Hydrophilic residues are crucial for ribosomal protein L11 (RPL11) interaction with zinc finger domain of MDM2 and p53 protein activation

Ribosomal protein L11 (RPL11) has been shown to activate p53 by binding to MDM2 and negating its p53 suppression activity in response to ribosomal stress. Although a mutation at Cys-305 within the zinc finger domain of MDM2 has been shown to drastically impair MDM2 interaction with RPL11 and thus escapes the inhibition by this ribosomal protein, it still remains elusive whether RPL11 inactivates MDM2 via direct action on this zinc finger domain and what is the chemical nature of this specific interaction. To define the roles of the MDM2 zinc finger in association with RPL11, we conducted hydrogen-deuterium exchange mass spectrometry, computational modeling, circular dichroism, and mutational analyses of the zinc finger domain of MDM2 and human RPL11. Our study reveals that RPL11 forms a stable complex with MDM2 in vitro through direct contact with its zinc finger. This binding is disrupted by single mutations of non-cysteine amino acids within the zinc finger domain of MDM2. Basic residues in RPL11 are crucial for the stable binding and RPL11 suppression of MDM2 activity toward p53. These results provide the first line of evidence for the specific interaction between RPL11 and the zinc finger of MDM2 via hydrophilic residues as well as a molecular foundation for better understanding RPL11 inhibition of MDM2 function.     

Regulation of the MDM2-p53 pathway by ribosomal protein L11 involves 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.     

Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2

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 MDM2 RING Domain and Central Acidic Domain Play Distinct Roles in MDM2 Protein Homodimerization and MDM2-MDMX Protein Heterodimerization

The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization.     

Interplay between ribosomal protein S27a and MDM2 protein in p53 activation in response to ribosomal stress

Ribosomal proteins play a critical role in tightly coordinating p53 signaling with ribosomal biogenesis. Several ribosomal proteins have been shown to induce and activate p53 via inhibition of MDM2. Here, we report that S27a, a small subunit ribosomal protein synthesized as an 80-amino acid ubiquitin C-terminal extension protein (CEP80), functions as a novel regulator of the MDM2-p53 loop. S27a interacts with MDM2 at the central acidic domain of MDM2 and suppresses MDM2-mediated p53 ubiquitination, leading to p53 activation and cell cycle arrest. Knockdown of S27a significantly attenuates the p53 activation in cells in response to treatment with ribosomal stress-inducing agent actinomycin D or 5-fluorouracil. Interestingly, MDM2 in turn ubiquitinates S27a and promotes proteasomal degradation of S27a in response to actinomycin D treatment, thus forming a mutual-regulatory loop. Altogether, our results reveal that S27a plays a non-redundant role in mediating p53 activation in response to ribosomal stress via interplaying with MDM2.     

The MDM2 oncoprotein binds specifically to RNA through its RING finger domain.

BACKGROUND: The cellular mdm2 gene has transforming activity when overexpressed and is amplified in a variety of human tumors. At least part of the transforming ability of the MDM2 protein is due to binding and inactivating the p53 tumor suppressor protein. Additionally, this protein forms a complex in vivo with the L5 ribosomal protein and its associated 5S ribosomal RNA and may be part of a ribosomal complex. MATERIALS AND METHODS: A RNA homopolymer binding assay and a SELEX procedure have been used to characterize the RNA-binding activity of MDM2. RESULTS: The MDM2 protein binds efficiently to the homopolyribonucleotide poly(G) but not to other homopolyribonucleotides. This binding is independent of the interaction of MDM2 with the L5 protein, which occurs through the central acidic domain of MDM2. An RNA SELEX procedure was performed to identify specific RNA ligands that bind with high affinity to the human MDM2 (HDM2) protein. After 10 rounds of selection and amplification, a subset of RNA molecules that bound efficiently to HDM2 was isolated from a randomized pool. Sequencing of these selected ligands revealed that a small number of sequence motifs were selected. The specific RNA binding occurs through the RING finger domain of the protein. Furthermore, a single amino acid substitution in the RING finger domain, G446S, completely abolishes the specific RNA binding. CONCLUSIONS: These observations, showing that MDM2 binds the L5/5S ribosomal ribonucleoprotein particle and can also bind to specific RNA sequences or structures, suggest a role for MDM2 in translational regulation in a cell.     

