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• W2045971301 abstract "Inhibition of the MDM2-p53 feedback loop is critical for p53 activation in response to cellular stresses. The ribosomal proteins L5, L11, and L23 can block this loop by inhibiting MDM2-mediated p53 ubiquitination and degradation in response to ribosomal stress. Here, we show that L11, but not L5 and L23, leads to a drastic accumulation of ubiquitinated and native MDM2. This effect is dependent on the ubiquitin ligase activity of MDM2, but not p53, and requires the central MDM2 binding domain (residues 51–108) of L11. We further show that L11 inhibited 26 S proteasome-mediated degradation of ubiquitinated MDM2 in vitro and consistently prolonged the half-life of MDM2 in cells. These results suggest that L11, unlike L5 and L23, differentially regulates the levels of ubiquitinated p53 and MDM2 and inhibits the turnover and activity of MDM2 through a post-ubiquitination mechanism. Inhibition of the MDM2-p53 feedback loop is critical for p53 activation in response to cellular stresses. The ribosomal proteins L5, L11, and L23 can block this loop by inhibiting MDM2-mediated p53 ubiquitination and degradation in response to ribosomal stress. Here, we show that L11, but not L5 and L23, leads to a drastic accumulation of ubiquitinated and native MDM2. This effect is dependent on the ubiquitin ligase activity of MDM2, but not p53, and requires the central MDM2 binding domain (residues 51–108) of L11. We further show that L11 inhibited 26 S proteasome-mediated degradation of ubiquitinated MDM2 in vitro and consistently prolonged the half-life of MDM2 in cells. These results suggest that L11, unlike L5 and L23, differentially regulates the levels of ubiquitinated p53 and MDM2 and inhibits the turnover and activity of MDM2 through a post-ubiquitination mechanism. The tumor suppressor protein p53 is a transcription factor activated in response to stress to induce expression of its target genes. The proteins encoded by these genes then mediate multiple cellular responses, such as cell cycle arrest, apoptosis, differentiation, cell senescence, or DNA repair (1Oren M. Cell Death Differ. 2003; 10: 431-442Crossref PubMed Scopus (892) Google Scholar). Also, p53 can directly trigger mitochondria-mediated apoptosis in response to DNA damage (2Dumont P. Leu J.I. Della Pietra III, A.C. George D.L. Murphy M. Nat. Genet. 2003; 33: 357-365Crossref PubMed Scopus (1092) Google Scholar, 3Mihara M. Erster S. Zaika A. Petrenko O. Chittenden T. Pancoska P. Moll U.M. Mol. Cell. 2003; 11: 577-590Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar, 4Chipuk J.E. Kuwana T. Bouchier-Hayes L. Droin N.M. Newmeyer D.D. Schuler M. Green D.R. Science. 2004; 303: 1010-1014Crossref PubMed Scopus (1630) Google Scholar). The tumor suppression function of p53 is well reflected in the fact that more than half of human tumors harbor mutations in the p53 gene, and many others retain impaired function of the p53 pathway (5Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5707) Google Scholar, 6Soussi T. Dehouche K. Beroud C. Hum. Mutat. 2000; 15: 105-113Crossref PubMed Scopus (246) Google Scholar). Because of its inhibitory effect on cell growth, p53 is maintained at a low steady-state level and in an inert form in physiological conditions. This duty is mainly fulfilled by the E3 2The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; ARF, alternative reading frame; E1, ubiquitin activating enzyme (UBA); E2, ubiquitin carrier protein; HA, hemagglutinin; Ni-NTA, nickel nitrilotriacetic acid; GFP, green fluorescent protein; MEF, murine embryonic fibroblast cell. 2The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; ARF, alternative reading frame; E1, ubiquitin activating enzyme (UBA); E2, ubiquitin carrier protein; HA, hemagglutinin; Ni-NTA, nickel nitrilotriacetic acid; GFP, green fluorescent protein; MEF, murine embryonic fibroblast cell. ubiquitin ligase MDM2 that mediates p53 constant degradation through a ubiquitin-dependent proteasome pathway (7Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3629) Google Scholar, 8Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2798) Google Scholar). The mdm2 gene itself is a downstream target of p53, thus forming a tight autoregulatory feedback loop (9Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1165) Google Scholar, 10Picksley S.M. Lane D.P. BioEssays. 