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 tumor suppressor ARF induces a p53-dependent and -independent cell cycle arrest. Unlike the nucleoplasmic MDM2 and p53, ARF localizes in the nucleolus. The role of ARF in the nucleolus, the molecular target, and the mechanism of its p53-independent function remains unclear. Here we show that ARF interacts with B23, a multifunctional nucleolar protein involved in ribosome biogenesis, and promotes its polyubiquitination and degradation. Overexpression of B23 induces a cell cycle arrest in normal fibroblasts, whereas in cells lacking p53 it promotes S phase entry. Conversely, knocking down B23 inhibits the processing of preribosomal RNA and induces cell death. Further, oncogenic Ras induces B23 only in ARF null cells, but not in cells that retain wild-type ARF. Together, our results reveal a molecular mechanism of ARF in regulating ribosome biogenesis and cell proliferation via inhibiting B23, and suggest a nucleolar role of ARF in surveillance of oncogenic insults.

The importance of coordinating cell growth with proliferation has been recognized for a long time. The molecular basis of this relationship, however, is poorly understood. Here we show that the ribosomal protein L23 interacts with HDM2. The interaction involves the central acidic domain of HDM2 and an N-terminal domain of L23. L23 and L11, another HDM2-interacting ribosomal protein, can simultaneously yet distinctly interact with HDM2 together to form a ternary complex. We show that, when overexpressed, L23 inhibits HDM2-induced p53 polyubiquitination and degradation and causes a p53-dependent cell cycle arrest. On the other hand, knocking down L23 causes nucleolar stress and triggers translocation of B23 from the nucleolus to the nucleoplasm, leading to stabilization and activation of p53. Our data suggest that cells may maintain a steady-state level of L23 during normal growth; alternating the levels of L23 in response to changing growth conditions could impinge on the HDM2-p53 pathway by interrupting the integrity of the nucleolus.The tumor suppressor protein p53 plays a pivotal role in preventing damaged and abnormal cells from becoming malignant, and its loss of function is associated with a majority of human cancers (26,36,37). The activity of p53 is not required for normal cell growth, and the protein is kept at low levels and inactive. This is accomplished by the proto-oncoprotein HDM2 (human counterpart of MDM2 in mice) through either ubiquitin-dependent p53 degradation in the cytoplasm (6, 7, 11) or repression of p53 transcriptional activity in the nucleus (17, 35). The HDM2 gene can, in turn, be transcriptionally activated by p53, constituting a feedback regulatory loop (1, 40). The current understanding of the major mechanisms of p53 activation includes one that is triggered by DNA damage and induces p53 phosphorylation through a cascade of protein kinases (5, 15, 32) and one that is triggered by aberrant oncogenic signals and is mediated by p14 ARF (p19 ARF in mice) (31). Little is known about the connection between p53 and ribosomal biogenesis, even though ribosomal biogenesis occupies the major part of a cell's life cycle. Whether and how p53 may be involved in checking ribosomal stress and the integrity of ribosomal biogenesis remains elusive (28).Previous studies have identified the interaction of the ribosomal protein L5 with HDM2 (14). Recently, it has been shown that the ribosomal protein L11 also interacts with HDM2, and through this interaction, L11 stabilizes and activates p53 and induces a cell cycle arrest (13,41). This is achieved, at least in part, through L11 inhibiting the E3 ligase activity of HDM2 (41). Based on the evidence that low levels of actinomycin D, which selectively inhibits rRNA transcription, enhances L11-HDM2 interaction, it has been proposed that L11 plays a role in the response to ribosomal perturbations to induce p53 and cell cycle arrest. In this study, we describe functional interactions of HDM2 with L23, a protein component in the 60S large ribosomal subunit. The...

