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: Identification of a novel TRF1-binding protein, TAP68. A, mitotic HeLa cell extracts were applied to a TRF1 peptide antibody affinity column. After binding, columns were extensively washed, and bound proteins were eluted and separated by SDS-PAGE. The indicated proteins were extracted from the gel and digested with trypsin, and the amino acid sequence of the peptides was determined by MALDI-TOF and LC-MS/MS mass spectrometry. P1 is TAP68. B, protein band of P1 was extracted from the acrylamide gel and digested with trypsin, and the resulting peptide fragments were subjected to mass spectrometric analysis. C, P1 peptides match a previously uncharacterized protein with a 593-amino acid open reading frame of unknown function (AAH03618). D, schematic representation of the TAP68 domain organization predicted by NCBI's on-line conserved domain search tool. E, TRF1-TAP68 interaction was confirmed by immunoprecipitation assay. The anti-TRF1 antibody binding on protein A/G beads was incubated with mitotic cell lysate prepared in high stringency lysis buffer containing 1% Nonidet P-40 and 0.5% deoxycholate. Under this condition, TAP68 remained firmly bound to TRF1, whereas the majority of other TRF1-binding proteins, such as TRF2 and tankyrase, were absent from the co-immunoprecipitates.

Article Snippet: Mitotic extracts from HeLa S3 cells (1 × 10 9 ) were prepared in lysis buffer (25 m m HEPES, pH 6.9, 150 m m NaCl, 2 m m EGTA, 5 m m MgCl 2 , 0.2% Triton X-100, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml chymostatin, and 10 μg/ml pepstatin A) and incubated with 100 μl of protein A-Sepharose beads coupled with a rabbit anti-TRF1 antibody (Ab13792; Novus Biologicals, Inc.) using a previously described protocol ( 24 ).

Techniques: Binding Assay, Affinity Column, SDS Page, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Acrylamide Gel Assay, Immunoprecipitation, Incubation, Lysis

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: Biochemical characterization of TRF1-TAP68 interaction. A, exogenous TRF1 interacts with TAP68 in HeLa cells transfected with FLAG-tagged TRF1 and either GFP vector or GFP-tagged TAP68. After 36 h, HeLa cells were then extracted with 1% Nonidet P-40 plus 0.5% deoxycholate, and FLAG-tagged TRF1 protein and its binding proteins were immunoprecipitated (IP) with 20 μl of FLAG-M2 antibody-conjugated beads. Co-precipitated proteins were separated by SDS-PAGE and then immunoblotted for the presence of FLAG-TRF1 (upper panel) or GFP-TAP68 proteins (lower panel). B, exogenous TAP68 interacts with TRF1 in HeLa cells transfected with FLAG-tagged TRF1 and either GFP or GFP-tagged TAP68. After 36 h, HeLa cells were then extracted with 1% Nonidet P-40 plus 0.5% deoxycholate, and GFP-tagged TAP68 protein and its associated proteins were precipitated with 20 μl of GFP antibody-conjugated beads. Co-precipitated proteins were then detected by immunoblot for the presence of GFP-TAP68 (upper panel) or FLAG-TRF1 proteins (lower panel). C, schematic representation of different TAP68 truncation constructs. D, HeLa cells were transfected with full-length or different truncations of GFP-tagged TAP68 along with FLAG-tagged TRF1. After 36 h, HeLa cells were then extracted with 1% Nonidet P-40 plus 0.5% deoxycholate, and FLAG-tagged TRF1 and its accessory proteins were precipitated with 20 μl of rabbit TRF1 antibody-conjugated beads. Co-precipitated proteins were then immunoblotted for the presence of GFP-TAP68 proteins (upper panel) or FLAG-TRF1 (lower panel).

Article Snippet: Mitotic extracts from HeLa S3 cells (1 × 10 9 ) were prepared in lysis buffer (25 m m HEPES, pH 6.9, 150 m m NaCl, 2 m m EGTA, 5 m m MgCl 2 , 0.2% Triton X-100, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml chymostatin, and 10 μg/ml pepstatin A) and incubated with 100 μl of protein A-Sepharose beads coupled with a rabbit anti-TRF1 antibody (Ab13792; Novus Biologicals, Inc.) using a previously described protocol ( 24 ).