1993; 15: 689-690Crossref PubMed Scopus (171) Google Scholar, 11Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1614) Google Scholar). Consistent with this notion, gene amplification and overexpression of MDM2 have also been shown in a variety of tumors, particularly in soft tissue sarcomas, lymphomas, and breast and lung cancers (12Momand J. Jung D. Wilczynski S. Niland J. Nucleic Acids Res. 1998; 26: 3453-3459Crossref PubMed Scopus (802) Google Scholar, 13Deb S.P. Mol. Cancer Res. 2003; 1: 1009-1016PubMed Google Scholar, 14Cordon-Cardo C. Latres E. Drobnjak M. Oliva M.R. Pollack D. Woodruff J.M. Marechal V. Chen J. Brennan M.F. Levine A.J. Cancer Res. 1994; 54: 794-799PubMed Google Scholar, 15Dworakowska D. Jassem E. Jassem J. Peters B. Dziadziuszko R. Zylicz M. Jakobkiewicz-Banecka J. Kobierska-Gulida G. Szymanowska A. Skokowski J. Roessner A. Schneider-Stock R. Lung Cancer. 2004; 43: 285-295Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 16Watanabe T. Ichikawa A. Saito H. Hotta T. Leuk. Lymphoma. 1996; 21: 391-397Crossref PubMed Scopus (63) Google Scholar). Interfering with the MDM2-p53 feedback loop leads to p53 activation, ultimately preventing neoplasia. One example of this regulation is alternative reading frame (ARF) (p14ARF in human, p19ARF in mouse)-mediated inhibition of this loop in response to overexpression of oncogenes such as c-Myc and RAS (17Palmero I. Pantoja C. Serrano M. Nature. 1998; 395: 125-126Crossref PubMed Scopus (540) Google Scholar, 18Zindy F. Eischen C.M. Randle D.H. Kamijo T. Cleveland J.L. Sherr C.J. Roussel M.F. Genes Dev. 1998; 12: 2424-2433Crossref PubMed Scopus (1053) Google Scholar). Also, in response to DNA-damaging agents, N-terminal serine/threonine phosphorylation at the MDM2 binding domain of p53 prevents MDM2-p53 interaction and activates p53 (19Banin S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1688) Google Scholar, 20Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1684) Google Scholar, 21Maya R. Balass M. Kim S.T. Shkedy D. Leal J.F. Shifman O. Moas M. Buschmann T. Ronai Z. Shiloh Y. Kastan M.B. Katzir E. Oren M. Genes Dev. 2001; 15: 1067-1077Crossref PubMed Scopus (523) Google Scholar, 22Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (707) Google Scholar, 23Schon O. Friedler A. Bycroft M. Freund S.M. Fersht A.R. J. Mol. Biol. 2002; 323: 491-501Crossref PubMed Scopus (269) Google Scholar). Hence, the MDM2-p53 loop presents as a central regulatory point for the cellular response to a multitude of environmental as well as internal stressors. Increasing evidence shows that the MDM2-p53 feedback loop can also be regulated by ribosomal stress. Fine coordination between ribosomal biogenesis and other cellular functions such as the cell cycle and differentiation is important for normal cell growth (24Rudra D. Warner J.R. Genes Dev. 2004; 18: 2431-2436Crossref PubMed Scopus (175) Google Scholar, 25Moss T. Curr. Opin. Genet. Dev. 2004; 14: 210-217Crossref PubMed Scopus (131) Google Scholar). Impeding ribosomal biogenesis would generate ribosomal stress that activates p53 to stop cell growth. Examples of such stress include inhibition of ribosomal RNA (rRNA) synthesis and processing by a low dose (<5 nm) of actinomycin D or overexpression of a dominant-negative mutant of the rRNA processing factor Bop1 (26Ashcroft M. Taya Y. Vousden K.H. Mol. Cell. Biol. 2000; 20: 3224-3233Crossref PubMed Scopus (318) Google Scholar, 27Pestov D.G. Strezoska Z. Lau L.F. Mol. Cell. Biol. 2001; 21: 4246-4255Crossref PubMed Scopus (281) Google Scholar). Malfunctions of ribosomal biogenesis have also been proposed to correlate to human cancers (28Ruggero D. Pandolfi P.P. Nat. Rev. Cancer. 