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• W2020124633 abstract "MDM2 associates with ribosomal protein S7, and this interaction is required to inhibit MDM2's E3 ligase activity, leading to stabilization of MDM2 and p53. Notably, the MDM2 homolog MDMX facilitates the inhibition of MDM2 E3 ligase activity by S7. Further, ablation of S7 inhibits MDM2 and p53 accumulation induced by different stress signals in some cell types. Thus, ribosomal/nucleolar stress is likely a key integrating event in DNA damage signaling to p53. Interestingly, S7 is itself a substrate for MDM2 E3 ligase activity both in vitro and in vivo. An S7-ubiquitin fusion protein (S7-Ub) selectively inhibits MDM2 degradation of p53 and is unaffected by MDMX. S7-Ub promotes apoptosis to a greater extent than S7 alone. This indicates that MDM2 ubiquitination of S7 is involved in sustaining the p53 response. Thus, S7 functions as both effector and affector of MDM2 to ensure a proper cellular response to different stress signals. MDM2 associates with ribosomal protein S7, and this interaction is required to inhibit MDM2's E3 ligase activity, leading to stabilization of MDM2 and p53. Notably, the MDM2 homolog MDMX facilitates the inhibition of MDM2 E3 ligase activity by S7. Further, ablation of S7 inhibits MDM2 and p53 accumulation induced by different stress signals in some cell types. Thus, ribosomal/nucleolar stress is likely a key integrating event in DNA damage signaling to p53. Interestingly, S7 is itself a substrate for MDM2 E3 ligase activity both in vitro and in vivo. An S7-ubiquitin fusion protein (S7-Ub) selectively inhibits MDM2 degradation of p53 and is unaffected by MDMX. S7-Ub promotes apoptosis to a greater extent than S7 alone. This indicates that MDM2 ubiquitination of S7 is involved in sustaining the p53 response. Thus, S7 functions as both effector and affector of MDM2 to ensure a proper cellular response to different stress signals. MDM2 is a crucial negative regulator of the p53 tumor suppressor protein (Brooks and Gu, 2006Brooks C.L. Gu W. p53 ubiquitination: Mdm2 and beyond.Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, Poyurovsky and Prives, 2006Poyurovsky M.V. Prives C. Unleashing the power of p53: lessons from mice and men.Genes Dev. 2006; 20: 125-131Crossref PubMed Scopus (93) Google Scholar, Toledo and Wahl, 2006Toledo F. Wahl G.M. Regulating the p53 pathway: in vitro hypotheses, in vivo veritas.Nat. Rev. Cancer. 2006; 6: 909-923Crossref PubMed Scopus (1000) Google Scholar). Multiple functional domains of MDM2 are involved in p53 regulation. The N terminus of MDM2 directly binds to the transactivation domain of p53, leading to inhibiton of p53 target gene transcription. The C-terminal RING domain is required for the activity of MDM2 as an E3 ubiquitin ligase promoting ubiquitination and proteasomal degradation of p53 and itself. Additionally, the central region of MDM2 is required for its ability to degrade p53 in response to various stress signals. Multiple mechanisms including different posttranslational modifications, changes in subcellular localization, and modulation of MDM2 stability are employed to rapidly uncouple the MDM2-p53 interaction and consequently stabilize and activate p53, leading to the transcriptional regulation of a variety of genes including the mdm2 gene itself (Meek and Knippschild, 2003Meek D.W. Knippschild U. Posttranslational modification of MDM2.Mol. Cancer Res. 2003; 1: 1017-1026PubMed Google Scholar). Thus, MDM2 forms an autoregulatory feedback loop with p53 to control both the level and the activity of p53. Besides p53, an increasing number of proteins have been shown to interact with MDM2 protein, functioning either as affectors (regulating MDM2 function) or effectors (regulated by MDM2 function) or both (Ganguli and Wasylyk, 2003Ganguli G. Wasylyk B. p53-independent functions of MDM2.Mol. Cancer Res. 2003; 1: 1027-1035PubMed Google Scholar, Iwakuma and Lozano, 2003Iwakuma T. Lozano G. MDM2, an introduction.Mol. Cancer Res. 2003; 1: 993-1000PubMed Google Scholar). MDM2-interacting proteins can thus have controlling effects on the MDM2/p53 circuit through their association