Opin

Opin. knockdown of L11 or L5. Consistently, knockdown of L29 or L30 enhanced the interaction of MDM2 with L11 and L5 and markedly inhibited MDM2-mediated p53 ubiquitination, suggesting that direct perturbation of 60 S ribosomal biogenesis activates p53 via L11- and L5-mediated MDM2 suppression. Mechanistically, knockdown of L30 or L29 significantly increased the NEDDylation and nuclear retention of L11. Knocking down endogenous NEDD8 suppressed p53 activation induced by knockdown of L30. These results demonstrate that NEDDylation of L11 plays a critical role in mediating p53 activation in response to perturbation of ribosomal biogenesis. gene rescues the lethal phenotype of knock-out mice (9, 10). The importance of the MDM2-p53 feedback loop is also evident from the fact that diverse stressors activate p53 by interfering with this loop. For example, DNA damage, such as that induced by ionizing radiation and UV irradiation, triggers phosphorylation of both p53 and MDM2, blocking their physical and functional interaction and alleviating the inhibition of p53 by MDM2 (2). Aberrant proliferating signals induced by overexpression of oncogenes induce the expression of the ARF tumor suppressor (11). ARF binds to the central acidic domain of MDM2 and inhibits its ubiquitin E3 ligase activity toward p53, leading to p53 activation (11, 12). Recently, it has been shown that p53 is also activated by nucleolar stress (also called ribosomal stress) via inhibition of MDM2. This type of stress is induced by perturbation of ribosomal biogenesis, a multistep cellular process for making the ribosome, including ribosomal RNA synthesis, processing, and ribosomal assembly in the nucleolus as well as ribosome subunit export into the cytoplasm (13, 14). Ribosomal biogenesis is vital for cell growth and must be tightly coordinated with cell cycle progression. Deregulation of ribosomal biogenesis contributes to tumorigenesis (14, PLX51107 15). Accumulating evidence points to a key role for p53 in sensing ribosomal stress. Examples of such stress conditions include treatment of cells with a low dose of actinomycin D (Act D) (16), ARHGEF11 5-fluorouracil (17, 18), or mycophenolic acid (MPA) (19), expression of dominant-negative mutant of the ribosomal RNA processing factor Bop1 (20), serum starvation or contact inhibition (21), genetic disruption of the PLX51107 polymerase I transcription initiation factor TIF-IA (22), or knockdown of either ribosomal protein S6 (23), or nucleostemin (24). Mechanistically, it has been shown that several ribosomal proteins, including L5, L11, L23, and S7, activate p53 by binding to MDM2 and inhibiting MDM2-mediated p53 ubiquitination and degradation in response to nucleolar stress (25,C32). Reduction of these proteins by siRNA significantly attenuated the p53 activation induced by nucleolar stress. Interestingly, it has recently been shown that L11 and S7 are also required for p53 activation induced by DNA-damaging agents (32), suggesting that ribosomal proteins may play a crucial PLX51107 role in p53 activation in response to diverse stressors. Relevantly, mutations or deletions of ribosomal protein genes leading to haploinsufficiency of individual ribosomal proteins, including L5 and L11, contribute to Diamond-Blackfan anemia, a rare inherited anemia syndrome with increased incidence of tumors (15, 33, 34). Haploinsufficiency of several ribosomal proteins in zebrafish develop tumors as well (35), implying that these ribosomal proteins may possess intrinsic tumor suppressor function. Currently, it is not known why multiple ribosomal proteins regulate the MDM2-p53 pathway. It is tempting to speculate that these proteins may act using different mechanisms or in concert with each other while controlling MDM2. Supporting the collaborative role of these ribosomal proteins is that L5 and L11 synergistically inhibit MDM2, leading to a robust activation of p53 compared with individual expression of L5 or L11 (36). Also, these ribosomal proteins appear to bind to different domains at the central region of MDM2 (27, 28, 37, 38), suggesting that they may form a multiprotein complex with MDM2. Another unanswered question is whether the ribosomal protein regulation of the MDM2-p53 pathway is specific to some, but not all, ribosomal proteins. In this study, we show that two ribosomal proteins from the large ribosome subunit, L29 and L30, do not bind to MDM2 and do not inhibit MDM2-mediated p53 suppression, demonstrating that the ribosomal protein regulation of the MDM2-p53 pathway is specific. Interestingly, perturbation of 60 S ribosomal biogenesis by knocking down either L29 or L30 significantly induced p53 activity. This p53 activation requires L5 and.