Identification and Characterization of a New Tubulin-Binding

We studied the mechanism of action of 3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (JG-03-14) and found that it is a potent microtubule depolymerizer. JG-03-14 caused a dose-dependent loss of cellular microtubules, formation of aberrant mitotic spindles, accumulation of cells in the G2/M phase of the cell cycle, and Bcl-2 phosphorylation. These events culminated in the initiation of apoptosis, as evidenced by the caspase 3-dependent cleavage of poly(ADP-ribose) polymerase (PARP). JG-03-14 has antiproliferative activity against a wide range of cancer cell lines, with an average IC50 value of 62 nM, and it is a poor substrate for transport by P-glycoprotein. JG-03-14 inhibited the polymerization of purified tubulin in vitro, consistent with a direct interaction between the compound and tubulin. JG-03-14 potently inhibited the binding of [3H]colchicine to tubulin, suggesting that it bound to tubulin at a site overlapping the colchicine site. JG-03-14 had antitumor effects in the PC3 xenograft model, in which it caused greater than 50% reduction in tumor burden after 14 days of treatment. Molecular modeling studies indicated that the dimethoxyphenyl group of JG-03-14 occupies a space similar to that of the trimethoxyphenyl group of colchicine. However, the 2,3,5-trisubstituted pyrrole group, which is connected to the dimethoxyphenyl moiety, interacted with both α and β tubulin in space not shared with colchicine, suggesting significant differences compared with colchicine in the mechanism of binding to tubulin. Our results suggest that this tetransubstituted pyrrole represents a new, biologically active chemotype for the colchicine site on tubulin

Dynamic Phosphorylation of NudC by Aurora B in Cytokinesis.

Nuclear distribution protein C (NudC) is a mitotic regulator that plays a role in cytokinesis. However, how NudC is regulated during cytokinesis remains unclear. Here, we show that NudC is phosphorylated by Aurora B, a kinase critical for cell abscission. NudC is co-localized with Aurora B at the midbody and co-immunoprecipitated with Aurora B in mitosis. Inhibition of Aurora B by ZM447439 reduced NudC phosphorylation, suggesting that NudC is an Aurora B substrate in vivo. We identified T40 on NudC as an Aurora B phosphorylation site. NudC depletion resulted in cytokinesis failure with a dramatic elongation of the intercellular bridge between daughter cells, sustained Aurora B activity at the midbody, and reduced cell abscission. These cytokinetic defects can be rescued by the ectopic expression of wild-type NudC. Reconstitution with T40A phospho-defective NudC was found to rescue the cytokinesis defect. In contrast, reconstitution with the T40D phospho-mimetic NudC was inefficient in supporting the completion of cytokinesis. These results suggest that that dynamic phosphorylation of NudC by Aurora B regulates cytokinesis

PCR primers for GST-human NudC truncation constructs.

<p>PCR primers for GST-human NudC truncation constructs.</p

NudC is phosphorylated by Aurora B <i>in vitro</i> and <i>in vivo</i>.

<p>(A) HeLa cells were transfected with FLAG-Aurora B wild type (WT) or a kinase dead (K106R) mutant Aurora B for 24 h. Aurora B was immunoprecipitated using anti-FLAG antibody and used in IP kinase assays. Substrates used were GST-NudC (lanes 4–6), histone H3 (lanes 1–3) as a positive control, and GST (lane 7) as a negative control. Aurora B WT was also incubated with 2 μM of ZM447439 as a specificity control (lanes 3 and 6). Samples were transferred to a filter, stained by Ponceau S (lower panel) and analyzed by autoradiography (upper panel). *, degradation product. Data are reproducible in 3 independent experiments. (B) HeLa cells were synchronized by an overnight incubation with 100 ng/ml nocodazole (M, mitotic) as indicated. Cells (1 X 10⁶) were labeled with ³²P orthophosphate for 4 h in the presence or absence of 2 μM ZM447439 (ZM). Cell lysates (300 μg at 1 mg/ml) were immunoprecipitated for NudC, transferred to a filter, analyzed by autoradiography, and immunoblotted for NudC. ³²P-NudC was quantified as ³²P-NudC/total immunoprecipitated NudC and normalized against NudC signals in asynchronously cycling (Asy) cells.</p

Aurora B localization at the kinetochore is not affected in NudC-deficient cells.