Techniques: Transfection, Plasmid Preparation, Binding Assay, Immunoprecipitation, SDS Page, Western Blot, Construct

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: TAP68 mediates TRF1 localization to the centrosome. A, TAP68 and TRF1 share similar subcellular localization. Nuclear and post-nuclear fractions were generated from interphase HeLa cell homogenates. Equal amounts of proteins (35 μg) from various fractions were separated by 5–15% gradient SDS-PAGE and detected by Western blot analyses using antibodies against TAP68, TRF1, and β-tubulin, respectively. B, representative images of interphase (top panels), late prophase/early prometaphase (middle panels), and metaphase (bottom panels) HeLa cells stained for TRF1 (green), TAP68 (red), DAPI (blue), and γ-tubulin (white). In interphase cells, TRF1 (panel a) and TAP68 (panel b) are readily seen in the nucleus as speckles and dots in the merge panel (panel d). In late prophase or the earliest prometaphase cells marked by nuclear membrane fragmentation, TAP68 appears at the centrosome (panel f, arrows), and TRF1 remains as speckles in the partially condensed chromosome (panel e, arrows). Both TAP68 and TRF1 become co-localized to the centrosome as the chromosomes align or after sister chromatid separation (panels i and j, arrows). A merged image shows the co-localization of TRF1 and TAP68 to the centrosome of mitotic cells (panel l). Bars, 10 μm. C, immunoelectron microscopy of late prophase cells indicated that TAP68 localizes to the pericentrioles. D, representative immunofluorescence images of HeLa cells treated with nocodazole or DMSO. 10 h after drug treatment, the cells were fixed and stained for tubulin (green), TAP68 (red), and DAPI (blue). TAP68 remained centrosome-associated in the absence of microtubules (panel h, arrows). E, ectopic expression of different GFP-tagged TAP68 proteins. HeLa cells were transfected GFP-tagged full-length TAP68 or its deletion mutants. After 36 h, HeLa cells were then harvested for Western blot analysis using a GFP antibody. F, representative images collected from metaphase HeLa cells transiently transfected with GFP-tagged TAP68 and its deletion mutants and stained for TRF1 (red), TAP68 (green), and DAPI (blue). In cells expressing full-length TAP68, both TAP68 and TRF1 are co-localized to the centrosome (panels a, b, and d, arrows). A similar co-distribution of TRF1 with TAP68 was observed in TAP68ΔC mutant-expressing cells (panels e, f, and h) but not TAP68MC-expressing cells (panels i, j, and l), demonstrating that the centrosomal localization region of TAP68 is independent of its TRF1-binding domain. Bars, 10 μm. G, schematic representation of the regions of TAP68, which specifies its centrosomal localization. H, representative images of HeLa cells transfected with different GFP-tagged TAP68 constructs. 24 h after transfection, cells were fix and co-stained for γ-tubulin (red) and DNA (blue). Bars, 10 μm.

Article Snippet: Mitotic extracts from HeLa S3 cells (1 × 10 9 ) were prepared in lysis buffer (25 m m HEPES, pH 6.9, 150 m m NaCl, 2 m m EGTA, 5 m m MgCl 2 , 0.2% Triton X-100, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml chymostatin, and 10 μg/ml pepstatin A) and incubated with 100 μl of protein A-Sepharose beads coupled with a rabbit anti-TRF1 antibody (Ab13792; Novus Biologicals, Inc.) using a previously described protocol ( 24 ).

