Mu Shui Dai

Ribosomal Protein L23 Activates p53 by Inhibiting MDM2 Function in Response to Ribosomal Perturbation but Not to Translation Inhibition

ABSTRACT The p53-MDM2 feedback loop is vital for cell growth control and is subjected to multiple regulations in response to various stress signals. Here we report another regulator of this loop. Using an immunoaffinity method, we purified an MDM2-associated protein complex that contains the ribosomal protein L23. L23 interacted with MDM2, forming a complex independent of the 80S ribosome and polysome. The interaction of L23 with MDM2 was enhanced by treatment with actinomycin D but not by gamma-irradiation, leading to p53 activation. This activation was inhibited by small interfering RNA against L23. Ectopic expression of L23 reduced MDM2-mediated p53 ubiquitination and also induced p53 activity and G1 arrest in p53-proficient U2OS cells but not in p53-deficient Saos-2 cells. These results reveal that L23 is another regulator of the p53-MDM2 feedback regulation.

MDM2 promotes p21waf1/cip1 proteasomal turnover independently of ubiquitylation.

The CDK inhibitor p21waf1/cip1 is degraded by a ubiquitin‐independent proteolytic pathway. Here, we show that MDM2 mediates this degradation process. Overexpression of wild‐type or ring finger‐deleted, but not nuclear localization signal (NLS)‐deleted, MDM2 decreased p21waf1/cip1 levels without ubiquitylating this protein and affecting its mRNA level in p53−/− cells. This decrease was reversed by the proteasome inhibitors MG132 and lactacystin, by p19arf, and by small interfering RNA (siRNA) against MDM2. p21waf1/cip1 bound to MDM2 in vitro and in cells. The p21waf1/cip1‐binding‐defective mutant of MDM2 was unable to degrade p21waf1/cip1. MDM2 shortened the half‐life of both exogenous and endogenous p21waf1/cip1 by 50% and led to the degradation of its lysine‐free mutant. Consequently, MDM2 suppressed p21waf1/cip1‐induced cell growth arrest of human p53−/− and p53−/−/Rb−/− cells. These results demonstrate that MDM2 directly inhibits p21waf1/cip1 function by reducing p21waf1/cip1 stability in a ubiquitin‐independent fashion.

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

ABSTRACT 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 G1 cell cycle arrest, and inhibits cell proliferation. Interestingly, the knockdown of NS by small interfering RNA also activates p53 and induces G1 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.

Inhibition of c-Myc activity by ribosomal protein L11.

The c‐Myc oncoprotein promotes cell growth by enhancing ribosomal biogenesis through upregulation of RNA polymerases I‐, II‐, and III‐dependent transcription. Overexpression of c‐Myc and aberrant ribosomal biogenesis leads to deregulated cell growth and tumorigenesis. Hence, c‐Myc activity and ribosomal biogenesis must be regulated in cells. Here, we show that ribosomal protein L11, a component of the large subunit of the ribosome, controls c‐Myc function through a negative feedback mechanism. L11 is transcriptionally induced by c‐Myc, and overexpression of L11 inhibits c‐Myc‐induced transcription and cell proliferation. Conversely, reduction of endogenous L11 by siRNA increases these c‐Myc activities. Mechanistically, L11 binds to the Myc box II (MB II), inhibits the recruitment of the coactivator TRRAP, and reduces histone H4 acetylation at c‐Myc target gene promoters. In response to serum stimulation or serum starvation, L11 and TRRAP display inverse promoter‐binding profiles. In addition, L11 regulates c‐Myc levels. These results identify L11 as a feedback inhibitor of c‐Myc and suggest a novel role for L11 in regulating c‐Myc‐enhanced ribosomal biogenesis.

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.

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.