<p>(A) HeLa cells were transfected with siLuc or siNudC oligos for 72 h. NudC knockdown was examined by western blotting for NudC. β-tubulin was used as a loading control. (B) Prometaphase cells treated with siRNAs as in (A) were stained for pS326-NudC (red) and Aurora B (green) (enlarged in inset), or with Spc25 (green), and counterstained with DAPI for DNA (blue). In initial experiments, siGLO was co-transfected as an indicator for siRNA oligo uptake. (C) For quantification, cells treated as in (B) were also co-stained with the CREST autoserum to mark the kinetochores. For Aurora B or Spc25 staining, maximum-intensity projections of deconvolved images were measured using AutoDeblur/AutoVisualize software, and their fluorescence intensities (average ± s.d.) relative to that of CREST staining at the kinetochore were quantified, using 10 randomly chosen kinetochores from at least 10 siLuc or siNudC prometaphase cells. n.s., not significant.</p

NudC co-localizes with Aurora B in mitosis.

<p>(A) Unperturbed mitotic HeLa cells were stained for NudC (green), Aurora B (red) and counterstained with DAPI (blue). Bar, 10 μm. (B) HeLa cells were transfected with Myc-NudC and FLAG-Aurora B (left) or EGFP-NudC and FLAG-Aurora B (right) for 24 h. Cell lysates (1 mg in 250 μl) were immunoprecipitated with anti-Myc antibody and blotted for Aurora B followed by reblotting for NudC (left). A reciprocal immunoprecipitation was performed, in which cell lysates (500 μg in 250 μl, 1 mg in 250 μl or 2 mg in 500 μl) were immunoprecipitated with anti-FLAG antibody followed by blotting for NudC and reblotting for Aurora B (right). Immunoprecipitation with either anti-Myc or anti-FLAG antibody using non-transfected cell lysates was used as a negative control. β-tubulin was used as a loading control. Input, 20 μg total cell lysates. Data are representative of n = 5 independent experiments.</p

NudC interaction with Aurora B in mitosis.

<p>(A) HeLa cells were synchronized by a double thymidine block and release protocol as indicated. “P” (prometaphase and metaphase) and “A” (anaphase, telophase and cytokinesis) lysates were prepared from early versus late mitotic cells. Synchronization efficiency was confirmed by a cyclin B1 western blot. α-tubulin was used as a loading control. (B) Lysates from asynchronously cycling (Asy), P or A cells were incubated with GST-NudC fusion protein in GST pulldown assays. GST-NudC bound proteins were immunoblotted for Aurora B. GST binding to lysates from either Asy (this experiment), P or A cells (not shown), served as a negative control. Ponceau S staining showed equal GST-NudC fusion protein used in the pulldown assay. Aurora B binding was quantified as Aurora B signal/input Aurora B normalized against the P sample (mean ± s.e.m.) from 3 independent experiments. *, p < 0.05. (C) Lysates (2 mg in 500 μl) from Asy, P or A cells prepared as in (A) were immunoprecipitated with G1 goat NudC antibody, blotted for Aurora B and reblotted for NudC using 2D9 monoclonal antibody. Asy lysates were also immunoprecipitated with preimmune goat serum (IgG) as a negative control. β-tubulin was used as a loading control. (D) An immunoprecipitation using a different batch of A cell lysate (500 μg in 250 μl) was performed as in (C).</p

NudC is phosphorylated by Aurora B on T40.

<p>(A) A series of GST-NudC truncations were constructed based on functional domains in human NudC. N1 –N4, NudC truncations that retain the N terminal 49 amino acids (a.a.) but contain various deletions from the C terminus. C1 –C4, NudC truncations that retain most or the entire C terminal nuclear movement domain but contain various deletions from the N terminus. Numbers within brackets refer to amino acid residues in the human NudC protein. CC, coiled-coiled; AR, acidic rich; p23-like CHORD-Sgt domain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153455#pone.0153455.ref051" target="_blank">51</a>]; NMD, conserved nuclear movement domain. (B) GST-NudC full-length (FL), N- and C- terminal truncation series depicted in (A) were used in Aurora B IP kinase assays. Reactions were transferred to filters, analyzed by autoradiography, blotted for Aurora B, and stained by Ponceau S. Substrates used were GST-NudC (lanes 2–9), histone H3 (lane 1) as a positive control and GST (lane 10) as a negative control. Arrowheads, ³²P-labeled GST-NudC proteins in the autoradiogram corresponding to the GST-NudC proteins in the Ponceau stain. *, degradation product. The levels of ³²P-GST-NudC signals (autoradiogram)/total GST-NudC (Ponceau) normalized against that of GST-NudC full-length (set as 1) were quantified (mean ± s.e.m.) from 3 independent experiments, except for GST-NudC-N2 which was obtained from one experiment (data not shown). **, p < 0.001; ***, p < 0.04. (C) GST-NudC-N1 was used in Aurora B IP kinase assays. (D) NudC protein sequences from various species share a high degree of sequence homology surrounding amino acid T40. (E) GST-NudC-N1 wild type (WT) and GST-NudC-N1 containing T40A mutation were used in Aurora B IP kinase assays. GST, negative control. Data in C and E are representative of 3 independent experiments.</p