Journal: Redox Biology

Article Title: Covalent Aurora A regulation by the metabolic integrator coenzyme A

doi: 10.1016/j.redox.2019.101318

Figure Lengend Snippet: Kinase profiling of CoA, dpCoA and ADP. The ability of CoA, dpCoA or ADP (100 μM final concentration of each compound and the indicated concentrations of ATP) to inhibit the phosphotransferase activity of 117 kinases was determined using a radioactive filter-binding assay at the International Centre for Kinase Profiling, DSTT, University of Dundee. Kinase activity in the absence of inhibitor is taken as 100%.

Article Snippet: The following antibodies were employed: mouse anti-CoA antibody; mouse anti-FLAG M2 antibody (Sigma-Aldrich); rabbit anti-Aurora A and anti-Aurora B antibodies (Merck-Millipore); rabbit anti-pT288 Aurora A (Cell Signaling Technology), Alexa Fluor 680 goat anti-mouse IgG H&L (Life Technologies) and IRdye 800 CW goat anti-rabbit IgG H&L (LI-COR Biosciences).

Techniques: Concentration Assay, Activity Assay, Filter-binding Assay

Journal: Redox Biology

Article Title: Covalent Aurora A regulation by the metabolic integrator coenzyme A

doi: 10.1016/j.redox.2019.101318

Figure Lengend Snippet: Specificity of Aurora A interaction with CoA is controlled by Thr 217. (A) Amino acid conservation in vertebrate Aurora kinases. Thr 217 defines Aurora A, and is changed to a Glu residue in Aurora B and C. Cys 290 (boxed) is invariant in all Aurora kinases, and lies in the activation segment adjacent to the phosphorylated Thr 288 (human Aurora A numbering). (B) In vitro CoAlation of Aurora A. (C) In vitro CoAlation of Aurora A is abolished in the Thr217Glu mutant. (D) In vitro CoAlation is not detected with Aurora B. (E) The Glu161Thr Aurora B mutant is efficiently CoAlated. Experiments were performed with the indicated concentration of CoA. Reaction mixtures were separated by SDS-PAGE and immunoblotted with anti-CoA, Aurora A or Aurora B antibodies. (F) A thermal shift assay was employed to evaluate Aurora A binding to 5 mM ATP, 5 mM CoA or 0.1 mM MLN8237, in the presence and absence of 1 mM DTT, as indicated. Assays were performed with T 288 phosphorylated, active, Aurora A (open bars), kinase-dead, dephosphorylated Aurora A (Asp274Asn, green bars), Cys290Ala Aurora A (red bars) or Thr217Glu Aurora A (blue bars). Mean ΔT m values ± SD (n = 3) were calculated by subtracting the control T m value (buffer, no addition) from the measured T m value. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following antibodies were employed: mouse anti-CoA antibody; mouse anti-FLAG M2 antibody (Sigma-Aldrich); rabbit anti-Aurora A and anti-Aurora B antibodies (Merck-Millipore); rabbit anti-pT288 Aurora A (Cell Signaling Technology), Alexa Fluor 680 goat anti-mouse IgG H&L (Life Technologies) and IRdye 800 CW goat anti-rabbit IgG H&L (LI-COR Biosciences).

Techniques: Activation Assay, In Vitro, Mutagenesis, Concentration Assay, SDS Page, Thermal Shift Assay, Binding Assay

Journal: Redox Biology

Article Title: Covalent Aurora A regulation by the metabolic integrator coenzyme A

