Inhibition of HDM2 and activation of p53 by ribosomal protein L23 - PubMed (original) (raw)

Inhibition of HDM2 and activation of p53 by ribosomal protein L23

Aiwen Jin et al. Mol Cell Biol. 2004 Sep.

Abstract

The importance of coordinating cell growth with proliferation has been recognized for a long time. The molecular basis of this relationship, however, is poorly understood. Here we show that the ribosomal protein L23 interacts with HDM2. The interaction involves the central acidic domain of HDM2 and an N-terminal domain of L23. L23 and L11, another HDM2-interacting ribosomal protein, can simultaneously yet distinctly interact with HDM2 together to form a ternary complex. We show that, when overexpressed, L23 inhibits HDM2-induced p53 polyubiquitination and degradation and causes a p53-dependent cell cycle arrest. On the other hand, knocking down L23 causes nucleolar stress and triggers translocation of B23 from the nucleolus to the nucleoplasm, leading to stabilization and activation of p53. Our data suggest that cells may maintain a steady-state level of L23 during normal growth; alternating the levels of L23 in response to changing growth conditions could impinge on the HDM2-p53 pathway by interrupting the integrity of the nucleolus.

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Figures

FIG. 1.

HDM2 interacts with ribosomal protein L23. (A) Mass spectrometry identification of HDM2 binding proteins. Extracts of U2OS cells infected with the indicated Ad for 2 days were immunoprecipitated with HDM2 antibody 4B11 and were resolved on a silver-stained gel (SDS-12.5% PAGE). Three ribosomal proteins, L5, L11, and L23, were identified based on peptide sequences obtained from the mass spectrometry. αHDM2, anti-HDM2; IgG, immunoglobulin G; K, thousands. (B) Binding between ectopically expressed HDM2 and L23. HeLa cells were transfected with indicated plasmid DNA for 24 h. Each cell extract was immunoprecipitated (IP) with antibodies to either HDM2 (4B11, left panel) or myc (9E10, right panel), and the precipitates were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with antibodies to HDM2 (N20; Santa Cruz) and myc (A14; Santa Cruz). αmyc, anti-myc; +, present; −, absent. (C) Binding between endogenous HDM2 and L23. Endogenous HDM2 and L23 binding was detected by coIP from SJSA cells with antibodies to HDM2 (4B11) and L23, and Western blotting (WB) was performed as described above. Saos2 cells were served as a negative control. αL23, anti-L23.

FIG. 2.

Mapping of the HDM2 domain for L23 binding and the L23 domain for HDM2 binding. (A) Mapping of the HDM2 domain for L23 binding. Extracts from U2OS cells transfected with the indicated plasmid DNA encoding deletion mutants of HDM2 were immunoprecipitated with HDM2 antibodies (4B11 for lanes 1, 6, and 7; SMP14 for lane 2; 2A10 for lanes 3 and 5), and the precipitates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with a mixture of two rabbit anti-HDM2 (α-HDM2) antibodies (N20 and H228; Santa Cruz). A diagram of each deletion mutant is shown. WT, wild type; α-myc, anti-myc; WB, Western blotting; +, present; −, absent. (B) Mapping of the L23 domain for HDM2 binding. Extracts from U2OS cells transfected with the indicated plasmid DNA encoding deletion mutants of L23 were immunoprecipitated with HDM2 antibody 4B11, and the precipitates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with antibodies to HDM2 (N20) and myc (A14) as indicated. A diagram for the deletion mutants is shown.

FIG. 3.

L23 and L11 simultaneously interact with HDM2 to form ternary complexes. Extracts from U2OS cells infected with Ad expressing HDM2 for 2 days were immunoprecipitated (IP) with L23 antibodies, and Western blotting (WB) was performed with antibodies to HDM2 (4B11), L11, and L23. Endog, endogenous; α-L23, anti-L23; α-L11, anti-L11; α-HDM2, anti-HDM2.

FIG. 4.

L23 inhibits HDM2-mediated p53 polyubiquitination and degradation. (A) Ectopic expression of L23 stabilizes HDM2 and p53. U2OS cells were transfected with the indicated plasmid DNA for 2 days, and cell extracts were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with antibodies as indicated. Plasmid DNA expressing GFP was cotransfected as a control. +, present; −, absent; α-HDM2, anti-HDM2; α-p53, anti-p53; α-myc, anti-myc; α-GFP, anti-GFP. (B) Ectopic expression of L23 stabilizes HDM2 and p53 in normal human fibroblast cells. WI38 cells were infected with virus expressing HDM2 for 2 days, and cell extracts were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with antibodies as indicated. Virus expressing GFP was coinfected as a control. α-actin, anti-actin. (C) L23 inhibits HDM2-mediated p53 polyubiquitination. U2OS cells were transfected with the indicated plasmid DNA for 2 days, and the cells were treated with MG132 (25 μM) for 5 h before lysing. Cell extracts were analyzed by Western blotting with antibodies to p53 (D01) and myc (9E10) as indicated. (D) L23 stabilizes HDM2 independent of p53. WI38-E6 cells were infected with viruses expressing GFP, HDM2, and myc-L23 as indicated. Cells were lysed 2 days after infection, and the cell lysates were blotted as described above. Endog, endogenous; α-L23, anti-L23.

