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. 2010 Sep 3;9(9):4554-64.
doi: 10.1021/pr100281h.

Acetylation of RNA processing proteins and cell cycle proteins in mitosis

Carol Chuang  1 , Sue-Hwa Lin, Feilei Huang, Jing Pan, Djuro Josic, Li-yuan Yu-Lee
Affiliations

Acetylation of RNA processing proteins and cell cycle proteins in mitosis

Carol Chuang et al. J Proteome Res. .

Abstract

Mitosis is a highly regulated process in which errors can lead to genomic instability, a hallmark of cancer. During this phase of the cell cycle, transcription is silent and RNA translation is inhibited. Thus, mitosis is largely driven by post-translational modification of proteins, including phosphorylation, methylation, ubiquitination, and sumoylation. Here, we show that protein acetylation is prevalent during mitosis. To identify proteins that are acetylated, we synchronized HeLa cells in early prometaphase and immunoprecipitated lysine-acetylated proteins with antiacetyl-lysine antibody. The immunoprecipitated proteins were identified by LC-ESI-MS/MS analysis. These include proteins involved in RNA translation, RNA processing, cell cycle regulation, transcription, chaperone function, DNA damage repair, metabolism, immune response, and cell structure. Immunoprecipitation followed by Western blot analyses confirmed that two RNA processing proteins, eIF4G and RNA helicase A, and several cell cycle proteins, including APC1, anillin, and NudC, were acetylated in mitosis. We further showed that acetylation of APC1 and NudC was enhanced by apicidin treatment, suggesting that their acetylation was regulated by histone deacetylase. Moreover, treating mitotic cells with apicidin or trichostatin A induced spindle abnormalities and cytokinesis failure. These studies suggest that protein acetylation/deacetylation is likely an important regulatory mechanism in mitosis.

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Figures

Figure 1
Figure 1
Isolation of acetylated proteins from cells in mitosis. (A) Cell synchronization protocol. HeLa cells were synchronized in mitosis by double thymidine block (DTB) and release into 50 ng/ml nocodazole to enrich for mitotic cells. Phase contrast images show the mitotic population as rounded cells. Images were acquired using a Nikon TE2000 microscope system. Bars, 200 μm. Flow cytometry was performed to determine cell cycle distribution of (i) randomly cycling cells versus (ii) DTB synchronized mitotic cells. Percentage of cells in each cell cycle phase was determined by fitting the DNA content histogram to the Watson Pragmatic model using FlowJo with a root mean squared value of 6.98 and 21.29 for (i) and (ii), respectively. Randomly cycling cells: G1, 53.4%; S, 29.1%; G2/M, 12.5%. DTB synchronized cells: G1, 0.04%; S, 3.03%; G2/M, 83.1%. (B) A representative mitotic sample used for acetyl-lysine immunoprecipitation followed by mass spectrometry. Three samples prepared as in (A) (9 – 15 mg each) from two independent experiments were immunoprecipitated with anti-acetyl lysine polyclonal antibody (Millipore), resolved on SDS-PAGE, and stained with GelCode Blue. 24 gel slices were processed for LC-ESI-MS/MS analysis. HC, IgG heavy chain.
Figure 2
Figure 2
Acetylation of structural proteins in mitosis. Mitotic cell lysates (2 mg), prepared as in Fig. 1, were immunoprecipitated with a second anti-acetyl-lysine monoclonal antibody (Millipore) (Ac-K IP) and immunoblotted with specific antibodies (arrowhead) as shown. (A, left) Histone H3. (B, left) α-tubulin. For confirmation, reciprocal protein-specific immunoprecipitation of H3 (A, right) and α-tubulin (B, right) was performed. Mitotic cell lysates (2 mg) were prepared in a reducing condition as described in Materials and Methods, immunoprecipitated with anti-peptide antibodies against the protein of interest, immunoblotted with a third anti-acetyl-lysine monoclonal antibody (Cell Signaling) to show acetylation (arrowhead), and re-immunoblotted with the same anti-peptide antibodies to show efficiency of immunoprecipitation and protein loading. The data are reproducible in three independent experiments. Input lanes, 20 μg total mitotic cell lysates. Antibody alone was included as an IgG control. *, non-specific band; HC, IgG heavy chain; LC, IgG light chain.
Figure 3
Figure 3
Acetylation of RNA processing proteins in mitosis. The acetylation of RNA processing proteins eIF4G (A) and RNA helicase A (RHA) (B) was demonstrated by acetyl-lysine immunoprecipitation (Ac-K IP) (left panels) and by reciprocal protein-specific immunoprecipitation (right panels) as described in Fig. 2. The data are reproducible in three to six independent experiments. Input lanes, 20 μg total mitotic cell lysates. Antibody alone was included as an IgG control. *, non-specific band(s); HC, IgG heavy chain.
Figure 4
Figure 4
Acetylation of cell cycle proteins in mitosis. The acetylation of cell cycle proteins APC1 (A), anillin (B) and NudC (C) was demonstrated by acetyl-lysine immunoprecipitation (Ac-K IP) (left panels) and by reciprocal protein-specific immunoprecipitation (right panels) as described in Fig. 2. The data are reproducible in three to six independent experiments. Input lanes, 20 μg total mitotic cell lysates. Antibody alone was included as an IgG control. *, non-specific band(s); HC, IgG heavy chain.
Figure 5
Figure 5
Mitotic proteins are regulated by acetylation and deacetylation. Mitotic HeLa cells were synchronized as in Fig. 1 and treated with or without Apicidin (500 nM) for 3.5 h prior to harvest to block protein deacetylation during the peak of mitosis. Cell lysates were prepared as in Fig. 2B, immunoprecipitated with antibodies against each specific protein of interest, immunoblotted with a third anti-acetyl-lysine monoclonal antibody (Cell Signaling) (arrowhead), and re-immunoblotted with protein-specific antibodies to show efficiency of immunoprecipitation and protein loading. (A) Proteins whose acetylation is affected by apicidin treatment during mitosis. (B) Proteins whose acetylation is not affected by apicidin treatment during mitosis. The data are reproducible in three independent experiments.
Figure 6
Figure 6
Protein acetylation plays a role in mitosis and cytokinesis. HeLa cells were synchronized by a double thymidine block and released to enter into mitosis as described in Fig. 1. At 5 h after release at around the G2/M transition, cells were treated with isopropanol solvent control (A), 100 nM apicidin (B, E), 500 nM apicidin (C, F), or 660 nM TSA (D) for an additional 3.5 h during mitosis. Cells were stained with CREST antiserum (green) to label the inner centromere, anti-tubulin antibody (red) to label the mitotic spindle, and counterstained with DAPI (blue) to label the DNA. Tubulin and DNA staining are also shown in black and white for contrast. (E, F) show only DNA in black and white for contrast. Arrows, chromatin bridge in between two divided cells; M, multinucleation; arrowheads, micronucleation. Images were acquired using a Nikon TE2000 microscope system. Bars, 10 μm. (G) Early and late mitotic phenotypes observed in (A – F) were quantified. About 200 cells were counted for each condition for the early mitotic phenotypes (A – D) and 200 cells were counted for each condition for the late mitotic phenotypes (E, F). Api, 100 nM apicidin; TSA, 660 nM TSA. The graph shows the average (± SD) from three independent experiments. *, p < 0.025; **, p < 0.01; ***, p < 0.005; #, p < 0.0025; ##, p < 0.00025.
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