with MDM2. At the same time, in a p53-independent manner, MDM2 is able to participate in various cellular functions through its interaction with proteins such as RB, Numb, p21, and others, thereby contributing to cellular responses to different stimuli (Ganguli and Wasylyk, 2003Ganguli G. Wasylyk B. p53-independent functions of MDM2.Mol. Cancer Res. 2003; 1: 1027-1035PubMed Google Scholar). Adding to the complexity of the p53/Mdm2 circuitry is the fact that the homolog of MDM2, MDMX (also called MDM4), plays an important role in facilitating MDM2 downregulation of p53 (Marine et al., 2007Marine J.C. Dyer M.A. Jochemsen A.G. MDMX: from bench to bedside.J. Cell Sci. 2007; 120: 371-378Crossref PubMed Scopus (186) Google Scholar). While itself lacking E3 ligase activity, MDMX can both repress p53 transcriptionally and form oligomers with MDM2 that modulate its E3 ligase activity (Gu et al., 2002Gu J. Kawai H. Nie L. Kitao H. Wiederschain D. Jochemsen A.G. Parant J. Lozano G. Yuan Z.M. Mutual dependence of MDM2 and MDMX in their functional inactivation of p53.J. Biol. Chem. 2002; 277: 19251-19254Crossref PubMed Scopus (201) Google Scholar, Sharp et al., 1999Sharp D.A. Kratowicz S.A. Sank M.J. George D.L. Stabilization of the MDM2 oncoprotein by interaction with the structurally related MDMX protein.J. Biol. Chem. 1999; 274: 38189-38196Crossref PubMed Scopus (225) Google Scholar, Stad et al., 2001Stad R. Little N.A. Xirodimas D.P. Frenk R. van der Eb A.J. Lane D.P. Saville M.K. Jochemsen A.G. Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms.EMBO Rep. 2001; 2: 1029-1034Crossref PubMed Scopus (180) Google Scholar) Normally cytoplasmic, upon DNA damage, MDMX is recruited to the nucleus where it is degraded by MDM2, presumably to augment the function and stability of p53 under such conditions (Kawai et al., 2003Kawai H. Wiederschain D. Kitao H. Stuart J. Tsai K.K. Yuan Z.M. DNA damage-induced MDMX degradation is mediated by MDM2.J. Biol. Chem. 2003; 278: 45946-45953Crossref PubMed Scopus (140) Google Scholar, LeBron et al., 2006LeBron C. Chen L. Gilkes D.M. Chen J. Regulation of MDMX nuclear import and degradation by Chk2 and 14–3-3.EMBO J. 2006; 25: 1196-1206Crossref PubMed Scopus (92) Google Scholar, Pereg et al., 2005Pereg Y. Shkedy D. de Graaf P. Meulmeester E. Edelson-Averbukh M. Salek M. Biton S. Teunisse A.F. Lehmann W.D. Jochemsen A.G. Shiloh Y. Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage.Proc. Natl. Acad. Sci. USA. 2005; 102: 5056-5061Crossref PubMed Scopus (137) Google Scholar). Relevant to this study, an important connection between ribosomal stress and the MDM2/p53 circuit has been revealed. Three ribosomal large-subunit proteins, L5, L11, and L23, were shown to interact with and regulate MDM2 activity (Bhat et al., 2004Bhat K.P. Itahana K. Jin A. Zhang Y. Essential role of ribosomal protein L11 in mediating growth inhibition-induced p53 activation.EMBO J. 2004; 23: 2402-2412Crossref PubMed Scopus (199) Google Scholar, Dai and Lu, 2004Dai M.S. Lu H. Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5.J. Biol. Chem. 2004; 279: 44475-44482Crossref PubMed Scopus (402) Google Scholar, Dai et al., 2004Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition.Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (386) Google Scholar, Jin et al., 2004Jin A. Itahana K. O'Keefe K. Zhang Y. Inhibition of HDM2 and activation of p53 by ribosomal protein L23.Mol. Cell. Biol. 2004; 24: 7669-7680Crossref PubMed Scopus (289) Google Scholar, Lohrum et al., 2003Lohrum M.A. Ludwig R.L. Kubbutat M.H. Hanlon M. Vousden K.H. Regulation of HDM2 activity by the ribosomal protein L11.Cancer Cell. 2003; 3: 577-587Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, Marechal et al., 1994Marechal V. Elenbaas B. Piette J. Nicolas J.C. Levine A.J. The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes.Mol. Cell. Biol. 1994; 14: 7414-7420Crossref PubMed Scopus (283) Google Scholar). Each, when overexpressed, can activate p53 by inhibiting MDM2-mediated p53 degradation and, when ablated by siRNA knockdown, can attenuate the p53 response to low-dose actinomycin D (ActD) and 5-fluorouracil (5-FU) treatments. In fact, cancer-associated missense mutations (C305F, C308Y) disrupt the interaction of MDM2 with L5 and L11, and these mutant MDM2 proteins are impaired in undergoing nuclear export, proteasomal degradation, and promoting p53 degradation (Lindstrom et al., 2007aLindstrom M.S. Deisenroth C. Zhang Y. Putting a finger on growth surveillance: insight into MDM2 zinc finger-ribosomal protein interactions.Cell Cycle. 