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, 10 (22), 4087-97

p53-Dependent Subcellular Proteome Localization Following DNA Damage

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p53-Dependent Subcellular Proteome Localization Following DNA Damage

François-Michel Boisvert et al. Proteomics.

Abstract

The nucleolus is involved in regulating several aspects of stress responses and cell cycle arrest through the tumor suppressor p53. Under normal conditions, p53 is a short-lived protein that is present in cells at a barely detectable level. Upon exposure of cells to various forms of exogenous stress, such as DNA damage, there is a stabilization of p53 which is then responsible for an ensuing cascade of events. To further investigate the effect of p53 activation, we used a MS-based proteomics method to provide an unbiased, quantitative and high-throughput approach for measuring the subcellular distribution of the proteome that is dependent on p53. The spatial proteomics method analyses a whole cell extract created by recombining differentially labeled subcellular fractions derived from cells in which proteins have been mass labeled with heavy isotopes [Boisvert, F.-M., Lam, Y. W., Lamont, D., Lamond, A. I., Mol. Cell. Proteomics 2010, 9, 457-470]. This was used here to measure the relative distribution between cytoplasm, nucleus and nucleolus of around 2000 proteins in HCT116 cells that are either expressing wild-type p53 or null for p53. Spatial proteomics also facilitates a proteome-wide comparison of changes in protein localization in response to a wide range of physiological and experimental perturbations. We used this method to study differences in protein localization in HCT116 cells either with or without p53, and studied the differences in cellular response to DNA damage following treatment of HCT116 cells with etoposide in both p53 wild-type and null genetic backgrounds.

Figures

Figure 1
Figure 1
Spatial Proteomics Method (A) Human colon carcinoma HCT116 cells were grown in DMEM containing either the normal “light” isotopes of carbon and nitrogen (i.e. 12C14N) (light) or l-arginine-formula image and l-lysine-2H4 (medium) or l-arginine- 13C6-15N4 and l-lysine-13C6-15N2 (heavy). Separate cytoplasmic, nuclear and nucleolar fractions were isolated from each labeled cell population. Equal amounts of total protein from each fraction were then recombined to recreate a whole cell extract, but with Cyto, Nuc and No arising from cells with different isotope labels. The recombined whole cell extract mixture was solubilized with loading buffer, proteins separated using SDS-PAGE and the resulting gel cut into eight equal pieces, trypsin digested and analyzed by LC-MS/MS using an LTQ Orbitrap. To compare different genotype or conditions, cells are analyzed as described above, and changes in localization under different conditions will result in changes in SILAC ratios under normal conditions (A) and following treatment or when studying a different condition (C).
Figure 2
Figure 2
Comparison of HCT116 p53+/+ and p53−/− cells. Graph represents the proteins (x axis) versus the log base two of the SILAC ratio corresponding to the (A), (C) nucleoplasmic/cytoplasmic (medium over light, M/L) or the (B), (D) nucleolar/cytoplasmic (heavy over light, H/L) in the y axis. The plots correspond to the cellular distribution of proteins from WT cells (A and B) or from the p53 knock-out cells (C and D). The order of the proteins shown is sorted according to their localization ratio from the WT cells, and the proteins are displayed in the same order in the p53 knock-out cells.
Figure 3
Figure 3
DNA damage response and p53. Visualization of the spatial proteomics data by graphical representation, plotting the log base two Nucleoplasmic/Cytoplasmic ratio on the x axis and log base two Nucleolar/Cytoplasmic ratio on the y axis of either mock treated (A) wild-type and (B) p53 knock-out cells or of Eto-treated (C) wild-type and (D) p53 knock-out cells.
Figure 4
Figure 4
Effect of p53 on the ribosomal proteins response to DNA damage. Visualization of the spatial proteomics data of ribosomal proteins by graphical representation, plotting the log base two Nucleoplasmic/Cytoplasmic ratio on the x axis and log base two Nucleolar/Cytoplasmic ratio on the y axis of (A) wild-type mock treated (blue) and wild-type Eto-treated cells (red), (B) wild-type mock treated (blue) and p53 knock-out mock treated cells (red), (C) p53 knock-out mock treated (blue) and p53 knock-out Eto-treated cells (red) and (D) wild-type Eto treated (blue) and p53 knock-out Eto-treated cells (red). Visualization of the spatial proteomics data of ribosomal proteins by graphical representation, plotting the log base two Nucleolar/Nucleoplasmic ratio of mock-treated cells on the x axis and of Eto-treated cells on the y axis for the (E) wild-type cells and (F) p53 knock-out cells.
Figure 5
Figure 5
Western blot analysis of ribosomal proteom response. (A) Equal amount (10 μg) of total cell lysates (lanes 1 and 5) or of extracts from the cytoplasmic (Cyto, lanes 2 and 6), nuclear (Nuc, lanes 3 and 7) or nucleolar (No, lanes 4 and 8) fractions of HCT116 wild-type cells (top) or p53 knock-out (bottom) that were either mock-treated (1–4) or treated with 50 μM Eto for 1 h (5–8) were separated by SDS-PAGE and stained with Coomassie Blue. The same fractions were separated by SDS-PAGE, transferred to nitrocellulose prior to Western blotting with an RPL11 antibody (B) or a B23-nucleophosmin antibody (C).
Figure 6
Figure 6
Immunofluorescence analysis of ribosomal protein response. HCT116 wild-type (A–F) and p53 knock-out (G–L) were cultured on coverslips and either mock-treated (A–C and G–I) or treated with 50 μM Eto (D–F and J–L) for 1 h. Cells were then fixed with 3.7% paraformaldehyde in CSK buffer for 10 min, permeabilized and labeled for immunofluorescence using an antibody recognizing RPL11 (B–E–H–K). DNA was visualized using DAPI (A–D–G–J). Scale bars represent 15 μm.
Figure 7
Figure 7
p53-dependent protein induced by DNA damage. Example of two proteins (Phosphoserine Phosphatase and ATP-dependent DNA helicase Q1) whose intensity was increased following Eto treatment in wild-type cells (compare intensity WT versus intensity WTE), but not in p53 knock-out cells (compare intensity p53 versus intensity p53E).

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References

    1. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature. 2000;408:307–310. - PubMed
    1. Prives C. Signaling to p53: breaking the MDM2-p53 circuit. Cell. 1998;95:5–8. - PubMed
    1. Wsierska-Gadek J, Horky M. How the nucleolar sequestration of p53 protein or its interplayers contributes to its (re)-activation. Ann. N. Y. Acad. Sci. 2003;1010:266–272. - PubMed
    1. Bertwistle D, Sugimoto M, Sherr CJ. Physical and functional interactions of the Arf tumor suppressor protein with nucleophosmin/B23. Mol. Cell. Biol. 2004;24:985–996. - PMC - PubMed
    1. Rubbi CP, Milner J. Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses. EMBO J. 2003;22:6068–6077. - PMC - PubMed

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