doi: 10.1016/j.redox.2019.101318

Figure Lengend Snippet: Oxidative stress induces Aurora A CoAlation in human cells. (A) Aurora A CoAlation is induced in cellular response to H 2 O 2 . FLAG-tagged WT Aurora A and WT Aurora B were transiently overexpressed in HEK293/Pank1β. Transfected cells were treated for 30 min with 0.25 mM H 2 O 2 . Overexpressed proteins were immunoprecipitated with an anti-FLAG antibody and the immune complexes immunoblotted with anti-CoA and anti-FLAG antibodies. (B) Oxidising agents promote Aurora A CoAlation in vivo . FLAG-tagged WT Aurora A was transiently over expressed in HEK293/Pank1β. Transfected cells were treated for 30 min with a panel of oxidising agents (250 μM H 2 O 2 , 500 μM diamide, 50 μM menadione, 10 μM phenylarsine oxide and 1 μM rotenone). Transiently expressed proteins were immunoprecipitated with an anti-FLAG antibody, separated by SDS-PAGE under non-reducing conditions and immunoblotted with anti-CoA antibodies. (C) Phosphorylation at Thr 288 and dimerization of Aurora A are induced in cells by oxidising agents. FLAG-tagged WT Aurora A was transiently overexpressed in HEK293/Pank1β. Transfected cells were treated for 30 min with a panel of oxidising agents (250 μM H 2 O 2 , 500 μM diamide, 50 μM menadione, 10 μM phenylarsine oxide and 1 μM rotenone). Transiently expressed proteins were immunoprecipitated with an anti-FLAG antibody, separated by SDS-PAGE under reducing and non-reducing conditions and immunoblotted with anti-FLAG and anti-pT288 Aurora A antibodies. (D) Schematic illustration showing the key features of the ‘dual anchor’ mechanism for interaction of CoA with Thr 217 and Cys 290 in Aurora A.

Article Snippet: The following antibodies were employed: mouse anti-CoA antibody; mouse anti-FLAG M2 antibody (Sigma-Aldrich); rabbit anti-Aurora A and anti-Aurora B antibodies (Merck-Millipore); rabbit anti-pT288 Aurora A (Cell Signaling Technology), Alexa Fluor 680 goat anti-mouse IgG H&L (Life Technologies) and IRdye 800 CW goat anti-rabbit IgG H&L (LI-COR Biosciences).

Techniques: Transfection, Immunoprecipitation, In Vivo, SDS Page

Journal: PLoS ONE

Article Title: Tripolin A, a Novel Small-Molecule Inhibitor of Aurora A Kinase, Reveals New Regulation of HURP's Distribution on Microtubules

doi: 10.1371/journal.pone.0058485

Figure Lengend Snippet: ( A) Chemical structure of Tripolin A and Tripolin B. (B) Graph showing IC 50 values (in µM) of Tripolin A (red) and Tripolin B (green) in the presence of different ATP concentrations, using an in vitro kinase assay. (C) Differential Scanning Fluorimetry results for Aurora A in the presence and absence of the inhibitors. Blue curve determines the melting temperature of Aurora A alone (45°C), red in the presence of Tripolin A (47°C) and green in the presence of Tripolin B (53°C).

Article Snippet: Cells were blocked in PBS/5% w/v BSA pH 7.4 and stained with various combinations of: anti-Aurora A pT288 rabbit polyclonal antibody (1∶100; Cell Signaling Technology), anti-Aurora A mouse monoclonal antibody (1∶2000; Abcam), anti-AIM-1/Aurora B mouse monoclonal antibody (1∶500; BD Biosciences), anti-pHistoneH3 (Ser10) rabbit polyclonal antibody (1∶1000; Millipore), anti-pericentrin rabbit polyclonal antibody (1∶2000; abcam), anti-α-tubulin mouse monoclonal antibody clone GTU-88 (1∶500; Sigma-Aldrich), anti-α-tubulin mouse monoclonal antibody (1∶1000; Santa Cruz Biotechnology), rabbit polyclonal antisera against HURP and TPX2 , , for 1 h at room temperature.

Techniques: In Vitro, Kinase Assay

Journal: PLoS ONE

Article Title: Tripolin A, a Novel Small-Molecule Inhibitor of Aurora A Kinase, Reveals New Regulation of HURP's Distribution on Microtubules

