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. 2013 Jan 17;32(3):388-96.
doi: 10.1038/onc.2012.63. Epub 2012 Mar 5.

Ribosomal protein S14 unties the MDM2-p53 loop upon ribosomal stress

X Zhou  1 , Q Hao, J Liao, Q Zhang, H Lu
Affiliations

Ribosomal protein S14 unties the MDM2-p53 loop upon ribosomal stress

X Zhou et al. Oncogene. .

Abstract

The MDM2-p53 feedback loop is crucially important for restricting p53 level and activity during normal cell growth and proliferation, and is thus subjected to dynamic regulation in order for cells to activate p53 upon various stress signals. Several ribosomal proteins, such as RPL11, RPL5, RPL23, RPL26 or RPS7, have been shown to have a role in regulation of this feedback loop in response to ribosomal stress. Here, we identify another ribosomal protein S14, which is highly associated with 5q-syndrome, as a novel activator of p53 by inhibiting MDM2 activity. We found that RPS14, but not RPS19, binds to the central acidic domain of MDM2, similar to RPL5 and RPL23, and inhibits its E3 ubiquitin ligase activity toward p53. This RPS14-MDM2 binding was induced upon ribosomal stress caused by actinomycin D or mycophenolic acid. Overexpression of RPS14, but not RPS19, elevated p53 level and activity, leading to G1 or G2 arrest. Conversely, knockdown of RPS14 alleviated p53 induction by these two reagents. Interestingly, knockdown of either RPS14 or RPS19 caused a ribosomal stress that led to p53 activation, which was impaired by further knocking down the level of RPL11 or RPL5. Together, our results demonstrate that RPS14 and RPS19 have distinct roles in regulating the MDM2-p53 feedback loop in response to ribosomal stress.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. RPS14 interacts with MDM2 in cells
(A) (B) Exogenous RPS14 interacts with exogenous MDM2 in 293 cells. Cells were transfected with HA-MDM2, Flag-RPS14, or Flag-RPS19 plasmids and harvested for IP (0.5 mg total proteins) using anti-Flag, anti-HA, or mouse IgG, followed by IB with the antibodies as indicated. (C) Endogenous RPS14 interacts with endogenous MDM2 in HCT116 cells. Cells lysates (1 mg) were prepared from HCT116 cells after treatment with Act D for 8 h. IP was conducted with anti-RPS14 or rabbit IgG followed by IB with anti-MDM2 or anti-RPS14. (D) Exogenous RPS14 does not interact with exogenous MDMX in H1299 cells. Cells were transfected with combinations of HA-MDM2, Myc-MDMX, Flag-S14 or Flag-L11 plasmids and harvested for co-IP-IB assays using the antibodies as indicated. All the experiments were repeated more than 2 times and true to the rest of data.
Figure 2
Figure 2. Mapping the MDM2-RPS14 binding domains
(A) RPS14 directly binds to the central acidic domain of MDM2 in vitro. Purified GST, GST-MDM2, or GST-MDM2-fragments immobilized on glutathione beads were incubated with 500 ng of bacterially purified His-RPS14. Bound proteins were blotted with anti-His. Arrows indicate the GST-MDM2 or GST-MDM2-fragments. (B) RPS14 binds to the central acidic domain of MDM2 in cells. Cells were transfected with Flag-RPS14, V5-MDM2, or deletion mutants of V5-MDM2 plasmids and harvested for IP (0.5 mg total proteins) with anti-V5, followed by IB with the antibodies as indicated. Arrows indicate the V5-MDM2, V5-MDM2-fragments or heavy chains (HC). (C) MDM2 binds to the C-terminus of RPS14 in cells. Cells were transfected with HA-MDM2, Flag-RPS14, or deletion mutants of Flag-RPS14 plasmids and harvested for IP (0.5 mg total proteins) with anti-Flag, followed by IB with the antibodies as indicated. (D) Schematic presentation of the MDM2 domain(s) that binds to RPS14. (E) Schematic presentation of the RPS14 domain(s) that binds to MDM2.
Figure 3
Figure 3. RPS14 stabilizes p53 by inhibiting MDM2-mediated p53 ubiquitination