Summary In vitro studies have shown that inhibition of ribosomal biogenesis can activate p53 through ribosomal protein (RP)-mediated suppression of Mdm2 E3 ligase activity. To study the physiological significance of the RP-Mdm2 interaction, we generated mice carrying a cancer-associated cysteine-to-phenylalanine substitution in the zinc finger of Mdm2 that disrupted its binding to RPL5 and RPL11. Mice harboring this mutation, although retain normal p53 response to DNA damage, lack p53 response to perturbations in ribosome biogenesis. Loss of RP-Mdm2 interaction significantly accelerates Eμ-Myc induced lymphomagenesis. Furthermore, ribosomal perturbation induced p53 response does not require tumor suppressor p19Arf. Collectively, our findings establish RP-Mdm2 interaction as a genuine p53 stress-signaling pathway activated by aberrant ribosomal biogenesis and essential for safeguarding against oncogenic c-Myc-induced tumorigenesis.

The ribosomal protein L11 binds to and suppresses the E3 ligase function of HDM2, thus activating p53. Despite being abundant as a component of the 60S large ribosomal subunit, L11 does not induce p53 under normal growth conditions. In search of mechanisms controlling L11-HDM2 interaction, we found that the induction of p53 under growth inhibitory conditions, such as low dose of actinomycin D or serum depletion, can be significantly attenuated by knocking down L11, indicating the importance of L11 in mediating these growth inhibitory signals to p53. We show that L11 is not regulated by transcription or protein stability and its level remains relatively constant during serum starvation. However, serum starvation induces translocation of L11 from the nucleolus to the nucleoplasm, where it participates in a complex with HDM2. We propose that the nucleolus acts as a barrier to prevent L11 interacting with HDM2 during normal growth. Growth inhibition, presumably through suppression of rRNA production in the nucleolus, facilitates translocation of L11 to the nucleoplasm, thus activating p53 through inhibiting HDM2.

It is believed that Mdm2 suppresses p53 in two ways: transcriptional inhibition by direct binding, and degradation via its E3 ligase activity. To study these functions physiologically, we generated mice bearing a single-residue substitution (C462A) abolishing the E3 function without affecting p53 binding. Unexpectedly, homozygous mutant mice died before E7.5, and deletion of p53 rescued the lethality. Furthermore, reintroducing a switchable p53 by crossing with p53ER(TAM) mice surprisingly demonstrated that the mutant Mdm2(C462A) was rapidly degraded in a manner indistinguishable from that of the wild-type Mdm2. Hence, our data indicate that (1) the Mdm2-p53 physical interaction, without Mdm2-mediated p53 ubiquitination, cannot control p53 activity sufficiently to allow early mouse embryonic development, and (2) Mdm2's E3 function is not required for Mdm2 degradation.

The p53-inhibitory function of the oncoprotein MDM2 is regulated by a number of MDM2-binding proteins, including ARF and ribosomal proteins L5, L11, and L23, which bind the central acidic domain of MDM2 and inhibit its E3 ubiquitin ligase activity. Various human cancer-associated MDM2 alterations targeting the central acidic domain have been reported, yet the functional significance of these mutations in tumor development has remained unclear. Here, we show that cancer-associated missense mutations targeting MDM2's central zinc finger disrupt the interaction of MDM2 with L5 and L11. We found that the zinc finger mutant MDM2 is impaired in undergoing nuclear export and proteasomal degradation as well as in promoting p53 degradation, yet retains the function of suppressing p53 transcriptional activity. Unlike the wild-type MDM2, whose p53-suppressive activity can be inhibited by L11, the MDM2 zinc finger mutant escapes L11 inhibition. Hence, the MDM2 central zinc finger plays a critical role in mediating MDM2's interaction with ribosomal proteins and its ability to degrade p53, and these roles are disrupted by human cancer-associated MDM2 mutations.The mammalian p53 transcription factor mediates a major tumor suppression pathway that is negatively controlled by the proto-oncoprotein MDM2 (HDM2 in humans; henceforth denoted MDM2) and is altered in most, if not all, human cancers. The gene for mouse Mdm2 (murine double minute 2) was originally identified in a spontaneously transformed mouse BALB/c cell line (13). The Mdm2 protein was found to be responsible for transformation of NIH 3T3 and Rat2 cells when overexpressed (13), and this transforming function is believed to stem from its ability to bind with and inhibit the transactivation activity of p53 (39). Subsequently, the HDM2 gene, the human homologue of Mdm2, was found to be amplified in over one-third of those human sarcomas that still retain wild-type p53 (41), suggesting that overexpression of MDM2 could be a common mechanism by which cells inactivate p53. Mice with targeted deletion of the Mdm2 gene die during early embryonic development, and this lethality can be rescued by concomitant deletion of p53, indicating that a major in vivo function of MDM2 is to keep p53 activity in check (27,33).It is believed that MDM2 controls p53 through two mechanisms: inhibition of the transcriptional activity of p53 (39) and promotion of p53 ubiquitination and degradation (18,29). Mdm2 binds to and masks the N-terminal transactivation domain of p53 by directly interfering with the interaction between p53 and the basal transcriptional machinery (42, 54). Mdm2 belongs to a large family of RING finger ubiquitin ligases (25). Studies have demonstrated that Mdm2 is a ubiquitin ligase (19) and that the ubiquitin ligase activity of Mdm2 is responsible for degradation of p53 both in vitro (14,20) and in transfected cells (14). MDM2-mediated p53 degradation also depends on its ability to promote p53 nuclear export (46). Mutation of a nuclear export signal (NES) in MDM2 abolishes ...