doi: 10.1371/journal.pone.0058485

Figure Lengend Snippet: (A) Representative immunofluorescence images of HeLa cells in metaphase treated with solvent control (DMSO), 20 µM Tripolin A or Tripolin B for 5 h and 24 h. In the merged images Aurora A is pseudocolored red, pAurora T288 green, DNA blue. (Scale bars, 5 µm). (B) Fluorescence intensity (% percentage) of pAurora A T288 on centrosomes and total Aurora A on spindles were quantified in control metaphase cells or cells treated with Tripolin A or Tripolin B (n≥20 cells for each group, from at least two independent experiments). **: 0.0010.05; (Mann-Whitney test, two-tailed). Error bars represent SEM. (C) Western Blot analysis for Aurora A, Aurora B and pHistone H3 Ser10 in Tripolin A and Tripolin B-treated mitotic cells. α-tubulin was used as a loading control. (D) Representative immunofluorescence images of bipolar metaphase HeLa cells treated with solvent control (DMSO), 20 µM Tripolin A or Tripolin B for 24 h. In the merged images pHistone H3 Ser10 is pseudocolored red, Aurora B green, DNA blue. (Scale bars, 5 µm).

Article Snippet: Cells were blocked in PBS/5% w/v BSA pH 7.4 and stained with various combinations of: anti-Aurora A pT288 rabbit polyclonal antibody (1∶100; Cell Signaling Technology), anti-Aurora A mouse monoclonal antibody (1∶2000; Abcam), anti-AIM-1/Aurora B mouse monoclonal antibody (1∶500; BD Biosciences), anti-pHistoneH3 (Ser10) rabbit polyclonal antibody (1∶1000; Millipore), anti-pericentrin rabbit polyclonal antibody (1∶2000; abcam), anti-α-tubulin mouse monoclonal antibody clone GTU-88 (1∶500; Sigma-Aldrich), anti-α-tubulin mouse monoclonal antibody (1∶1000; Santa Cruz Biotechnology), rabbit polyclonal antisera against HURP and TPX2 , , for 1 h at room temperature.

Techniques: Immunofluorescence, Fluorescence, MANN-WHITNEY, Two Tailed Test, Western Blot

Journal: PLoS ONE

Article Title: Tripolin A, a Novel Small-Molecule Inhibitor of Aurora A Kinase, Reveals New Regulation of HURP's Distribution on Microtubules

doi: 10.1371/journal.pone.0058485

Figure Lengend Snippet: (A) Representative immunofluorescence images of mitotic HeLa cells treated with DMSO, 20 µM Tripolin A for 24 h, 100 nM MLN8237 for 24 h or Aurora A siRNAs. In the merged images α-tubulin is pseudocolored red, DNA blue. (Scale bars, 5 µm). (B) Graph showing the percentage of normal, multipolar, misaligned, disorganized and monopolar figures in control mitotic cells (DMSO or control siRNAs) and mitotic cells treated with Tripolin A, MLN8237 or Aurora A siRNA (n = 300 cells for each group, from three independent experiments). (C) Western Blot analysis for Aurora A levels in Aurora A siRNA treated cells. α-tubulin was used as a loading control. (D) Images of mitotic HeLa cells treated with DMSO, 20 µM Tripolin A for 5 h and 24 h or Aurora A siRNA. In the merged images Aurora A is pseudocolored red, pericentrin green, DNA blue. (Scale bar 5 µm). (E) Graph showing the percentage of mitotic cells with fragmented centrosomes (up), or acentrosomal poles (down) in control mitotic cells (DMSO or control siRNA) and mitotic cells treated with Tripolin A, or Aurora A siRNA (n = 150 cells for each group, from three independent experiments).

Article Snippet: Cells were blocked in PBS/5% w/v BSA pH 7.4 and stained with various combinations of: anti-Aurora A pT288 rabbit polyclonal antibody (1∶100; Cell Signaling Technology), anti-Aurora A mouse monoclonal antibody (1∶2000; Abcam), anti-AIM-1/Aurora B mouse monoclonal antibody (1∶500; BD Biosciences), anti-pHistoneH3 (Ser10) rabbit polyclonal antibody (1∶1000; Millipore), anti-pericentrin rabbit polyclonal antibody (1∶2000; abcam), anti-α-tubulin mouse monoclonal antibody clone GTU-88 (1∶500; Sigma-Aldrich), anti-α-tubulin mouse monoclonal antibody (1∶1000; Santa Cruz Biotechnology), rabbit polyclonal antisera against HURP and TPX2 , , for 1 h at room temperature.

Techniques: Immunofluorescence, Western Blot