Techniques: Generated, SDS Page, Western Blot, Staining, Membrane, Immuno-Electron Microscopy, Immunofluorescence, Expressing, Transfection, Mutagenesis, Binding Assay, Construct

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: TAP68 mediates TRF1 translocation to the centrosome and mitotic regulation. A, efficient siRNA-mediated suppression of TAP68. HeLa cells were transfected with a siRNA duplex specific for TAP68 and a scrambled control siRNA duplex. After 48 h, HeLa cells were then harvested for Western blot analysis using antibodies specific for TAP68, TRF1, tankyrase 1 (TANKS1), and NuMA. The analyses indicate the specificity and efficiency of the targeted protein suppression. B, representative immunofluorescence images of HeLa cells transfected with control or TAP68-specific siRNA duplex. 48 h post-transfection, cells were stained for the indicated antibodies and DNA, respectively. In the control duplex-transfected cells, NuMA, TAP68, and tankyrase 1 are all co-localized to the centrosome (panels a–c). In TAP68 siRNA duplex-treated cells, TAP68 protein expression was suppressed (panels f and j), and localization of TRF1 to the centrosome was diminished, although TRF1 distribution on chromosome remained (panel g). The distribution of tankyrase 1 to the centrosome was also eliminated in TAP68-depleted cells (panel k). However, the spindle pole localization of NuMA was not altered by the loss of TAP68 (panel e). Tetrapolar spindle in TAP68-depleted cells was revealed by NuMA staining (panel e). Bars, 10 μm. C, quantitative analyses of the mitotic spindle after TAP68 knockdown. HeLa cells were transfected with TAP68 siRNA oligonucleotide and control oligonucleotide for 36–48 h followed by fixation and staining for DNA, TAP68, and NuMA. After examination under a fluorescence microscope, the centrosome instability phenotypes were categorized as bipolar, tripolar, or tetrapolar spindles. Data represent the percentage of total mitotic cells evaluated. An average of 100 cells from three separate experiments was counted. * indicates a significant difference between the TAP68 siRNA- and control siRNA-transfected cells.

Article Snippet: Mitotic extracts from HeLa S3 cells (1 × 10 9 ) were prepared in lysis buffer (25 m m HEPES, pH 6.9, 150 m m NaCl, 2 m m EGTA, 5 m m MgCl 2 , 0.2% Triton X-100, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml chymostatin, and 10 μg/ml pepstatin A) and incubated with 100 μl of protein A-Sepharose beads coupled with a rabbit anti-TRF1 antibody (Ab13792; Novus Biologicals, Inc.) using a previously described protocol ( 24 ).

Techniques: Translocation Assay, Transfection, Control, Western Blot, Immunofluorescence, Staining, Expressing, Knockdown, Fluorescence, Microscopy

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: Mitotic extracts from HeLa S3 cells (1 × 10 9 ) were prepared in lysis buffer (25 m m HEPES, pH 6.9, 150 m m NaCl, 2 m m EGTA, 5 m m MgCl 2 , 0.2% Triton X-100, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml chymostatin, and 10 μg/ml pepstatin A) and incubated with 100 μl of protein A-Sepharose beads coupled with a rabbit anti-TRF1 antibody (Ab13792; Novus Biologicals, Inc.) using a previously described protocol ( 24 ).

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

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: Identification of a novel TRF1-binding protein, TAP68. A, mitotic HeLa cell extracts were applied to a TRF1 peptide antibody affinity column. After binding, columns were extensively washed, and bound proteins were eluted and separated by SDS-PAGE. The indicated proteins were extracted from the gel and digested with trypsin, and the amino acid sequence of the peptides was determined by MALDI-TOF and LC-MS/MS mass spectrometry. P1 is TAP68. B, protein band of P1 was extracted from the acrylamide gel and digested with trypsin, and the resulting peptide fragments were subjected to mass spectrometric analysis. C, P1 peptides match a previously uncharacterized protein with a 593-amino acid open reading frame of unknown function (AAH03618). D, schematic representation of the TAP68 domain organization predicted by NCBI's on-line conserved domain search tool. E, TRF1-TAP68 interaction was confirmed by immunoprecipitation assay. The anti-TRF1 antibody binding on protein A/G beads was incubated with mitotic cell lysate prepared in high stringency lysis buffer containing 1% Nonidet P-40 and 0.5% deoxycholate. Under this condition, TAP68 remained firmly bound to TRF1, whereas the majority of other TRF1-binding proteins, such as TRF2 and tankyrase, were absent from the co-immunoprecipitates.