14-3-3γ binds to MDMX that is phosphorylated by UV-activated Chk1, resulting in p53 activation

It has been shown that MDMX inhibits the activity of the tumor suppressor p53 by primarily cooperating with the p53 feedback regulator MDM2. Here, our study shows that this inhibition can be overcome by 14‐3‐3γ and Chk1. 14‐3‐3γ was identified as an MDMX‐associated protein via an immuno‐affinity purification‐coupled mass spectrometry. Consistently, 14‐3‐3γ directly interacted with MDMX in vitro, and this interaction was stimulated by MDMX phosphorylation in vitro and in cells. Interestingly, in response to UV irradiation, the wild‐type, but not the kinase‐dead mutant, Chk1 phosphorylated MDMX at serine 367, enhanced the 14‐3‐3γ‐MDMX binding and the cytoplasmic retaining of MDMX. The Chk1 specific inhibitor UCN‐01 repressed all of these effects. Moreover, overexpression of 14‐3‐3γ, but not its mutant K50E, which did not bind to MDMX, suppressed MDMX‐enhanced p53 ubiquitination, leading to p53 stabilization and activation. Finally, ablation of 14‐3‐3γ by siRNA reduced UV‐induced p53 level and G1 arrest. Thus, these results demonstrate 14‐3‐3γ and Chk1 as two novel regulators of MDMX in response to UV irradiation.

Crosstalk between c‐Myc and ribosome in ribosomal biogenesis and cancer

Protein production is driven by protein translation and relies on ribosomal biogenesis, globally essential for cell growth, proliferation, and animal development. Deregulation of these sophisticated cellular processes leads to abnormal homeostasis and carcinogenesis. Thus, their tight regulation is vitally important for a cell to warrant normal growth and proliferation. One newly identified key regulator for ribosomal biogenesis and translation is the oncoprotein c‐Myc, whose aberrantly excessive level and activity are highly associated with human cancers, too. Recently, we have shown that ribosomal protein L11 functions as a feedback regulator of c‐Myc. Hence, in this review, we will provide some prospects on the interplay between c‐Myc and ribosomal proteins during ribosomal biogenesis and discuss its implications in cancer. J. Cell. Biochem. 105: 670–677, 2008.

Positive regulation of p53 stability and activity by the deubiquitinating enzyme Otubain 1

The ubiquitin (Ub)–proteasome system plays a pivotal role in the regulation of p53 protein stability and activity. p53 is ubiquitinated and destabilized by MDM2 and several other Ub E3s, whereas it is deubiquitinated and stabilized by Ub‐specific protease (USP)7 and USP10. Here we show that the ovarian tumour domain‐containing Ub aldehyde‐binding protein 1 (Otub1) is a novel p53 regulator. Otub1 directly suppresses MDM2‐mediated p53 ubiquitination in cells and in vitro. Overexpression of Otub1 drastically stabilizes and activates p53, leading to apoptosis and marked inhibition of cell proliferation in a p53‐dependent manner. These effects are independent of its catalytic activity but require residue Asp88. Mutation of Asp88 to Ala (Otub1D88A) abolishes activity of Otub1 to suppress p53 ubiquitination. Further, wild‐type Otub1 and its catalytic mutant (Otub1C91S), but not Otub1D88A, bind to the MDM2 cognate E2, UbcH5, and suppress its Ub‐conjugating activity in vitro. Overexpression of Otub1D88A or ablation of endogenous Otub1 by siRNA markedly impaired p53 stabilization and activation in response to DNA damage. Together, these results reveal a novel function for Otub1 in regulating p53 stability and activity.

Mycophenolic acid activation of p53 requires ribosomal proteins L5 and L11.

Mycophenolate mofetil (MMF), a prodrug of mycophenolic acid (MPA), is widely used as an immunosuppressive agent. MPA selectively inhibits inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme for the de novo synthesis of guanine nucleotides, leading to depletion of the guanine nucleotide pool. Its chemotherapeutic effects have been attributed to its ability to induce cell cycle arrest and apoptosis. MPA treatment has also been shown to induce and activate p53. However, the mechanism underlying the p53 activation pathway is still unclear. Here, we show that MPA treatment results in inhibition of pre-rRNA synthesis and disruption of the nucleolus. This treatment enhances the interaction of MDM2 with L5 and L11. Interestingly, knockdown of endogenous L5 or L11 markedly impairs the induction of p53 and G1 cell cycle arrest induced by MPA. These results suggest that MPA may trigger a nucleolar stress that induces p53 activation via inhibition of MDM2 by ribosomal proteins L5 and L11.