The oncoprotein MDM2 associates with ribosomal proteins L5, L11, and L23. Both L11 and L23 have been shown to activate p53 by inhibiting MDM2-mediated p53 suppression. Here we have shown that L5 also activates p53. Overexpression of L5 stabilized ectopic p53 in H1299 cells and endogenous p53 in U2OS cells. Consequently, L5 enhanced p53 transcriptional activity and induced p53-dependent G 1 cell cycle arrest. Furthermore, like L11 and L23, L5 also remarkably inhibited MDM2-mediated p53 ubiquitination. The interaction of L5 with MDM2 was also enhanced by treatment with a low dose of actinomycin D. Actinomycin D-induced p53 was inhibited by small interference RNA against L5. By reciprocal co-immunoprecipitation, we further showed that there were at least two MDM2-ribosomal protein complexes in cells: MDM2-L5-L11-L23 and p53-MDM2-L5-L11-L23. We propose that the MDM2-L5-L11-L23 complex functions to inhibit MDM2-mediated p53 ubiquitination and thus activates p53.

The p53-MDM2 feedback loop is vital for cell growth control and is subjected to multiple regulations in response to various stress signals. Here we report another regulator of this loop. Using an immunoaffinity method, we purified an MDM2-associated protein complex that contains the ribosomal protein L23. L23 interacted with MDM2, forming a complex independent of the 80S ribosome and polysome. The interaction of L23 with MDM2 was enhanced by treatment with actinomycin D but not by gamma-irradiation, leading to p53 activation. This activation was inhibited by small interfering RNA against L23. Ectopic expression of L23 reduced MDM2-mediated p53 ubiquitination and also induced p53 activity and G 1 arrest in p53-proficient U2OS cells but not in p53-deficient Saos-2 cells. These results reveal that L23 is another regulator of the p53-MDM2 feedback regulation.

The nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis. Both depletion and overexpression of NS reduce cell proliferation. However, the mechanisms underlying this regulation are still unclear. Here, we show that NS regulates p53 activity through the inhibition of MDM2. NS binds to the central acidic domain of MDM2 and inhibits MDM2-mediated p53 ubiquitylation and degradation. Consequently, ectopic overexpression of NS activates p53, induces G 1 cell cycle arrest, and inhibits cell proliferation. Interestingly, the knockdown of NS by small interfering RNA also activates p53 and induces G 1 arrest. These effects require the ribosomal proteins L5 and L11, since the depletion of NS enhanced their interactions with MDM2 and the knockdown of L5 or L11 abrogated the NS depletion-induced p53 activation and cell cycle arrest. These results suggest that a p53-dependent cell cycle checkpoint monitors changes of cellular NS levels via the impediment of MDM2 function.The tumor suppressor protein p53 responds to diverse stresses to regulate many target genes whose protein products induce cell cycle arrest, apoptosis, senescence, and DNA repair (28,48). In unstressed cells, p53 is maintained at low levels by its inhibitor, MDM2, an E3 ubiquitin ligase that ubiquitylates and targets p53 for proteasome-mediated degradation (12,15,16,20) through a feedback mechanism (33, 49). In response to various stresses, p53 is stabilized and activated largely through the inhibition of MDM2. These stresses include DNA damage and oncogenic and nucleolar stresses (22). DNA damage induces phosphorylation of both p53 and MDM2, unties the p53-MDM2 feedback loop, and consequently activates p53 (2,5,25,39,41). Oncogenic stress induces the expression of the tumor suppressor protein ARF, which in turn prevents MDM2 from targeting p53, leading to p53 activation (29,40,55). Recently, a number of external and internal insults were shown to induce nucleolar stress by disrupting the nucleolar structure, activating p53 (37). Also, the perturbation of ribosomal biogenesis by interfering with rRNA synthesis, processing, and ribosome assembly, as exemplified by the treatment of actinomycin D or fluorouracil (5-FU) (1, 13, 43), serum starvation (4), expression of dominant-negative mutant Bop1 (32), or genetic disruption of ribosomal proteins S6 and TIF-IA (31, 51), can cause ribosomal stress and subsequent p53 activation. It is now believed that in response to ribosomal stress caused by some of the aforementioned agents, several nucleolar proteins, including B23 (also called nucleophosmin) (21) and C23 (also called nucleolin) (38), and ribosomal proteins, such as L5, L11, L23, and S7 (6, 8-10, 18, 23, 52), can bind to MDM2 directly and inhibit its activity toward p53, resulting in p53 activation. Hence, these findings demonstrate a critical role for these nucleolar proteins in transmitting nucleolar stress signals to p53 and initiating p53-dependent cell growth arrest or apoptosis.Interestingly, another recently iden...

The ubiquitin (Ub)-proteasome system plays a pivotal role in the regulation of p53 protein stability and activity. p53 is ubiquitinated and destabilized by MDM2 and several other Ub E3s, whereas it is deubiquitinated and stabilized by Ub-specific protease (USP)7 and USP10. Here we show that the ovarian tumour domain-containing Ub aldehydebinding protein 1 (Otub1) is a novel p53 regulator. Otub1 directly suppresses MDM2-mediated p53 ubiquitination in cells and in vitro. Overexpression of Otub1 drastically stabilizes and activates p53, leading to apoptosis and marked inhibition of cell proliferation in a p53-dependent manner. These effects are independent of its catalytic activity but require residue Asp88. Mutation of Asp88 to Ala (Otub1 D88A ) abolishes activity of Otub1 to suppress p53 ubiquitination. Further, wild-type Otub1 and its catalytic mutant (Otub1 C91S ), but not Otub1 D88A , bind to the MDM2 cognate E2, UbcH5, and suppress its Ub-conjugating activity in vitro. Overexpression of Otub1 D88A or ablation of endogenous Otub1 by siRNA markedly impaired p53 stabilization and activation in response to DNA damage. Together, these results reveal a novel function for Otub1 in regulating p53 stability and activity.

The CDK inhibitor p21waf1/cip1 is degraded by a ubiquitin-independent proteolytic pathway. Here, we show that MDM2 mediates this degradation process. Overexpression of wild-type or ring finger-deleted, but not nuclear localization signal (NLS)-deleted, MDM2 decreased p21waf1/cip1 levels without ubiquitylating this protein and affecting its mRNA level in p53(-/-) cells. This decrease was reversed by the proteasome inhibitors MG132 and lactacystin, by p19(arf), and by small interfering RNA (siRNA) against MDM2. p21waf1/cip1 bound to MDM2 in vitro and in cells. The p21waf1/cip1-binding-defective mutant of MDM2 was unable to degrade p21waf1/cip1. MDM2 shortened the half-life of both exogenous and endogenous p21waf1/cip1 by 50% and led to the degradation of its lysine-free mutant. Consequently, MDM2 suppressed p21waf1/cip1-induced cell growth arrest of human p53(-/-) and p53(-/-)/Rb(-/-)cells. These results demonstrate that MDM2 directly inhibits p21waf1/cip1 function by reducing p21waf1/cip1 stability in a ubiquitin-independent fashion.