2007; 6: 434-437Crossref PubMed Scopus (51) Google Scholar). Furthermore, exogenous L11 stimulates MDMX polyubiquitination by MDM2, while knockdown of L11 by siRNA reduces the ability of ActD to downregulate MDMX (Gilkes et al., 2006Gilkes D.M. Chen L. Chen J. MDMX regulation of p53 response to ribosomal stress.EMBO J. 2006; 25: 5614-5625Crossref PubMed Scopus (103) Google Scholar). Ribosomal proteins are not the only nucleolar-associated proteins that have been shown to interact with MDM2. Nucleophosmin (Colombo et al., 2002Colombo E. Marine J.C. Danovi D. Falini B. Pelicci P.G. Nucleophosmin regulates the stability and transcriptional activity of p53.Nat. Cell Biol. 2002; 4: 529-533Crossref PubMed Scopus (418) Google Scholar, Kurki et al., 2004Kurki S. Peltonen K. Latonen L. Kiviharju T.M. Ojala P.M. Meek D. Laiho M. Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation.Cancer Cell. 2004; 5: 465-475Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) and nucleolin (Saxena et al., 2006Saxena A. Rorie C.J. Dimitrova D. Daniely Y. Borowiec J.A. Nucleolin inhibits Hdm2 by multiple pathways leading to p53 stabilization.Oncogene. 2006; 25: 7274-7288Crossref PubMed Scopus (68) Google Scholar) have also been reported to bind and regulate MDM2 functions. Further, one of the mechanisms by which the nucleolar-associated p19ARF protein can counteract the repressive effect of MDM2 is to relocalize it to nucleoli (Lohrum et al., 2000Lohrum M.A. Ashcroft M. Kubbutat M.H. Vousden K.H. Identification of a cryptic nucleolar-localization signal in MDM2.Nat. Cell Biol. 2000; 2: 179-181Crossref PubMed Scopus (168) Google Scholar, Weber et al., 1999Weber J.D. Taylor L.J. Roussel M.F. Sherr C.J. Bar-Sagi D. Nucleolar Arf sequesters Mdm2 and activates p53.Nat. Cell Biol. 1999; 1: 20-26Crossref PubMed Scopus (772) Google Scholar). Here we have identified ribosomal small subunit protein 7 (S7) as an MDM2-interacting protein that is required for regulating the stability of MDM2. There are several features of our observations with S7 that go beyond previous findings with other ribosomal proteins. First, its requirement for counteracting MDM2 is very broad, since virtually every stress inducer we have tested requires S7. Second, there is an important and selective role for MDMX in this process. Third, S7 is itself a substrate of MDM2, and S7 ubiquitination may serve to extend the p53 stress response and facilitate cell death. Our results strengthen the hypothesis that interference with ribosomal and nucleolar integrity is the key integrator of the p53-MDM2 stress response. During our studies, another group reported that S7 binds MDM2 and described the consequences of their interaction (Chen et al., 2007Chen D. Zhang Z. Li M. Wang W. Li Y. Rayburn E.R. Hill D.L. Wang H. Zhang R. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function.Oncogene. 2007; 26: 5029-5037Crossref PubMed Scopus (194) Google Scholar). Similarities and differences between their results and ours are addressed in the Discussion. Using a construct expressing MDM2 (amino acids 4–491) as bait, a high-throughput yeast two-hybrid screen was carried out to identify MDM2-interacting proteins. Multiple clones identified from three different cDNA libraries encoded the ribosomal protein S7. The interaction between MDM2 and S7 was confirmed by coimmunoprecipitation of the two proteins from extracts of H1299 (Figure 1A) or U2OS (data not shown) cells transfected with constructs expressing S7 and MDM2. The association of endogenous S7 and MDM2 was also demonstrated by using cell lysates from HCT116 cells (Figure 1B). Levels of MDM2 detected in α-S7 immunoprecipitates were increased when cells were treated with a low dose of ActD or with the proteasome inhibitor MG132. Combining ActD and MG132 did not further enhance the interaction between S7 and MDM2 (Figure 1C). Although the increased association of S7 and MDM2 is likely due to the increased cellular MDM2 levels caused by these treatments, it is also possible that relocalization of S7 or MDM2 in response to these agents is involved in this response. Indeed, MDM2 associates with nucleoli in response to MG132 treatment (Klibanov et al., 2001Klibanov S.A. O'Hagan H.M. Ljungman M. Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress.J. Cell Sci. 2001; 114: 1867-1873Crossref PubMed Google Scholar, Latonen et al., 2003Latonen L. Kurki S. Pitkanen K. Laiho M. p53 and MDM2 are regulated by PI-3-kinases on multiple levels under stress induced by UV radiation and proteasome dysfunction.Cell. Signal. 2003; 15: 95-102Crossref PubMed Scopus (31) Google Scholar) (data not shown). In addition, while ectopic S7 accumulated in nucleoli in unstressed U2OS cells, a low dose of ActD (Figure 1D) or daunorubicin (data not shown) led to redistribution of a significant proportion of S7 into the nucleoplasm (Figure 1D). p53 was also found in α-S7 immunoprecipitates, suggesting that it formed a complex with S7 and MDM2. Further, using a series of Flag-MDM2 mutants coexpressed with Myc-S7 in H1299 cells, a sequence within amino acids 273–339 of MDM2 was found to be required for S7 binding (Figure 1E and see Figure S1 available online). This region is within the central portion of MDM2 that is required for its degradation of p53 and also overlaps sequences shown previously to interact with other ribosomal proteins, L5, L11, and L23 (Bhat et al., 2004Bhat K.P. Itahana K. Jin A. Zhang Y. Essential role of ribosomal protein L11 in mediating growth inhibition-induced p53 activation.EMBO J. 2004; 23: 2402-2412Crossref PubMed Scopus (199) Google Scholar, Dai and Lu, 2004Dai M.S. Lu H. Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5.J. Biol. Chem. 2004; 279: 44475-44482Crossref PubMed Scopus (402) Google Scholar, Dai et al., 2004Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition.Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (386) Google Scholar, Jin et al., 2004Jin A. Itahana K. O'Keefe K. Zhang Y. Inhibition of HDM2 and activation of p53 by ribosomal protein L23.Mol. Cell. Biol. 2004; 24: 7669-7680Crossref PubMed Scopus (289) Google Scholar, Lohrum et al., 2003Lohrum M.A. Ludwig R.L. Kubbutat M.H. Hanlon M. Vousden K.H. Regulation of HDM2 activity by the ribosomal protein L11.Cancer Cell. 2003; 3: 577-587Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, Marechal et al., 1994Marechal V. Elenbaas B. Piette J. Nicolas J.C. Levine A.J. The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes.Mol. Cell. Biol. 1994; 14: 7414-7420Crossref PubMed Scopus (283) Google Scholar). To elucidate the impact of S7 on MDM2, we coexpressed S7 with MDM2 and p53 in U2OS cells. MDM2-mediated degradation of p53 was greatly inhibited by S7 (Figure 2A). More significantly, the levels of endogenous p53, MDM2, and p21 in U2OS cells were elevated following S7 expression (Figure 2B). Consistent with this observation, overexpression of S7 led to cell-cycle arrest in U2OS cells (Figure S2). The increase in MDM2 was not solely due to augmentation of its expression resulting from increased p53 protein, because expression of S7 also resulted in stabilization of MDM2 in H1299 cells that lack p53 (Figure 2C). The ability of S7 to inhibit MDM2-induced p53 degradation is most likely related to its ability to inhibit both MDM2-mediated p53 ubiquitination and MDM2 autoubiquitination in vivo (Figure 2D). Under some circumstances, the ability of S7 to relocalize MDM2 to nucleoli (in a relatively small proportion of the cells) may also contribute to its inhibitory effects (Figure S3). To gain further insight into S7 inhibition of the E3 ligase activity of MDM2, an in vitro ubiquitination assay was carried out by using purified S7, MDM2, and p53 proteins. S7 was found to inhibit MDM2-mediated p53 ubiquitination but had no impact on MDM2 autoubiquitination in vitro (Figure 3B compare lanes 1 and 2 with lanes 5 and 6, and see Figure 6A, below). We considered the possibility that MDMX