2003; 3: 179-192Crossref PubMed Scopus (764) Google Scholar). Recently, we and others reported that ribosomal proteins L5, L11, and L23 interacted with MDM2 and inhibited the MDM2-p53 feedback loop in response to ribosomal stress, such as treatment with low dose actinomycin D, serum starvation, or possibly small interference RNA-induced reduction of L23 (29Zhang Y. Wolf G.W. Bhat K. Jin A. Allio T. Burkhart W.A. Xiong Y. Mol. Cell. Biol. 2003; 23: 8902-8912Crossref PubMed Scopus (441) Google Scholar, 30Lohrum M.A. Ludwig R.L. Kubbutat M.H. Hanlon M. Vousden K.H. Cancer Cell. 2003; 3: 577-587Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 31Jin A. Itahana K. O'Keefe K. Zhang Y. Mol. Cell. Biol. 2004; 24: 7669-7680Crossref PubMed Scopus (300) Google Scholar, 32Dai M.S. Lu H. J. Biol. Chem. 2004; 279: 44475-44482Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar, 34Bhat K.P. Itahana K. Jin A. Zhang Y. EMBO J. 2004; 23: 2402-2412Crossref PubMed Scopus (206) Google Scholar). Interestingly, these L proteins as well as the tumor suppressor protein ARF are primarily nucleolar proteins. Disruption of the nucleolus appears to be a common event in stress-induced p53 activation pathways (35Rubbi C.P. Milner J. EMBO J. 2003; 22: 6068-6077Crossref PubMed Scopus (635) Google Scholar). Thus, releasing small protein molecules such as the ribosomal L proteins from the nucleolus leads to p53 activation in response to ribosomal stress. Although ectopic expression of L5, L11, or L23 can inhibit MDM2-mediated p53 ubiquitination and degradation (29Zhang Y. Wolf G.W. Bhat K. Jin A. Allio T. Burkhart W.A. Xiong Y. Mol. Cell. Biol. 2003; 23: 8902-8912Crossref PubMed Scopus (441) Google Scholar, 30Lohrum M.A. Ludwig R.L. Kubbutat M.H. Hanlon M. Vousden K.H. Cancer Cell. 2003; 3: 577-587Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 31Jin A. Itahana K. O'Keefe K. Zhang Y. Mol. Cell. Biol. 2004; 24: 7669-7680Crossref PubMed Scopus (300) Google Scholar, 32Dai M.S. Lu H. J. Biol. Chem. 2004; 279: 44475-44482Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar), the detailed mechanisms underlying this effect remain undetermined. Also, little is known about the effect of these L proteins on MDM2 stability and ubiquitination. We have begun to address these issues by performing a series of cellular and biochemical analyses. In this study, we found that unlike L5 and L23, which drastically inhibited ubiquitination of both p53 and MDM2, L11 slightly inhibited MDM2-mediated p53 ubiquitination but markedly increased the ubiquitinated species and the steady-state level of MDM2 in cells. This effect was dependent on the ubiquitin ligase activity of MDM2, but not p53, and required the central MDM2 -binding domain of L11. Interestingly, L11 inhibited proteasome-mediated degradation of ubiquitinated MDM2 in vitro. These results suggest that L11 differentially regulates the levels of ubiquitinated p53 and MDM2 through a post-ubiquitination and proteasome-dependent mechanism. Cell Lines, Plasmids, and Antibodies—Human lung small cell adenocarcinoma H1299 cells, human osteosarcoma U2OS cells, and mouse p53–/–/mdm2–/– MEFs were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 50 units/ml penicillin, and 0.1 mg/ml streptomycin at 37 °C in a 5% CO2 humidified atmosphere as previously described (33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar). The FLAG-tagged L5, L11, and L23 expression plasmids have been described previously (30Lohrum M.A. Ludwig R.L. Kubbutat M.H. Hanlon M. Vousden K.H. Cancer Cell. 2003; 3: 577-587Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 32Dai M.S. Lu H. J. Biol. Chem. 2004; 279: 44475-44482Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar). All the deletion mutants of L11 were generated using PCR and cloned into pcDNA3-2-FLAG vector. The HA-MDM2 expression vector has been described (33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar). The full-length p14ARF (ARF, hereafter) was amplified using PCR and inserted into the pcDNA3-V5 vector at BamHI and EcoRI sites to generate pcDNA3-V5-ARF. The primers were 5′-CGCGGATTCATGGTGCGCAGGTTCTTGGTG-3′ and 5′-CCGGAATTCTCAGCCAGGTCCACGGGCAGAC-3′. The MDM2 mutant with a point mutation at position 464 from cysteine to alanine (MDM2C464A) was generously provided by Dr. Karen H. Vousden (36Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar). Anti-FLAG (Sigma), anti-p21 (NeoMarkers), anti-MDM2 (N20), and anti-p53 (DO-1, Santa Cruz) were purchased. Anti-MDM2 (2A10) and anti-HA (12CA5) have been described (33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar). Purified Proteins for in Vitro Ubiquitination Assay—Recombinant FLAG-MDM2 full-length protein was purified from baculovirus-infected SF9 insect cells as described (37Grossman S.R. Deato M.E. Brignone C. Chan H.M. Kung A.L. Tagami H. Nakatani Y. Livingston D.M. Science. 2003; 300: 342-344Crossref PubMed Scopus (381) Google Scholar). His-tagged L11, L5, and L23 were expressed in Escherichia coli and purified through a nickel nitrilotriacetic acid (Ni-NTA, Qiagen) column as previously described (32Dai M.S. Lu H. J. Biol. Chem. 2004; 279: 44475-44482Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). Purified rabbit ubiquitin activating enzyme (UBA) E1, purified human recombinant E2 (UbcH5a), and recombinant human ubiquitin were purchased from Boston Biochem. Cotransfection, Immunoblot, and Co-immunoprecipitation Analyses—H1299, U2OS, or p53–/–/mdm2–/– MEFs cells were transfected with plasmids as indicated in each figure legend using Lipofectin following the manufacturer's protocol (Invitrogen). Cells were harvested at 48 h post-transfection and lysed in lysis buffer consisting of 50 mm Tris/HCl (pH 8.0), 0.5% Nonidet P-40, 1 mm EDTA, 150 mm NaCl, and 1 mm phenylmethylsulfonyl fluoride. Equal amounts of clear cell lysate were used for immunoblot analysis as described previously (33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar). Immunoprecipitation was conducted using antibodies as indicated in the figure legends and described previously (33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar). Beads were washed with lysis buffer twice, once with SNNTE buffer (50 mm Tris/HCl (pH 7.4), 5 mm EDTA, 1% Nonidet P-40, 500 mm NaCl, and 5% sucrose), and once with radioimmune precipitation assay buffer (50 mm Tris/HCl (pH 7.4), 150 mm NaCl, 1% Triton X-100, 0.1% SDS, and 1% (w/v) sodium deoxycholate). Bound proteins were detected by immunoblot using antibodies as indicated in the figure legends. In Vivo Ubiquitination Assay—In vivo ubiquitination assays were conducted as previously described (33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar). Briefly, H1299 cells or p53–/–/mdm2–/– MEFs (60% confluence/100-mm plate) were transfected with combinations of the following plasmids as indicated in the figure legends: His6-ubiquitin (2 μg), p53 (1 μg), HA-MDM2 (2 μg), FLAG-L5 (2 μg), FLAG-L11 (2 μg), FLAG-L23 (2 μg), V5-ARF (2 μg) using Lipofectin (for H1299 cells), or Lipofectamine 2000 (for p53–/–/mdm2–/– MEFs) (Invitrogen). For inhibition of proteasome-mediated protein degradation, the cells were treated with 20 μm MG132 for 8 h before harvest. Forty-eight hours after transfection cells from each plate were harvested and split into two aliquots, one for immunoblot and the other for ubiquitination assays. Cell pellets were lysed in buffer I (6 m guanidinium-HCl, 0.1 mol/liter Na2HPO4/NaH2PO4, 10 mmol/liter Tris-HCl (pH 8.0), 10 mmol/liter β-mercaptoethanol) and incubated with Ni-NTA beads at room temperature for 4 h. Beads were washed once each with buffer I, buffer II (8 mol/liter urea, 0.1 mol/liter Na2HPO4/NaH2PO4, 10 mmol/liter Tris-HCl (pH 8.0), 10 mmol/liter β-mercaptoethanol), and buffer III (8 mol/liter urea, 0.1 mol/liter Na2HPO4/NaH2PO4, 10 mmol/liter Tris-HCl (pH 6.3), 10 mmol/liter β-mercaptoethanol). Proteins were eluted from