Article Snippet: The specificity of the TRF1-TAP68 interaction was validated using a different TRF1 antibody Ab1423 (Novus Biologicals, Inc) in high stringency lysis buffer containing 1% Nonidet P-40 and 0.5% deoxycholate.

Techniques: Binding Assay, Affinity Column, SDS Page, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Acrylamide Gel Assay, Immunoprecipitation, Incubation, Lysis

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: Biochemical characterization of TRF1-TAP68 interaction. A, exogenous TRF1 interacts with TAP68 in HeLa cells transfected with FLAG-tagged TRF1 and either GFP vector or GFP-tagged TAP68. After 36 h, HeLa cells were then extracted with 1% Nonidet P-40 plus 0.5% deoxycholate, and FLAG-tagged TRF1 protein and its binding proteins were immunoprecipitated (IP) with 20 μl of FLAG-M2 antibody-conjugated beads. Co-precipitated proteins were separated by SDS-PAGE and then immunoblotted for the presence of FLAG-TRF1 (upper panel) or GFP-TAP68 proteins (lower panel). B, exogenous TAP68 interacts with TRF1 in HeLa cells transfected with FLAG-tagged TRF1 and either GFP or GFP-tagged TAP68. After 36 h, HeLa cells were then extracted with 1% Nonidet P-40 plus 0.5% deoxycholate, and GFP-tagged TAP68 protein and its associated proteins were precipitated with 20 μl of GFP antibody-conjugated beads. Co-precipitated proteins were then detected by immunoblot for the presence of GFP-TAP68 (upper panel) or FLAG-TRF1 proteins (lower panel). C, schematic representation of different TAP68 truncation constructs. D, HeLa cells were transfected with full-length or different truncations of GFP-tagged TAP68 along with FLAG-tagged TRF1. After 36 h, HeLa cells were then extracted with 1% Nonidet P-40 plus 0.5% deoxycholate, and FLAG-tagged TRF1 and its accessory proteins were precipitated with 20 μl of rabbit TRF1 antibody-conjugated beads. Co-precipitated proteins were then immunoblotted for the presence of GFP-TAP68 proteins (upper panel) or FLAG-TRF1 (lower panel).

Article Snippet: The specificity of the TRF1-TAP68 interaction was validated using a different TRF1 antibody Ab1423 (Novus Biologicals, Inc) in high stringency lysis buffer containing 1% Nonidet P-40 and 0.5% deoxycholate.

Techniques: Transfection, Plasmid Preparation, Binding Assay, Immunoprecipitation, SDS Page, Western Blot, Construct

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: TAP68 mediates TRF1 localization to the centrosome. A, TAP68 and TRF1 share similar subcellular localization. Nuclear and post-nuclear fractions were generated from interphase HeLa cell homogenates. Equal amounts of proteins (35 μg) from various fractions were separated by 5–15% gradient SDS-PAGE and detected by Western blot analyses using antibodies against TAP68, TRF1, and β-tubulin, respectively. B, representative images of interphase (top panels), late prophase/early prometaphase (middle panels), and metaphase (bottom panels) HeLa cells stained for TRF1 (green), TAP68 (red), DAPI (blue), and γ-tubulin (white). In interphase cells, TRF1 (panel a) and TAP68 (panel b) are readily seen in the nucleus as speckles and dots in the merge panel (panel d). In late prophase or the earliest prometaphase cells marked by nuclear membrane fragmentation, TAP68 appears at the centrosome (panel f, arrows), and TRF1 remains as speckles in the partially condensed chromosome (panel e, arrows). Both TAP68 and TRF1 become co-localized to the centrosome as the chromosomes align or after sister chromatid separation (panels i and j, arrows). A merged image shows the co-localization of TRF1 and TAP68 to the centrosome of mitotic cells (panel l). Bars, 10 μm. C, immunoelectron microscopy of late prophase cells indicated that TAP68 localizes to the pericentrioles. D, representative immunofluorescence images of HeLa cells treated with nocodazole or DMSO. 10 h after drug treatment, the cells were fixed and stained for tubulin (green), TAP68 (red), and DAPI (blue). TAP68 remained centrosome-associated in the absence of microtubules (panel h, arrows). E, ectopic expression of different GFP-tagged TAP68 proteins. HeLa cells were transfected GFP-tagged full-length TAP68 or its deletion mutants. After 36 h, HeLa cells were then harvested for Western blot analysis using a GFP antibody. F, representative images collected from metaphase HeLa cells transiently transfected with GFP-tagged TAP68 and its deletion mutants and stained for TRF1 (red), TAP68 (green), and DAPI (blue). In cells expressing full-length TAP68, both TAP68 and TRF1 are co-localized to the centrosome (panels a, b, and d, arrows). A similar co-distribution of TRF1 with TAP68 was observed in TAP68ΔC mutant-expressing cells (panels e, f, and h) but not TAP68MC-expressing cells (panels i, j, and l), demonstrating that the centrosomal localization region of TAP68 is independent of its TRF1-binding domain. Bars, 10 μm. G, schematic representation of the regions of TAP68, which specifies its centrosomal localization. H, representative images of HeLa cells transfected with different GFP-tagged TAP68 constructs. 24 h after transfection, cells were fix and co-stained for γ-tubulin (red) and DNA (blue). Bars, 10 μm.