(A) Ectopic expression of RPS14 inhibits MDM2-mediated p53 ubiquitination in cells. H1299 cells were transfected with combinations of plasmids encoding p53, HA-MDM2 or Flag-RPS14 in the presence of the His-Ub plasmid. Cells were treated with MG132 for 8 h before harvesting. Cell lysates were subjected to an ubiquitination assay followed by IB with anti-p53. The expression levels of p53, HA-MDM2 and Flag-RPS14 are shown in the lower panels. (B) Ectopic expression of RPS14 stabilizes exogenous p53 in H1299 cells. Cells were transfected with combinations of p53, HA-MDM2 or Flag-RPS14 in the presence of pEGFP plasmids as a control and harvested 36 h post-transfection for IB with antibodies as indicated. (C) RPS14 stabilizes endogenous p53 in A549 cell. Cells were transfected with an increasing amount of the Flag-RPS14 plasmid and harvested 36 h after transfection for IB with antibodies as indicated. (D) RPS19 does not stabilize endogenous p53 in A549 cell. The assay as that in panel C was conducted except the RPS19 plasmid was used here. (E) RPS14 extends the half-life of endogenous p53. A549 cells were transfected with pcDNA3 or Flag-RPS14 and treated with cycloheximide (CHX) at 36 h post-transfection, and harvested at different time points for IB with antibodies as indicated. (F) A graphic presentation of the result from panel E. (G) wild-type RPS14 and its C-terminus (amino acids 51–151), but not its N-terminus (aa 1–51), drastically induced p53 in A549 cells. Cells were transfected with pcDNA3, Flag-RPS14/1–50, Flag-RPS14/51–151, or Flag-RPS14 plasmids and harvested 36 h after transfection for IB with antibodies as indicated. (H) RPS14 slightly induces a p53 apoptotic target, Bax, but not the cleaved-Parp. A549 cells were transfected with pcDNA3 or Flag-S14 plasmids and harvested for IB with antibodies as indicated.
Figure 4
Figure 4. RPS14 induces p53-dependent cell cycle arrest
(A) A549 cells were transfected with pcDNA3 or Flag-RPS14 and harvested 30 h after transfection for FACS analysis. The mean percentage of cells arrested in the G1 and G2 phases obtained from three separate experiments is presented. (B) H1299 cells were transfected with pcDNA3 or Flag-RPS14 and harvested 30 h after transfection for FACS analysis. The mean percentage of cells in each phases obtained from three separate experiments is presented. The same experiments as shown in (A) and (B) were performed in HCT116p53+/+ cells (C) and HCT116p53−/− cells (D) except the pEGFP and pEGFP-RPS14 plasmids were used here. The mean percentages of cells in each phases obtained from three separate experiments are presented. (E) RPS14 induced p53-dependent growth arrest was determined by BrdU incorporation assays (left panel). Cells were transfected with pcDNA3 or Flag-RPS14 and treated with 10 µM BrdU for 5 h, followed by BrdU and DAPI staining. The percentage of BrdU-positive cells is shown in the right panel. (F) (G) RPS14 inhibits cell growth as measured in cell viability assays. A549 cells (F) or H1299 (G) cells were transfected with pcDNA3 or Flag-S14 plasmids and seeded into 96-well plates at 24 h post-transfection. WST-8 was added to each well and cells were incubated at 37 °C for additional 2–3 h before absorbance at 450 nm was measured using a microplate reader every 24 h during 5-days long culture. Bars indicate standard deviations. “*” indicates P<0.05, “**” indicates P<0.005.
Figure 5
Figure 5. Regulation of p53 activity by knocking down endogenous RPS14 or RPS19
(A) and (B) RPS14 knockdown activates p53 but also inhibits ribosomal stress-induced p53 activation. HCT116p53+/+ cells (A) and A549 cells (B) were transfected with scrambled siRNAs or RPS14 siRNAs. Cells were treated with 5 nM actinomycin D (Act D) or 10 µM mycophenolic acid (MPA) before harvesting for IB with antibodies as indicated. (C) RPS14 knockdown results in RPL5- and RPL11-dependent p53 activation. A549 cells were transfected with combinations of scrambled siRNAs, RPS14 siRNAs, RPL5 siRNAs or RPL11 siRNAs. Expression of p53, p21, RPS14, RPL11 and β-actin were detected by IB using antibodies as indicated. Messenger RNA levels of RPL5 and GAPDH were detected by RT-PCR. (D) Knockdown of RPS19 results in RPL5- and RPL11-dependent p53 activation. The same experiment as shown in panel C was conducted except the RPS19 siRNAs were used here. Expression of p53, MDM2, RPS19, RPL11 and β-actin were detected by IB using antibodies as indicated. Messenger RNA levels of RPL5 and GAPDH were detected by RT-PCR. (E) (F) Knockdown of RPS14 causes p53-dependent cell cycle arrest. HCT116p53+/+ cells (E) and HCT116p53−/− cells (F) were transfected with scrambled siRNAs or RPS14 siRNAs for 48 h, followed by FACS analysis. The mean percentages of cells in each phase obtained from three separate experiments are presented. Bars indicate standard deviations. “*” indicates P<0.05. (G) A schematic model for p53 activation by general ribosomal stress (upper panel) or 40S subunit perturbation (lower panel).
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