The tumor suppressor p53 has recently been shown to regulate energy metabolism through multiple mechanisms. However, the in vivo signaling pathways related to p53-mediated metabolic regulation remain largely uncharacterized. By using mice bearing a single amino acid substitution at cysteine residue 305 of mouse double minute 2 (Mdm2 C305F ), which renders Mdm2 deficient in binding ribosomal proteins (RPs) RPL11 and RPL5, we show that the RP-Mdm2-p53 signaling pathway is critical for sensing nutrient deprivation and maintaining liver lipid homeostasis. Although the Mdm2 C305F mutation does not significantly affect growth and development in mice, this mutation promotes fat accumulation under normal feeding conditions and hepatosteatosis under acute fasting conditions. We show that nutrient deprivation inhibits rRNA biosynthesis, increases RP-Mdm2 interaction, and induces p53-mediated transactivation of malonyl-CoA decarboxylase (MCD), which catalyzes the degradation of malonyl-CoA to acetyl-CoA, thus modulating lipid partitioning. Fasted Mdm2 C305F mice demonstrate attenuated MCD induction and enhanced malonylCoA accumulation in addition to decreased oxidative respiration and increased fatty acid accumulation in the liver. Thus, the RPMdm2-p53 pathway appears to function as an endogenous sensor responsible for stimulating fatty acid oxidation in response to nutrient depletion.T he dynamic process of cell growth and division is under constant surveillance. As one of the primary "gatekeepers" of the cell, p53 plays a major role in sensing and responding to a variety of internal and external stressors to maintain cellular homeostasis. In addition to its conventional roles in promoting cell cycle arrest, senescence, and apoptosis, p53 has recently been shown to regulate metabolism through the transcriptional activation of genes involved in glucose transport, glycolysis, oxidative phosphorylation (OXPHOS), and glutamine hydrolysis as well as in the activation of genes upstream of the mammalian target of rapamycin and autophagic pathways (reviewed in ref.1). Virtually all cancers show metabolic changes that result in enhanced glucose consumption and elevated glycolytic activity known as the Warburg effect (2). Because cells continuously undergo metabolic perturbations as a result of constantly changing physiological and environmental cues such as daily feeding/fasting cycles, p53 may act in this context as a metabolic stress regulator altering cellular metabolic programs under nonlethal or "low-stress" conditions. Recent studies have shown that inhibition of ribosomal biogenesis can activate p53 through the ribosomal protein (RP)-mediated suppression of mouse double minute 2 (Mdm2) in the RP-Mdm2-p53 stress response pathway (3). Detailed analysis revealed that several RPs require the Mdm2 central zinc finger motif for efficient Mdm2 binding. Interestingly, cancer-associated MDM2 mutations have been reported to affect the central zinc finger motif (4, 5) and can specifically disrupt RP binding (6). To study the physiolog...