might be required for S7 to block MDM2 autoubiquitination. In fact, when we compared the autoubiquitination of MDM2 alone with that of a purified preformed MDM2/MDMX complex (see Figure 3A), S7 was only able to inhibit the heteromeric complex (Figure 3B, compare lanes 3 and 4 with lanes 7 and 8). Furthermore, coexpression of MDMX and S7 led to markedly greater stabilization of MDM2 than either of these two proteins alone (Figure 3C). We next evaluated how MDM2 is regulated by endogenously expressed S7. To approach this, we examined the effect of downregulation of S7 on the function of MDM2 and p53 using two different siRNAs (Figure 4). In both U2OS and SAOS-2 cells, even though S7 could be lowered to more than half of its original level, there was no discernable effect on cell proliferation under the conditions used, i.e., with up to 200 nM siRNA and 48 hr after siRNA treatment (data not shown). Furthermore, ablation of S7 in unstressed U2OS cells did not lead to increased levels of p53 and MDM2 (Figure 4A). It was shown previously that low doses of ActD or 5-FU trigger ribosomal stress and consequent activation of p53 (Bhat et al., 2004Bhat K.P. Itahana K. Jin A. Zhang Y. Essential role of ribosomal protein L11 in mediating growth inhibition-induced p53 activation.EMBO J. 2004; 23: 2402-2412Crossref PubMed Scopus (199) Google Scholar, Dai et al., 2004Dai M.S. Zeng S.X. Jin Y. Sun X.X. David L. Lu H. Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition.Mol. Cell. Biol. 2004; 24: 7654-7668Crossref PubMed Scopus (386) Google Scholar, Gilkes et al., 2006Gilkes D.M. Chen L. Chen J. MDMX regulation of p53 response to ribosomal stress.EMBO J. 2006; 25: 5614-5625Crossref PubMed Scopus (103) Google Scholar, Lindstrom et al., 2007bLindstrom M.S. Jin A. Deisenroth C. White Wolf G. Zhang Y. Cancer-associated mutations in the MDM2 zinc finger domain disrupt ribosomal protein interaction and attenuate MDM2-induced p53 degradation.Mol. Cell. Biol. 2007; 27: 1056-1068Crossref PubMed Scopus (106) Google Scholar, Sun et al., 2007Sun X.X. Dai M.S. Lu H. 5-fluorouracil activation of p53 involves an MDM2-ribosomal protein interaction.J. Biol. Chem. 2007; 282: 8052-8059Crossref PubMed Scopus (124) Google Scholar). When we introduced S7 siRNAs into U2OS cells, ActD-induced p53 stabilization was attenuated, and the levels of MDM2 were significantly decreased (Figure 4A). Incubating cells with MG132 abrogated this effect, showing that S7 modulates proteasome-mediated p53 and MDM2 degradation. In HCT116 p53−/− cells S7 reduction by siRNA also resulted in destabilized MDM2 after ActD treatment, indicating that the lower levels of MDM2 were due to its increased rate of degradation (Figure 4B). This was further supported by a cycloheximide half-life assay in U2OS cells which showed that S7 ablation significantly increased MDM2's turnover (Figures 4C and 4D). These results indicate that normal levels of S7 are critical to MDM2 stability under conditions of ribosomal stress. It should be mentioned that these observations were not obtained in all cell lines tested. In some cell lines, S7 ablation by siRNA led to p53-dependent and -independent cell-cycle arrest (Figure S4 and data not shown). We do not know the basis for cell-specific responses to reduced S7 but speculate that in some cells the ensuing nucleolar stress may lead to relocalization of proteins that are capable of counteracting MDM2 and thereby stabilizing p53 even in the absence of S7, while in others (e.g., U2OS and SAOS-2), such proteins either do not accumulate to sufficiently high levels or other mechanisms are involved. We anticipated that agents specifically known to induce ribosomal stress would require S7 to inhibit MDM2 and stabilize p53. It was therefore surprising that DNA-damaging agents that elicit a variety of different genotoxic assaults including doxorubicin, daunorubicin, hydoxylurea, deferoxamine mesylate, and neocarzinostatin gave essentially the same response of reducing MDM2 in response to ablation of S7 (Figures 5A and 5B). Stress-induced p53 stabilization was also attenuated by the various agents (to different extents) after S7 ablation. Note that these