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.     

Negative regulation of HDM2 to attenuate p53 degradation by ribosomal protein L26

HDM2 is a p53-specific E3 ubiquitin ligase. Its overexpression leads to excessive inactivation of tumor protein p53, diminishing its tumor suppressor function. HDM2 also affects the cell cycle, apoptosis and tumorigenesis through interacting with other molecules, including several ribosomal proteins. To identify novel HDM2 regulators, we performed a yeast two-hybrid screening using HDM2 as bait. Among the candidates, ribosomal protein L26 (RPL26) was characterized as a novel HDM2-interactor. The interaction between HDM2 and RPL26 was further validated by in vivo and in vitro assays. RPL26 modulates the HDM2–p53 interaction by forming a ternary complex among RPL26, HDM2 and p53, which stabilize p53 through inhibiting the ubiquitin ligase activity of HDM2. The ribosomal stress caused by a low dose of Act D enhances RPL26–HDM2 interaction and activates p53. Overexpression of RPL26 results in activating of p53, inhibits cell proliferation and induces a p53-dependent cell cycle arrest. These results provide a novel regulatory mechanism of RPL26 to activate p53 by inhibiting HDM2.     

An intact HDM2 RING-finger domain is required for nuclear exclusion of p53

The p53 tumour-suppressor protein is negatively regulated by HDM2. Recent reports indicate that the leucine-rich nuclear-export sequence (NES) of HDM2 enables it to shuttle to the cytoplasm, and that this activity is required for degradation of p53. However, it is unclear whether HDM2 is involved in nuclear export of p53, partly because p53 has itself been shown to contain a functional NES within its tetramerization domain. Here we show that co-expression of HDM2 with green fluorescent protein (GFP)-tagged p53 causes redistribution of p53 from the nucleus to the cytoplasm of the cell. This activity is dependent on binding of p53 to HDM2, and requires an intact p53 NES, but is independent of the HDM2 NES. A mutant of the HDM2 RING-finger domain that is unable to ubiquitinate p53 does not cause relocalization of p53, indicating that ubiquitin ligation or other activities of this region of HDM2 may be necessary for its regulation of p53 localization.     

Requirement for HDM2 activity in the rapid degradation of p53 in neuroblastoma

The wild type p53 tumor suppressor protein is rapidly degraded in normal cells by MDM2, the ubiquitin ligase that serves as the key regulator of p53 function by modulating protein stability. Cellular exposure to genotoxic stress triggers the stabilization of p53 by multiple pathways that converge upon interference with MDM2 function. In this study, we first investigated the ability of HDM2 (MDM2 human homologue) to degrade endogenous p53 in neuroblastoma (NB). Although the p53 protein in NB has been reported to be constitutively stabilized, we find that HDM2 in NB is functional and facilitates the rapid turnover of p53 in nonstressed cells via the proteasome pathway. Second, we examined the relationship between p53 and HDM2 in the adriamycin-mediated stabilization of p53 in NB. We demonstrate that while p53 stabilization depends neither upon the phosphorylation of specific N-terminal sites nor upon dissociation from HDM2, it requires inactivation of functional HDM2. In support of this notion, p53 stabilization following adriamycin resulted in an inhibition of both p53 ubiquitination and HDM2 ligase activity. Taken together, these data implicate a requirement for enzymatic inactivation of HDM2 as a novel mechanism for p53 stabilization in the DNA damage response pathway.     

Regulation of the HDM2-p53 pathway by ribosomal protein L6 in response to ribosomal stress

The HDM2-p53 loop is crucial for monitoring p53 level and human pathologies. Therefore, identification of novel molecules involved in this regulatory loop is necessary for understanding the dynamic regulation of p53 and treatment of human diseases. Here, we characterized that the ribosomal protein L6 binds to and suppresses the E3 ubiquitin ligase activity of HDM2, and subsequently attenuates HDM2-mediated p53 polyubiquitination and degradation. The enhanced p53 activity further slows down cell cycle progression and leads to cell growth inhibition. Conversely, the level of p53 is dramatically decreased upon the depletion of RPL6, indicating that RPL6 is essential for p53 stabilization. We also found that RPL6 translocalizes from the nucleolus to nucleoplasm under ribosomal stress, which facilitates its binding with HDM2. The interaction of RPL6 and HDM2 drives HDM2-mediated RPL6 polyubiquitination and proteasomal degradation. Longer treatment of actinomycin D increases RPL6 ubiquitination and destabilizes RPL6, and thereby putatively attenuates p53 response until the level of L6 subsides. Therefore, RPL6 and HDM2 form an autoregulatory feedback loop to monitor the level of p53 in response to ribosomal stress. Together, our study identifies the crucial function of RPL6 in regulating HDM2-p53 pathway, which highlights the importance of RPL6 in human genetic diseases and cancers.     

Inhibition of HDM2 and activation of p53 by ribosomal protein L23

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.     

p53 promotes its own polyubiquitination by enhancing the HDM2 and HDMX interaction

HDM2 and HDMX are two homologs essential for controlling p53 tumor suppressor activity under normal conditions. Both proteins bind different sites on the p53 N‐terminus, and while HDM2 has E3 ubiquitin ligase activity towards p53, HDMX does not. Nevertheless, HDMX is required for p53 polyubiquitination and degradation, but the underlying molecular mechanism remains unclear. Alone, HDMX and HDM2 interact via their respective C‐terminal RING domains but here we show that the presence of p53 induces an N‐terminal interface under normal cellular conditions. This results in an increase in HDM2‐mediated p53 polyubiquitination and degradation. The HDM2 inhibitor Nutlin‐3 binds the N‐terminal p53 binding pocket and is sufficient to induce the HDM2‐HDMX interaction, suggesting that the mechanism depends on allosteric changes that control the multiprotein complex formation. These results demonstrate an allosteric interchange between three different proteins (HDMX‐HDM2‐p53) and help to explain the molecular mechanisms of HDM2‐inhibitory drugs.     

Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization

p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.