Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway

Abstract

The gene encoding p53 mediates a major tumor suppression pathway that is frequently altered in human cancers. p53 function is kept at a low level during normal cell growth and is activated in response to various cellular stresses. The MDM2 oncoprotein plays a key role in negatively regulating p53 activity by either direct repression of p53 transactivation activity in the nucleus or promotion of p53 degradation in the cytoplasm. DNA damage and oncogenic insults, the two best-characterized p53-dependent checkpoint pathways, both activate p53 through inhibition of MDM2. Here we report that the human homologue of MDM2, HDM2, binds to ribosomal protein L11. L11 binds a central region in HDM2 that is distinct from the ARF binding site. We show that the functional consequence of L11-HDM2 association, like that with ARF, results in the prevention of HDM2-mediated p53 ubiquitination and degradation, subsequently restoring p53-mediated transactivation, accumulating p21 protein levels, and inducing a p53-dependent cell cycle arrest by canceling the inhibitory function of HDM2. Interference with ribosomal biogenesis by a low concentration of actinomycin D is associated with an increased L11-HDM2 interaction and subsequent p53 stabilization. We suggest that L11 functions as a negative regulator of HDM2 and that there might exist in vivo an L11-HDM2-p53 pathway for monitoring ribosomal integrity.

FIG. 1.

Analysis of HDM2 immunocomplex and identification of HDM2-L11 association. (A) HeLa cells were transiently transfected with plasmid DNA expressing HDM2. At 24 h after transfection, cells were metabolically labeled with [³⁵S]methionine and the lysates were immunoprecipitated with antibody to HDM2. Immunoprecipitated proteins were separated by SDS-PAGE and visualized by autoradiography. The molecular identities of L5 and L11 were determined by protein microsequencing following a preparative anti-HDM2 IP. The identity of the 32-kDa protein (p32) has not yet been established. (B) HDM2-associated p20 corresponds to human ribosomal protein L11. L11 protein contains a putative nuclear export signal (NES) and a nuclear and nucleolar localization signal (NLS/NoLS). Two peptide sequences were obtained from protein microsequencing; both matched perfectly with human L11.

FIG. 2.

HDM2 interacts with ribosomal protein L11. (A) U2OS cells were transiently transfected with plasmid expressing HDM2. Transfected cells were lysed in the presence (+) or absence (−) of RNaseA (10 μg/ml). HDM2-L11/L5 complexes were examined by IP with an α-HDM2 antibody. No obvious effect was observed on L11-Hdm2 association after the RNaseA treatment. (B) L11 associates with HDM2 in vivo. Asynchronously growing human SJSA cells or HDM2-negative Saos-2 cells were lysed and immunoprecipitated with antibodies recognizing L11 and HDM2.

FIG. 3.

L11 binds to a central domain in HDM2. (A) Mapping of L11 binding domain in HDM2. Wild-type HDM2-expressing plasmids were transiently transfected into U2OS cells. At 24 h after transfection, cells were metabolically labeled with [³⁵S]methionine and the lysates were immunoprecipitated with antibody to HDM2. (B) Mapping of HDM2 binding domain in L11. WT, wild type.

FIG. 4.

L11 can form quaternary complexes with HDM2, p53, and ARF. U2OS cells were transiently transfected with (+) or without (−) plasmid DNA expressing HDM2, p53, and HA-ARF as indicated. At 24 h after transfection, cells were lysed and immunoprecipitated with the indicated antibodies followed by immunoblotting.

FIG. 5.

L11 inhibits HDM2-mediated p53 ubiquitination and degradation. (A) U2OS cells were transfected with (+) or without (−) the indicated plasmids. At 36 h after transfection, cells were treated with proteasome inhibitor MG132 (50 μM) for 4 h prior to cell lysis. Clarified cell lysate was immunoprecipitated (IP) with anti-p53 antibody, and washed immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting with anti-HA antibody. Expression of individual transfected proteins was determined by direct immunoblotting (bottom panels). WB, Western blotting. (B) Total lysates prepared from cells transfected with (+) or without (−) the indicated plasmids were electrophoretically separated before immunoblotting with the indicated antibodies was performed.

FIG. 6.

L11 relieves HDM2-mediated repression of p53 transactivation. (A and B) U2OS cells were cotransfected with (+) or without (−) pGL13-Luc reporter plasmids and plasmids expressing indicated proteins. At 24 h after transfection, clarified cell lysates prepared from each transfected cell population were incubated with a luciferase assay buffer and the optical density at 595 nm was determined on a luminometer. The luciferase activity for each sample was normalized to β-galactosidase activity to control for transfection efficiency. The normalized luciferase activity of the pGL2-Basic plasmid was set to 1. (C) Total lysates prepared from cells transfected with the indicated plasmids were electrophoretically separated before immunoblotting with (+) or without (−) the indicated antibodies was performed. CMV, cytomegalovirus.

FIG. 7.

L11 induces a p53-dependent cell cycle arrest. (A) U2OS or Saos-2 cells were transfected with or without (Vector) a plasmid expressing a GFP marker and the indicated proteins. (B) U2OS cells were cotransfected with or without (Vector) a plasmid expressing a GFP marker and the indicated proteins. Transfected cells were sorted, and their cell cycle distribution characteristics were determined by flow cytometry at 24 h after transfection. The proportions of cells in S phase in each transfected cell population were compared using bar graphs. A minimum of 10,000 GFP-positive cells were analyzed for each transfection.

FIG. 8.

Perturbation of ribosomal biogenesis by actinomycin D (ActD) increases L11-HDM2-p53 complex levels. (A and B) WI38 cells were treated with 5 nM actinomycin D for 24 h (+) or left untreated (−) before being lysed for analysis for the expression of various proteins and protein complex formation. Approximately 500 ug of total protein was utilized for each IP. (C) WI38 cells were treated with the proteasome inhibitor MG132 for 6 h before lysis to stabilize HDM2 in the presence (+) or absence (−) of 5 nM actinomycin D. A total of 200 ug of protein was used in each IP. (D) WI38 cells were treated with UV (50 J/m²) (+) at 24 h before harvesting or left untreated (−), and IP was performed as described above.

FIG. 9.

A schematic model for the function of the L11-MDM2-p53 pathway. (See text for discussion.)