induced apoptosis based on PARP and caspase 3 cleavage. Scramble RNA oligo was transfected as a control. The efficacy of PLK1 inhibition on SF188 growth is exemplified in combination with 10 uM TMZ.

Article Snippet: PLK1 protein expression was evaluated using LSBio antibody (diluted 1:200, PLK1 rabbit anti-Human polyclonal Antibody LS-B4225- LSBio LifeSpan Bioscience, Seattle, WA).

Techniques: Solvent, Control, Western Blot, Quantitative RT-PCR, Inhibition, Transfection

Journal: Oncotarget

Article Title: Disulfiram, a drug widely used to control alcoholism, suppresses self-renewal of glioblastoma and overrides resistance to temozolomide

doi:

Figure Lengend Snippet: (A) U251 cells were exposed to DSF for 24 hrs using DMSO as a solvent control. Protein and transcript levels of PLK1 were assessed using immunoblotting or qRT-PCR. (B-C) Inhibiting PLK1 with BI-2536 or siRNA inhibits their growth. The efficacy of PLK1 inhibition on U251 growth is exemplified in combination with 10 uM TMZ. (D) Likewise, DSF inhibits the growth of U251 cells in a dose-dependent manner using DMSO as a solvent control.

Article Snippet: PLK1 protein expression was evaluated using LSBio antibody (diluted 1:200, PLK1 rabbit anti-Human polyclonal Antibody LS-B4225- LSBio LifeSpan Bioscience, Seattle, WA).

Techniques: Solvent, Control, Western Blot, Quantitative RT-PCR, Inhibition

Journal: Oncotarget

Article Title: Disulfiram, a drug widely used to control alcoholism, suppresses self-renewal of glioblastoma and overrides resistance to temozolomide

doi:

Figure Lengend Snippet: (A) U251 cells have lower PLK1 transcript expression than TMZ resistant SF188 cells. U251 cells were treated with 10 uM TMZ every 2 days for a total of 7 days and (B) partial TMZ sensitivity is demonstrated in a growth assay. (C) Immunoblot demonstrating an increase in PLK1 protein levels in TMZ treated U251 cells compared to untreated and DMSO after 24 hours. Actin is used as a loading control protein. (D) The surviving TMZ resistant cells were re-plated and treated with increasing concentrations of BI-2536 for 5 days.

Article Snippet: PLK1 protein expression was evaluated using LSBio antibody (diluted 1:200, PLK1 rabbit anti-Human polyclonal Antibody LS-B4225- LSBio LifeSpan Bioscience, Seattle, WA).

Techniques: Expressing, Growth Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Disulfiram, a drug widely used to control alcoholism, suppresses self-renewal of glioblastoma and overrides resistance to temozolomide

doi:

Figure Lengend Snippet: (A) BT74 cells are resistant to TMZ yet sensitive to PLK1 inhibition with BI-2536. (B) BT241 cells are a second example to which the cells are TMZ resistant yet sensitive to PLK1 inhibition. Both models are maintained as primary isolates and only cultured as neurospheres. The combination of TMZ and BI-2536 did not further improve self-renewal inhibition. Scale bar = 500 um.

Article Snippet: PLK1 protein expression was evaluated using LSBio antibody (diluted 1:200, PLK1 rabbit anti-Human polyclonal Antibody LS-B4225- LSBio LifeSpan Bioscience, Seattle, WA).

Techniques: Inhibition, Cell Culture

Journal: Oncotarget

Article Title: Disulfiram, a drug widely used to control alcoholism, suppresses self-renewal of glioblastoma and overrides resistance to temozolomide

doi:

Figure Lengend Snippet: (A) PLK1 levels were assessed in aBT001 and aBT003 by immunostaining. Both cases express high levels of PLK1. (B-C) The PLK1 inhibitor BI-2536 suppressed the growth of aBT001 and induced cell death.

Article Snippet: PLK1 protein expression was evaluated using LSBio antibody (diluted 1:200, PLK1 rabbit anti-Human polyclonal Antibody LS-B4225- LSBio LifeSpan Bioscience, Seattle, WA).

Techniques: Immunostaining