NudC Deacetylation Regulates Mitotic Progression

<div><p>Mitosis is largely driven by posttranslational modifications of proteins. Recent studies suggest that protein acetylation is prevalent in mitosis, but how protein acetylation/deacetylation regulates mitotic progression remains unclear. Nuclear distribution protein C (NudC), a conserved protein that regulates cell division, was previously shown to be acetylated. We found that NudC acetylation was decreased during mitosis. Using mass spectrometry analysis, we identified K39 to be an acetylation site on NudC. Reconstitution of NudC-deficient cells with wild-type or K39R acetylation-defective NudC rescued mitotic phenotypes, including chromosome misalignment, chromosome missegregation, and reduced spindle width, observed after NudC protein knockdown. In contrast, the K39Q acetylation-mimetic NudC was unable to rescue these mitotic phenotypes, suggesting that NudC deacetylation is important for mitotic progression. To examine proteins that may play a role in NudC deacetylation during mitosis, we found that NudC co-localizes on the mitotic spindle with the histone deacetylase HDAC3, an HDAC shown to regulate mitotic spindle stability. Further, NudC co-immunoprecipitates with HDAC3 and loss of function of HDAC3 either by protein knockdown or inhibition with a small molecule inhibitor increased NudC acetylation. These observations suggest that HDAC3 may be involved in NudC deacetylation during mitosis. Cells with NudC or HDAC3 knockdown exhibited overlapping mitotic abnormalities, including chromosomes arranged in a “dome-like” configuration surrounding a collapsed mitotic spindle. Our studies suggest that NudC acetylation/deacetylation regulates mitotic progression and NudC deacetylation, likely through HDAC3, is critical for spindle function and chromosome congression.</p></div

NudC associates with HDAC3 in mitosis and a loss of HDAC3 increases NudC acetylation.

<p>(A) Cell cycle synchronization schematic. (B) Whole cell lysates (10 µg) from S, G2, early mitosis (prometaphase-like; P), late mitosis (anaphase and telophase; A), and G1 cell cycle phases are blotted with antibodies as indicated. (C) Lysates (2 mg) prepared as in (B) were immunoprecipitated (IP) for HDAC3 then blotted for NudC followed by HDAC3. IgG, antibody control. β-tubulin was used as a loading control. The association of NudC with HDAC3 is analyzed by ImageJ and presented as a ratio of NudC co-IP over HDAC3 IP. Mean ± SEM of three independent experiments. **, p<.02. (D) Cells were transfected with siRNA against Luciferase (siLuc) or HDAC3 (siHDAC3) for 48 h. Lysates (2 mg) were immunoprecipitated for NudC under denaturing conditions, then blotted for Ac-K and NudC. NudC acetylation is quantified by ImageJ as a ratio of Ac-NudC over NudC and normalized to siLuc (fold changes in red). IgG, antibody control. Data represent three independent experiments.</p

NudC acetylation at K39 regulates mitotic progression.

<p>(A) HeLa cells were transfected with siRNA against Luciferase (siLuc; i) or NudC (siNudC; ii–v) 48 h then synchronized by a single thymidine block and release. Cells were stained (left) with CREST (green) to visualize centromeres and β-tubulin (red) for the mitotic spindle and counterstained with DAPI (blue) for DNA. Chromosome phenotypes were observed: “dome-like” configuration of chromosomes that (ii) surround the mitotic spindle or (iii) are involved in lateral, side-on attachments to mitotic spindle; (iv) miscongressed chromosomes at metaphase (arrow); and (v) lagging chromosomes in anaphase/telophase (arrowhead). These chromosome phenotypes were quantified (bottom). Graph shows the mean (± SEM) % from three independent experiments; >200 cells were counted per siRNA oligo. *, p<0.05. (B) Western blot of NudC in NudC knockdown cells without or with EGFP WT, K39R or K39Q reconstitution. HeLa cells were transfected with siLuc or siNudC for 24 h then reconstituted with EGFP-NudC WT, K39R or K39Q lysine mutants for another 24 h. Cells were synchronized by a single thymidine block and release followed by a nocodazole block and release to enrich for mitotic cells. Tubulin was blotted as a loading control. (C) NudC-reconstituted GFP-positive green cells, prepared as in (B), were enriched in mitosis by a single-thymidine block and counterstained with DAPI (DNA; blue). Images represent three independent experiments. Cells in (C) were quantified for “dome-like” configuration of chromosomes (D) and lagging chromosomes (E) relative to siLuc control cells. Graphs show the mean (± SEM) % of three to four independent experiments; >200 cells were counted for each condition. *, p<0.05 and **, p<0.02 by ANOVA. n.s., not significant. (F) HeLa cells were prepared and stained as in (C) and spindle widths (between arrows) were measured. Dotplot shows the mean (± SEM) of ∼70 cells measured for each condition. ***, p<0.0001 by ANOVA. n.s., not significant. Images are representative cells with median spindle widths. All scale bars, 10 µm.</p

NudC and HDAC3 co-localize in early mitosis.

<p>HeLa cells were stained for NudC (A; red) or HDAC3 (B; red) and β-tubulin (A–B; green) then counterstained with DAPI (DNA; blue). (C) Cells were stained for NudC (red) and HDAC3 (green) then counterstained with DAPI (blue). In late mitosis, NudC translocates to the midzone and the midbody (asterisk), whereas HDAC3 remains on the mitotic spindle in anaphase and the minus-end (outer edges) of midzone microtubules in telophase (arrow). Cells were treated with Noc (D) or Taxol (E), stained for β-tubulin (D; green) or HDAC3 (E; green) and NudC (D–E; red), then counterstained with DAPI (DNA; blue). Images represent three independent experiments. All scale bars, 10 µm.</p

NudC is acetylated in interphase and deacetylated in mitosis.

<p>(A) Unperturbed HeLa cell lysates (1 mg) were immunoprecipitated (IP) for NudC under denaturing conditions (used subsequently to examine NudC acetylation) and blotted with anti-acetyl lysine (Ac-K) antibody then reblotted for total NudC. (B) Sequential IP (Seq IP) flow chart. Lysates (5 mg) from asynchronouse (Asy) and mitotic (M) cells are IP'd first with anti-Ac-K antibody followed by IP for NudC then blotted for NudC. NudC acetylation is analyzed by ImageJ as a ratio of NudC IP over Input and normalized to R (fold change in red). Mean ± SEM of three independent experiments. **, p<0.02. (C) Cells were synchronized by a double thymidine block and release (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073841#pone-0073841-g004" target="_blank">Figure 4A</a>) then enriched in mitosis with nocodazole (Noc) with or without incubation with increasing concentrations of apicidin. Lysates (2 mg) were IP'd for NudC, histone H3 (H3), or α-tubulin (α-Tub) as in (A), and blotted with anti-Ac-K then reblotted for NudC, H3, or α-Tub, respectively. NudC, H3, and α-Tub acetylation are analyzed by ImageJ as a ratio of Ac-protein over either input (NudC) or protein IP (H3, α-Tub) and normalized to 0 nM Apicidin treatment (fold change in red). Data represent two to three independent experiments.</p

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

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