FIG. 5.

L23 induces a p53-dependent cell cycle arrest. (A and B) L23 overexpression stabilizes and activates p53. Normal human fibroblast WI38 cells and isogenic mutant WI38-E6 cells were infected with the indicated Ad for 2 days. Western blotting was performed as described above. α-myc, anti-myc; α-HDM2, anti-HDM2; α-p53, anti-p53; α-p21, anti-p21; α-actin, anti-actin. (C and D) L23 induces a p53-dependent cell cycle arrest. WI38 and WI38-E6 cells were infected with the indicated viruses. Cells were harvested 2 days after infection, fixed with 70% ethanol for 2 h, and stained with propidium iodide for 1 h, and the cell cycle distribution was determined by flow cytometry. Cell populations in the S phase are indicated as percentages of total cells. (E) L23 interacts with HDM2 in the nucleoplasm. U2OS cells were singly infected with Ad expressing myc-L23 for 2 days. Cells were then fixed with 3% paraformaldehyde for 10 min and immunostained with a rabbit anti-myc antibody (9E10) and a mouse anti-HDM2 antibody (N20). Nuclei were visualized by 4′,6′-diamidino-2-phenylindole (DAPI) staining. Fluorescence images were captured with a cooled charge-coupled device color digital camera (model 2.2.0; Diagnostic) on an Olympus IX70 inverted microscope equipped with the appropriate fluorescence filters.

FIG. 6.

Knocking down L23, but not L11, activates p53 and induces a cell cycle arrest. (A and B) U2OS cells were either untreated (Buffer) or transfected with a control scrambled RNA duplex (siScr), L23 siRNA (siL23), or L11 siRNA (siL11) for 2 days. Cell extracts were collected and analyzed by Western blotting with the indicated antibodies. α-HDM2, anti-HDM2; α-p53, anti-p53; α-p21, anti-p21; α-L23, anti-L23; α-actin, anti-actin. (C and D) U2OS cells were transfected with siRNA as described for panels A and B. Cells were harvested 2 days after transfection, fixed with ethanol, and stained with propidium iodide, and their cell cycle distribution was determined by flow cytometry. Percentages of cells in S phase are shown. The averages of the results from two independent experiments are shown as bar graphs.

FIG. 7.

Down-regulation of L23-induced cell cycle arrest is dependent on the function of p53. (A and B) Normal human fibroblast WI38 cells and isogenic mutant WI38-E6 cells were transfected with either a control scrambled RNA duplex (siScr) or L23 siRNA (siL23) for 2 days, and cell extracts were analyzed by Western blotting with the indicated antibodies. α-HDM2, anti-HDM2; α-p53, anti-p53; α-p21, anti-p21; α-L23, anti-L23; α-actin, anti-actin. (C and D) WI38 and WI38-E6 cells were transfected siRNA as described for panels A and B. Cells were harvested 2 days after infection and stained with propidium iodide, and their cell cycle distribution was determined by flow cytometry. Percentages of cells in S phase are shown. (E) Down-regulation of L23 releases nucleolar B23. U2OS cells were transfected with the indicated siRNA for 2 days. The cells were then fixed and stained with a mouse anti-B23 (α-B23) antibody (Zymed) and an fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch). Fluorescence images were captured with a cooled charge-coupled device color digital camera (model 2.2.0; Diagnostic) on an Olympus IX70 inverted microscope equipped with the appropriate fluorescence filters.

FIG. 8.

Inhibition of ribosomal biogenesis decreases the protein level of L23. (A) Low concentrations of actinomycin D induce p53-dependent cell cycle arrest. U2OS cells were treated with the indicated concentrations of actinomycin D (Act D) for 24 h, and the cell lysates were analyzed by Western blotting as described above. α-HDM2, anti-HDM2; α-p53, anti-p53; α-L23, anti-L23; α-actin, anti-actin. (B) Time required for actinomycin D treatment to suppress L23. U2OS cells were treated with 5 nM actinomycin D for the indicated times, and the protein levels were analyzed as described above. (C) Inhibition of ribosomal biogenesis by 5 nM actinomycin D down-regulates L23 but not L11. U2OS cells were treated with 5 nM actinomycin D for 24 h before lysing, the cell lysates were resolved by SDS-PAGE, and Western blotting was performed as described above. α-L11, anti-L11. (D) Ectopic expression of L23 suppresses endogenous L23. U2OS cells were infected with the indicated viruses for 2 days, and cell extracts were harvested and resolved by SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and blotted with the indicated antibodies. α-myc, anti-myc. (E) Suppression of endogenous L23 by ectopically expressed myc-L23 was independent of HDM2 and p53. Normal human fibroblast WI38 cells were infected with the indicated viruses for 2 days. Cell extracts were harvested and resolved by SDS-PAGE, and the proteins were analyzed as described above. +, present; −, absent.

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Inhibition of HDM2 and activation of p53 by ribosomal protein L23 - PubMed (original) (raw)