Aberrant Expression of Nucleostemin Activates p53 and Induces Cell Cycle Arrest via Inhibition of MDM2

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 identified nucleolar protein called nucleostemin (NS) has been shown to regulate cell cycle progression as well. NS possesses two putative GTP-binding motifs and can be readily detected in neural stem cells, embryonic stem cells, hematopoietic primitive cells, and tumor cell lines (46). However, the level of NS decreases drastically prior to cell cycle exit upon differentiation of the stem cells, suggesting that this protein may play a crucial role in stem cell proliferation (46). Indeed, NS is essential for early embryogenesis as deletion of this gene caused embryonic lethality at E3.5 (3, 54). Consistently, the depletion of NS levels by small interfering RNA (siRNA) in cultured cells also blocks cell cycle progression (24, 46). Astonishingly, the overexpression of NS in cultured neural stem cells, cancer cells, and mouse embryonic fibroblasts interferes with cell cycle progression as well. These studies suggest that the physiological level of NS is so finely monitored that its imbalance would be detrimental to cell proliferation (3, 46, 54). Interestingly, the overexpression of NS was shown to arrest cell proliferation possibly by acting on p53 via the N-terminal basic domain of NS and this NS domain was also required for NS-induced cell death (46). However, how NS activates p53 and whether it does so by suppressing MDM2 activity remain unknown. In addition, it is unclear how the reduction of NS levels brings about cell cycle arrest as well. In an attempt to address the above questions, we found that both the overexpression and the reduction of NS activate p53 through inhibition of MDM2 activity. On one hand, NS, when overexpressed, stabilized p53 and induced p53-dependent cell cycle arrest by directly binding to MDM2 and inhibiting its suppression activity toward p53. On the other hand, the knockdown of NS by siRNA induced p53 activation via enhancement of the binding of the ribosomal proteins L5 and L11 to MDM2. These results not only reveal a novel and critical role for NS in the regulation of MDM2 function but also divulge another role for L11 and L5 in transmitting the stress signal resulting from the loss of NS to the p53-MDM2 pathway.