Article Snippet: The specificity of the TRF1-TAP68 interaction was validated using a different TRF1 antibody Ab1423 (Novus Biologicals, Inc) in high stringency lysis buffer containing 1% Nonidet P-40 and 0.5% deoxycholate.

Techniques: Generated, SDS Page, Western Blot, Staining, Membrane, Immuno-Electron Microscopy, Immunofluorescence, Expressing, Transfection, Mutagenesis, Binding Assay, Construct

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: TAP68 mediates TRF1 translocation to the centrosome and mitotic regulation. A, efficient siRNA-mediated suppression of TAP68. HeLa cells were transfected with a siRNA duplex specific for TAP68 and a scrambled control siRNA duplex. After 48 h, HeLa cells were then harvested for Western blot analysis using antibodies specific for TAP68, TRF1, tankyrase 1 (TANKS1), and NuMA. The analyses indicate the specificity and efficiency of the targeted protein suppression. B, representative immunofluorescence images of HeLa cells transfected with control or TAP68-specific siRNA duplex. 48 h post-transfection, cells were stained for the indicated antibodies and DNA, respectively. In the control duplex-transfected cells, NuMA, TAP68, and tankyrase 1 are all co-localized to the centrosome (panels a–c). In TAP68 siRNA duplex-treated cells, TAP68 protein expression was suppressed (panels f and j), and localization of TRF1 to the centrosome was diminished, although TRF1 distribution on chromosome remained (panel g). The distribution of tankyrase 1 to the centrosome was also eliminated in TAP68-depleted cells (panel k). However, the spindle pole localization of NuMA was not altered by the loss of TAP68 (panel e). Tetrapolar spindle in TAP68-depleted cells was revealed by NuMA staining (panel e). Bars, 10 μm. C, quantitative analyses of the mitotic spindle after TAP68 knockdown. HeLa cells were transfected with TAP68 siRNA oligonucleotide and control oligonucleotide for 36–48 h followed by fixation and staining for DNA, TAP68, and NuMA. After examination under a fluorescence microscope, the centrosome instability phenotypes were categorized as bipolar, tripolar, or tetrapolar spindles. Data represent the percentage of total mitotic cells evaluated. An average of 100 cells from three separate experiments was counted. * indicates a significant difference between the TAP68 siRNA- and control siRNA-transfected cells.

Article Snippet: The specificity of the TRF1-TAP68 interaction was validated using a different TRF1 antibody Ab1423 (Novus Biologicals, Inc) in high stringency lysis buffer containing 1% Nonidet P-40 and 0.5% deoxycholate.

Techniques: Translocation Assay, Transfection, Control, Western Blot, Immunofluorescence, Staining, Expressing, Knockdown, Fluorescence, Microscopy

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: The specificity of the TRF1-TAP68 interaction was validated using a different TRF1 antibody Ab1423 (Novus Biologicals, Inc) in high stringency lysis buffer containing 1% Nonidet P-40 and 0.5% deoxycholate.

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