agents work by various distinct mechanisms and cause arrest at different stages of the cell cycle. We also ablated L11 using a previously reported siRNA (Bhat et al., 2004Bhat K.P. Itahana K. Jin A. Zhang Y. Essential role of ribosomal protein L11 in mediating growth inhibition-induced p53 activation.EMBO J. 2004; 23: 2402-2412Crossref PubMed Scopus (199) Google Scholar). As previously shown, downregulation of L11 led to decreased levels of p53 in U2OS cells even under unstressed condition and attenuated the impact of low-dose ActD- or 5-FU-induced p53 stabilization. Interestingly, like S7 ablation, L11 depletion also led to the inhibition of stress-induced MDM2 stabilization for all the other drugs that were tested (Figures 5A and 5B). This indicates that S7 and L11 do not compensate for each other in stabilization of MDM2. Based on these results, we propose that modulation of MDM2 function by MDM2-interacting ribosomal proteins is a common if not universal mechanism employed by cells in response to multiple stress stimuli. A large-scale analysis of the human ubiquitin-related proteome by mass spectrometric analysis revealed that many ribosomal proteins including S7 are ubiquitinated (Matsumoto et al., 2005Matsumoto M. Hatakeyama S. Oyamada K. Oda Y. Nishimura T. Nakayama K.I. Large-scale analysis of the human ubiquitin-related proteome.Proteomics. 2005; 5: 4145-4151Crossref PubMed Scopus (149) Google Scholar). Based on its apparently strong interaction with MDM2, we tested whether S7 can serve as a substrate for the E3 ligase activity of MDM2. Indeed, S7 was ubiquitinated by MDM2 in a dose-dependent manner in vitro (Figure 6A). Remarkably, under the conditions used, while S7 could inhibit p53 ubiquitination, p53 had no impact on the ability of MDM2 to ubiquitinate S7. In line with this, ubiquitination of ectopic S7 was also increased by coexpressed MDM2 in vivo both in H1299 and mouse 2KO (p53−/−, mdm2−/−) cells (Figure 6B). S7 ubiquitination by MDM2 requires the interaction between S7 and MDM2, as no ubiquitinated S7 species were detected in 2KO cells with a deletion mutant MDM2 (Δ23) that cannot bind S7 (Figure 6B). Moreover, ubiquitination of endogenous S7 was increased when U2OS cells were treated with either ActD or daunorubicin (Figure 6C). Similar results were obtained for HCT116 cells treated with ActD (data not shown). Since only polyubiquitinated S7 species were detected when cells were treated with these agents (Figure 6C), it is likely that polyubiquitination of S7 occurs rapidly, and there is very little steady-state monoubiquitinated S7. To examine the involvement of endogenous MDM2 in the ubiquitination and degradation of endogenous S7, we employed two different approaches using mouse embryonic fibroblasts (MEFs) from wild-type and Mdm2/p53 double null (2KO) mice. In the first case, we transfected His-tagged ubiquitin into the MEFs and performed Ni-NTA pull-downs from cell extracts followed by immunoblotting for S7. We detected a subtle but reproducible increase in endogenous polyubiquitinated S7 species after ActD treatment in wild-type, but not in 2KO cells (Figure 6D shows two separate experiments). Second, we subjected lysates of untreated or ActD-treated wild-type and 2KO MEFs to an in vitro degradation assay (Rape and Kirschner, 2004Rape M. Kirschner M.W. Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry.Nature. 2004; 432: 588-595Crossref PubMed Scopus (233) Google Scholar) in which extracts were supplemented with ubiquitin and UbcH5c known to be the E2 that works with MDM2 to degrade p53 (Saville et al., 2004Saville M.K. Sparks A. Xirodimas D.P. Wardrop J. Stevenson L.F. Bourdon J.C. Woods Y.L. Lane D.P. Regulation of p53 by the ubiquitin-conjugating enzymes UbcH5B/C in vivo.J. Biol. Chem. 2004; 279: 42169-42181Crossref PubMed Scopus (104) Google Scholar) (Figures 6E and 6F). We reasoned that if MDM2's E3 ligase activity is indeed involved in S7 ubiquitination, the addition of ubiquitin and specific E2 proteins (UbcH5c in this case) to cell extracts would very likely to drive S7 toward ubiquitin-mediated degradation. As shown in Figure 6E, extracts of ActD-treated wild-type MEF cells