Critical contribution of the MDM2 acidic domain to p53 ubiquitination

MDM2 is an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Recent studies have shown, however, that the ring-finger domain (RFD) of MDM2, where the ubiquitin E3 ligase activity resides, is necessary but not sufficient for p53 ubiquitination, suggesting that an additional activity of MDM2 might be required. To test this possibility, we generated a series of MDM2/MDMX chimeric proteins to assess the contribution of each domain of MDM2 to the ubiquitination process. MDMX is a close structural homolog of MDM2 that nevertheless lacks the E3 ligase activity in vivo. We demonstrate here that MDMX gains self-ubiquitination activity and becomes extremely unstable upon introduction of the MDM2 RFD, indicating that the RFD is essential for self-ubiquitination. This MDMX chimeric protein, however, is unable to ubiquitinate p53 in vivo despite its E3 ligase activity and binding to p53, separating the self-ubiquitination activity of MDM2 from its ability to ubiquitinate p53. Significantly, fusion of the central acidic domain (AD) of MDM2 to the MDMX chimeric protein renders the protein fully capable of ubiquitinating p53, and p53 ubiquitination is associated with p53 degradation and nuclear export. Moreover, the AD mini protein expressed in trans can functionally rescue the AD-lacking MDM2 mutant, further supporting a critical role for the AD in MDM2-mediated p53 ubiquitination.     

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

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.     

The RING finger domain of MDM2 is essential for MDM2-mediated TGF-beta resistance

In this study, we attempt to gain insights into the molecular mechanism underlying MDM2-mediated TGF-beta resistance. MDM2 renders cells refractory to TGF-beta by overcoming a TGF-beta-induced G1 cell cycle arrest. Because the TGF-beta resistant phenotype is reversible upon removal of MDM2, MDM2 likely confers TGF-beta resistance by directly targeting the cellular machinery involved in the growth inhibition by TGF-beta. Investigation of the structure-function relationship of MDM2 reveals three elements essential for MDM2 to confer TGF-beta resistance in both mink lung epithelial cells and human mammary epithelial cells. One of these elements is the C-terminal half of the p53-binding domain, which at least partially retained p53-binding and inhibitory activity. Second, the ability of MDM2 to mediate TGF-beta resistance is disrupted by mutation of the nuclear localization signal, but is restored upon coexpression of MDMX. Finally, mutations of the zinc coordination residues of the RING finger domain abrogates TGF-beta resistance, but not the ability of MDM2 to inhibit p53 activity or to bind MDMX. These data suggest that RING finger-mediated p53 inhibition and MDMX interaction are not sufficient to cause TGF-beta resistance and imply a crucial role of the E3 ubiquitin ligase activity of this domain in MDM2-mediated TGF-beta resistance.     

5-fluorouracil activation of p53 involves an MDM2-ribosomal protein interaction

5-fluorouracil (5-FU) is a widely used chemotherapeutic drug for the treatment of a variety of solid tumors. The anti-tumor activity of 5-FU has been attributed in part to its ability to induce p53-dependent cell growth arrest and apoptosis. However, the molecular mechanisms underlying p53 activation by 5-FU remain largely obscure. Here we report that 5-FU treatment leads to p53 stabilization and activation by blocking MDM2 feedback inhibition through ribosomal proteins. 5-FU treatment increased the fraction of ribosome-free L5, L11, and L23 ribosomal proteins and their interaction with MDM2, leading to p53 activation and G1/S arrest. Conversely, individual knockdown of these ribosomal proteins by small interfering RNA prevented the 5-FU-induced p53 activation and reversed the 5-FU-induced G1/S arrest. These results demonstrate that 5-FU treatment triggers a ribosomal stress response so that ribosomal proteins L5, L11, and L23 are released from ribosome to activate p53 by ablating the MDM2-p53 feedback circuit.     

Autoactivation of the MDM2 E3 Ligase by Intramolecular Interaction

The RING domain ubiquitin E3 ligase MDM2 is a key regulator of p53 degradation and a mediator of signals that stabilize p53. The current understanding of the mechanisms by which MDM2 posttranslational modifications and protein binding cause p53 stabilization remains incomplete. Here we present evidence that the MDM2 central acidic region is critical for activating RING domain E3 ligase activity. A 30-amino-acid minimal region of the acidic domain binds to the RING domain through intramolecular interactions and stimulates the catalytic function of the RING domain in promoting ubiquitin release from charged E2. The minimal activation sequence is also the binding site for the ARF tumor suppressor, which inhibits ubiquitination of p53. The acidic domain-RING domain intramolecular interaction is modulated by ATM-mediated phosphorylation near the RING domain or by binding of ARF. These results suggest that MDM2 phosphorylation and association with protein regulators share a mechanism in inhibiting the E3 ligase function and stabilizing p53 and suggest that targeting the MDM2 autoactivation mechanism may be useful for therapeutic modulation of p53 levels.     

Acidic domain is indispensable for MDM2 to negatively regulate the acetylation of p53

MDM2 is the most important negative regulator of tumor suppressor p53. Both RING finger domain and acidic domain of MDM2 contribute to the ubiquitination of p53. The crosstalk between ubiquitination and acetylation of p53 prompts us to examine whether acidic domain is essential for MDM2 to regulate the acetylation of p53. We find that the acidic domain of MDM2 is necessary to inhibit p300-mediated acetylation of p53 as well as to mediate the deacetylation of p53. Our results indicate that acidic domain of MDM2 provides essential information for acetyltransferase p300 and deacetylase HDAC1 and is indispensable for MDM2 to negatively regulate the acetylation of p53.