the beads in buffer IV (200 mmol/liter imidazole, 0.15 mol/liter Tris-HCl (pH 6.7), 30% (v/v) glycerol, 0.72 mol/liter β-mercaptoethanol, and 5% (w/v) SDS). Eluted proteins were analyzed by immunoblot with monoclonal anti-p53 (DO-1), anti-HA, or anti-MDM2 (2A10) antibodies. In Vitro Ubiquitination Reactions—MDM2 in vitro auto-ubiquitination reactions were performed in ubiquitination buffer (25 mm Hepes (pH 7.4), 10 mm NaCl, 3 mm MgCl2, 0.05% TritonX-100, freshly added 0.5 mm dithiothreitol) with 100 ng of UBA-E1, 25 ng of UbcH5a, and 5 ng of ubiquitin and were incubated for 120 min at 37 °C. A total of 10 ng of purified FLAG-MDM2 was used in each reaction. The mixture was either resolved by a SDS-PAGE gel followed by immunoblot using anti-MDM2 antibody or subjected to proteasome-dependent degradation assays. In Vitro 26 S Proteasome-dependent Degradation Assay—A total of 1 μg of purified 26 S proteasome (human erythrocytes, BIOMOL) preincubated with proteasome inhibitors (10 μm MG132, 10 μm lactacystin, 10 μm clasto-lactacystin-β-lactone) for 30 min at 4 °C where indicated was added to ubiquitination reactions along with 3 mm Mg-ATP (Boston Biochem) and further incubated at 37 °C for 5 h. To test the effect of L11, L5, and L23 on proteasome-dependent degradation of MDM2, ubiquitinated MDM2 was preincubated with purified L11, L5, and L23 protein for 30 min at 4 °C in ubiquitination buffer before the addition to the degradation reaction. Differential Effect of L11, L5, L23, and ARF on the Levels of Ubiquitinated MDM2 and p53—In an attempt to elucidate the mechanisms by which ribosomal proteins regulate the stability of MDM2 and p53, we determined the effect of ribosomal proteins L5, L11, and L23 on the ubiquitination of MDM2 and p53 in p53-deficient H1299 cells using ARF as a positive control for a factor known to stabilize p53 when exogenously introduced into cells. The cells were transfected with plasmids encoding MDM2 and p53 alone or with either one of the L proteins or ARF. As a control, the cells transfected with MDM2 and p53 only were treated with 20 μm concentrations of the proteasome inhibitor MG132 for 8 h before harvesting. The cells were harvested 48 h after transfection, and in vivo ubiquitination assays were conducted as described under “Materials and Methods.” As shown in Fig. 1A and as expected (36Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar, 38Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1576) Google Scholar), cotransfection of MDM2 with p53 resulted in p53 ubiquitination and drastic reduction of its protein level (compare lane 3 to lane 2). Consistent with our previous results (32Dai M.S. Lu H. J. Biol. Chem. 2004; 279: 44475-44482Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar), ectopic expression of L5 and L23 markedly inhibited the ubiquitination of both p53 and MDM2 and consequently protected p53 degradation by MDM2 (lanes 5 and 7 of Figs. 1, A and B). However, overexpression of L11 dramatically increased the ubiquitinated species of p53 and MDM2 as well as their protein levels (lane 6 of Figs. 1, A and B). This effect was similar to that of ARF (lane 4 of Figs. 1, A and B) as reported previously (39Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar). The enhancement of the level of ubiquitinated MDM2 and p53 species as well as their protein levels by L11 and ARF was not due to the variation in transfection efficiency because the GFP protein level in each transfectant was equivalent (Fig. 1C). Of note, the effect of L11 and ARF on stabilizing p53 and MDM2 was much more prominent than that of L5 and L23 (Fig. 1, A and B). Interestingly, the effect of L11 and ARF on the ubiquitination of MDM2 and p53 was similar to that of MG132 treatment (lane 8, Fig. 1, A and B), although to different extents. L11 was more effective than MG132 in enhancing the level of ubiquitinated MDM2 species. This might be due to the specific inhibition of 26 S proteasome-mediated degradation of ubiquitinated MDM2 by L11 (see Fig. 3C). These results suggest that these ribosomal proteins may utilize different mechanisms to regulate the MDM2 and p53 ubiquitination and proteasomal pathways although all these nucleolar proteins can stabilize p53 upon overexpression (Fig. 1).FIGURE 3L11 inhibits 26 S proteasome-mediated degradation of ubiquitinated MDM2 in vitro. A, in vitro auto-ubiquitination of MDM2. 