were capable of degrading endogenous S7, while extracts of untreated cells were unable to do so. Note that both wild-type cell extracts were able to degrade endogenous p53 in a UbcH5c-dependent manner. Extracts of 2KO cells were not able to degrade S7 significantly even when cells were treated with ActD (Figure 6F). Taken together, our data indicate that endogenous MDM2 plays a role in ubiqutination and degradation of endogenous S7. However, since a polypeptide species corresponding to monoubiquitinated S7 (indicated by an asterisk) was detected upon addition of the E2 even in 2KO cells, this indicates that MDM2 is not the only E3 ubiquitin ligase for S7. Note that cell lysates were centrifuged as part of the procedure leading to depletion of the rough-ER-associated ribosomes, so only a fraction of the total ribosomes are present in the reaction mixtures. ActD treatment disrupts nucleoli, and this may yield a lot more available free S7 for degradation. To gain further insight into the significance of S7 ubiquitination by MDM2, we followed an approach previously demonstrated to be instructive for understanding the impact of p53 ubiquitination in which a p53-ubiquitin fusion protein was utilized (Carter et al., 2007Carter S. Bischof O. Dejean A. Vousden K.H. C-terminal modifications regulate MDM2 dissociation and nuclear export of p53.Nat. Cell Biol. 2007; 9: 428-435Crossref PubMed Scopus (179) Google Scholar, Li et al., 2003Li M. Brooks C.L. Wu-Baer F. Chen D. Baer R. Gu W. Mono- versus polyubiquitination: differential control of p53 fate by Mdm2.Science. 2003; 302: 1972-1975Crossref PubMed Scopus (603) Google Scholar, Sasaki et al., 2007Sasaki M. Nie L. Maki C.G. MDM2 binding induces a conformational change in p53 that is opposed by heat-shock protein 90 and precedes p53 proteasomal degradation.J. Biol. Chem. 2007; 282: 14626-14634Crossref PubMed Scopus (46) Google Scholar). An S7-ubiquitin fusion protein (S7-Ub) was generated in which a single ubiquitin is joined to the C terminus of S7. When S7-Ub was introduced into H1299 cells, a fraction of it was retained in the cytoplasm and was extensively polyubiquitinated (Figures S5A and S5B). The portion of S7-Ub that localized to nuclei was largely unpolyubiquitinated. Importantly, when the impacts of S7 and S7-Ub on p53 and MDM2 protein levels were compared, while both versions of S7 could stabilize p53, only S7, but not S7-Ub, was capable of also stabilizing MDM2 (Figure 7B). Further, MDMX was unable to cooperate with S7-Ub to stabilize MDM2 (Figure 7C). Correspondingly, upon ectopic expression of S7 or S7-Ub in U2OS cells at similar levels, S7-Ub induced more apoptosis than S7 (Figure 7D). Based on these observations, we propose that MDM2 ubiquitination of S7 is a mechanism for extending the stabilization of p53 after conditions of ribosomal stress. Our studies have revealed a complex relationship between the ribosomal protein S7 and MDM2, which both extends previous observations with other ribosomal proteins and provides new insight into the role that nucleolar stress plays in stabilizing p53. (1) S7 levels in some cells are correlated with the stability of MDM2. When overexpressed, S7 stabilizes MDM2, while depletion of S7 leads to rapid MDM2 and p53 turnover after numerous types of genotoxic stress. In other cell types, ablation of S7 leads to p53-dependent and -independent cell-cycle arrest. (2) MDMX plays a critical and interesting role in allowing MDM2 to be stabilized by S7. (3) S7 and another well-studied ribosomal protein that interacts with MDM2, L11, are not mutually redundant in their impact on MDM2. (4) S7 itself is an excellent substrate for MDM2's E3 ligase activity, and S7 ubiquitination may serve to maintain the p53 response to DNA damage. Our findings not only characterize another ribosomal protein that intercepts the MDM2/p53 circuit, they implicate ribosomal stress as a critical integrating factor in regulating this circuit. Indeed, not only treatment with ActD (Figure 1D) but also Daunorubicin (data not shown) causes redistribution of ectopic S7 from nucleolus to the nucleoplasm. We" @default.
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