10 ng of purified FLAG-MDM2 was incubated in the ubiquitination reaction in the presence or absence of E1, UbcH5a (E2), and ubiquitin (Ub). Ubiquitinated MDM2 species (MDM2-(His-Ub)n) were detected by immunoblot using anti-MDM2 antibody. B, MDM2 ubiquitination is required for its proteasome degradation. Purified FLAG-MDM2 (10 ng) was incubated with buffer or purified 26 S proteasome, and then the reactions were assayed by anti-MDM2 immunoblot (N-20). C, L11 inhibits 26 S proteasome-mediated degradation of ubiquitinated MDM2. In vitro ubiquitinated FLAG-MDM2 (Ub-MDM2) was preincubated with or without His-L11, His-L5, or His-L23. 26 S proteasome (1 μg) or proteasome inhibitor (10 μm MG132, 10 μm lactacystin, 10 μm clasto-lactacystin-β-lactone)-treated 26 S proteasome was then added. Reactions were quenched in SDS-PAGE sample buffer and analyzed by immunoblot using anti-MDM2 (N20) antibody. A representative experiment was shown. Similar results were obtained in three independent experiments. The asterisk indicates nonspecific anti-N20 antibody-reacting bands.View Large Image Figure ViewerDownload Hi-res image Download (PPT) However, we were unsure whether the changes in ubiquitinated p53 or MDM2 species induced by the ribosomal L proteins were due to the direct effect of these proteins on the ubiquitin ligase activity of MDM2. To determine the effect of the L proteins and ARF on the MDM2 ubiquitin ligase activity with p53 and MDM2 as substrates, we performed a set of transfections similar to that in Fig. 1, but 20 μm MG132 was used to block proteasomal degradation so that we could compare ubiquitination at similar protein levels. As shown in Fig. 2, L5 and L23 again dramatically inhibited ubiquitination of both p53 and MDM2 (lanes 5 and 7, Fig. 2, A and B), indicating that these two L proteins may directly inhibit MDM2 ubiquitin ligase activity as previously described (32Dai M.S. Lu H. J. Biol. Chem. 2004; 279: 44475-44482Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 33Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (399) Google Scholar). By striking contrast, L11, like ARF (39Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar), which slightly reduced MDM2-mediated ubiquitinated species of p53 (lanes 4 and 6, Fig. 2A), still markedly increased the ubiquitinated species of MDM2 (lanes 4 and 6, Fig. 2B). This difference was not due to different transfection efficiencies among the different conditions, as all of the exogenous proteins were expressed equally well (Fig. 2C). These results suggest that L11 differentially regulates the levels of ubiquitinated species of p53 and MDM2, causing decreased p53 ubiquitinated species but increased MDM2 ubiquitinated species. L11 Inhibits in Vitro Degradation of Ubiquitinated MDM2 by 26 S Proteasome—Because overexpression of L11, but not L5 and L23, led to similar enhancing effects on the levels of ubiquitinated MDM2 as that of MG132 (Fig. 1) or overexpression of ARF (39Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar), we next wanted to determine whether L11, like MG132, could block proteasome-mediated degradation of ubiquitinated MDM2. To test this possibility, we performed an in vitro MDM2 autoubiquitination reaction as shown in Fig. 3A. In the presence of E1, E2, and ubiquitin, MDM2 efficiently ubiquitinated itself (lane 3) as expected (40Honda R. Yasuda H. EMBO J. 1999; 18: 22-27Crossref PubMed Scopus (610) Google Scholar, 41Midgley C.A. Desterro J.M. Saville M.K. Howard S. Sparks A. Hay R.T. Lane D.P. Oncogene. 2000; 19: 2312-2323Crossref PubMed Scopus (227) Google Scholar). To test if ubiquitination of MDM2 is required for its degradation by 26 S proteasome, we incubated purified MDM2 with 26 S proteasome or buffer and then assayed for the level of MDM2. As shown in Fig. 3B, purified MDM2 is not degraded by 26 S proteasome, indicating that ubiquitination is essential for proteasomal-mediated degradation of MDM2. Next, the ubiquitinated MDM2 was then used for 26 S proteasome-mediated degradation assays. As shown in Fig. 3C, the addition of purified 26 S proteasome caused a marked decrease in the amounts of MDM2-ubiquitin conjugates as well as a decrease of native (non-modified) MDM2 (compare lane 2 to lane 1). These effects were efficiently blocked by the addition of MG132 (lane 3), suggesting that the ubiquitinated MDM2 can be efficiently degraded through the proteasome system in our assays. Consistent with the above results, L11 (lanes 4–5), but not L5 (lanes 6–7) and L23 (lanes 8–9), drastically inhibited the degradation of ubiquitinated MDM2 by the 26 S proteasome. These results suggest that L11 directly blocked 26 S proteasome-mediated degradation of MDM2-ubiquitin conjugates. These results explain the accumulation of ubiquitinated species of MDM2 by L11 in cells (Fig. 2). L11 Also Stabilizes p53 in Cells—It has been shown that ectopic expression of L11 leads to elevated p53 protein levels and transcriptional activity in cells (29Zhang Y. Wolf G.W. Bhat K. Jin A. Allio T. Burkhart W.A. Xiong Y. Mol. Cell. Biol. 2003; 23: 8902-8912Crossref PubMed Scopus (441) Google Scholar, 30Lohrum M.A. Ludwig R.L. Kubbutat M.H. Hanlon M. Vousden K.H. Cancer Cell. 2003; 3: 577-587Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). Consistently, we also observed that both endogenous p53 and MDM2 proteins were dramatically increased upon L11 overexpression in p53 proficient U2OS cells (Fig. 4A). However, the effect of L11 on p53 stability has not been determined. Therefore, we transfected U2OS cells with or without FLAG-L11 plasmids. Forty-eight hours after transfection, the cells were treated with cyclohexamide to stop protein synthesis in cells. The cells were then harvested at different time points and subjected to immunoblot to determine endogenous p53 levels (Fig. 4B). The protein levels were determined by measuring the intensity of each band and normalized with expression of GFP. As shown in Figs. 4, B and C, the half-life of p53 prolonged from ∼25 min in empty vector transfected cells to more than 2 h in the cells overexpressing FLAG-L11. These results are consistent with the notion that L11 inhibited MDM2-mediated p53 ubiquitination (Fig. 2) and demonstrate that the increased level of p53 in the presence of ectopically expressed L11 is due to p53 stabilization by this ribosomal protein. We also observed that L11 significantly increased the half-life of MDM2 in the same assay (data not shown). Therefore, L11 can also stabilize MDM2 as well. Increment of Ubiquitinated Species and Stability of MDM2 by L11 Is p53-independent—L11 can directly bind to MDM2 (32Dai M.S. Lu H. J. Biol. Chem. 2004; 279: 44475-44482Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar) but not p53 (data not shown). Therefore, it is likely that stabilization of p53 by L11 is through the inhibitory effect of L11 on MDM2. However, is the L11-induced increase in ubiquitinated species and stability of MDM2 dependent on p53? To address this issue, we examined the effect of L11 on the half-life of MDM2 and its autoubiquitination in p53-deficient H1299 cells. Indeed, as shown in Fig. 5, A and B, the half-life of ectopic MDM2 protein increased from ∼20 min in the cells transfected with a control vector to ∼2 h in the FLAG" @default.
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• W2045971301 date "2006-08-01" @default.
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• W2045971301 title "Regulation of the MDM2-p53 Pathway by Ribosomal Protein L11 Involves a Post-ubiquitination Mechanism" @default.
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