Shelya X. Zeng

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.

p53 downregulates Down syndrome-associated DYRK1A through miR-1246

Several microRNAs mediate the functions of p53 family members. Here we characterize miR‐1246 as a new target of this family. In response to DNA damage, p53 induces the expression of miR‐1246 which, in turn, reduces the level of DYRK1A, a Down syndrome‐associated protein kinase. Knockdown of p53 has the opposite effect. Overexpression of miR‐1246 reduces DYRK1A levels and leads to the nuclear retention of NFATc1, a protein substrate of DYRK1A, and the induction of apoptosis, whereas a miR‐1246‐specific inhibitor prevented the nuclear import of NFATc1. Together, these results indicate that p53 inhibits DYRK1A expression through the induction of miR‐1246.

SSRP1 functions as a co-activator of the transcriptional activator p63.

The p53 homolog p63 is a transcriptional activator. Here, we describe the identification of an HMG1‐like protein SSRP1 as a co‐activator of p63. Over expression of wild‐type, but not deletion mutant, SSRP1 remarkably enhanced p63γ‐dependent luciferase activity, G1 arrest, apoptosis and expression of endogenous PIG3, p21Waf1/cip1 and MDM2 in human p53‐deficient lung carcinoma H1299 cells and mouse embryonic fibroblasts. Also, SSRP1 interacted to p63γ in vitro and in cells, and resided with p63γ at the p53‐responsive DNA element sites of the cellular endogenous MDM2 and p21Waf1/cip1 promoters. Moreover, N‐terminus‐deleted p63 (ΔN‐p63) bound to neither SSRP1 nor its central domain in vitro. Accordingly, SSRP1 was unable to stimulate ΔN‐p63‐mediated residual luciferase activity and apoptosis in cells. Finally, the ectopic expression of the central p63‐binding domain of SSRP1 inhibited p63‐dependent transcription in cells. Thus, these results suggest that SSRP1 stimulates p63 activity by associating with this activator at the promoter.

A small molecule Inauhzin inhibits SIRT1 activity and suppresses tumour growth through activation of p53

Although ∼50% of all types of human cancers harbour wild‐type TP53, this p53 tumour suppressor is often deactivated through a concerted action by its abnormally elevated suppressors, MDM2, MDMX or SIRT1. Here, we report a novel small molecule Inauhzin (INZ) that effectively reactivates p53 by inhibiting SIRT1 activity, promotes p53‐dependent apoptosis of human cancer cells without causing apparently genotoxic stress. Moreover, INZ stabilizes p53 by increasing p53 acetylation and preventing MDM2‐mediated ubiquitylation of p53 in cells, though not directly in vitro. Remarkably, INZ inhibits cell proliferation, induces senescence and tumour‐specific apoptosis, and represses the growth of xenograft tumours derived from p53‐harbouring H460 and HCT116 cells without causing apparent toxicity to normal tissues and the tumour‐bearing SCID mice. Hence, our study unearths INZ as a novel anti‐cancer therapeutic candidate that inhibits SIRT1 activity and activates p53.

UV Induces p21 Rapid Turnover Independently of Ubiquitin and Skp2

It was previously reported that low doses, but not high doses, of UV trigger the Skp2-mediated proteasomal degradation of the cyclin-dependent kinase inhibitor p21 in mammalian cells. Here we show that both UV-C and UV-B lead to decrease of p21 protein, but not mRNA, level in a dose-dependent fashion in all of six human cell lines and five mouse cell lines tested. Also, high doses of UV reduce the half-life of p21. High doses, but not low doses, of UV induced p21 degradation in both skp2-proficient and -deficient murine embryonic fibroblast cells. UV-induced p21 reduction was rescued by proteasome inhibitors in all human and mouse cell lines tested. Neither a caspase inhibitor nor small interfering RNA against skp2 had an effect on the UV-induced p21 decrease, suggesting that this p21 degradation pathway may not involve caspases, or Skp2. Finally, UV did not induce p21 ubiquitination but still induced its degradation when the E1-activating enzyme was inactivated in an E1 temperature-sensitive mouse embryonic fibroblast cell line. Altogether, these results demonstrate that UV induces p21 degradation through an Skp2and ubiquitin-independent pathway.

Human SSRP1 Has Spt16-dependent and -independent Roles in Gene Transcription

The facilitating chromatin transcription (FACT) complex, a heterodimer of SSRP1 and Spt16, has been shown to regulate transcription elongation through a chromatin template in vitro and on specific genes in cells. However, its global role in transcription regulation in human cells remains largely elusive. We conducted spotted microarray analyses using arrays harboring 8308 human genes to assess the gene expression profile after knocking down SSRP1 or Spt16 levels in human non-small cell lung carcinoma (H1299) cells. Although the changes of these transcripts were surprisingly subtle, there were ∼170 genes whose transcript levels were either reduced or induced >1.5-fold. Approximately 106 genes with >1.2-fold change at the level of transcripts were the common targets of both SSRP1 and Spt16 (∼1.3%). A subset of genes was regulated by SSRP1 independent of Spt16. Further analyses of some of these genes not only verified this observation but also identified the serum-responsive gene, egr1, as a novel target for both SSRP1 and Spt16. We further showed that SSRP1 and Spt16 are important for the progression of elongation RNA pol II on the egr1 gene. These results suggest that SSRP1 has Spt16-dependent and -independent roles in regulating gene transcription in human cells.

p300 Regulates p63 Transcriptional Activity

The transcriptional co-activator p300 has been reported to regulate the tumor suppressor p53 and its ortholog p73. Here we describe a study showing that this coactivator also regulates the transcriptional function of p63. p300 bound to the N-terminal domain of p63γ, and p63γ bound to the N terminus of p300 in vitro and in cells. p300, but not its acetylase-defective mutant AT2, stimulated p63γ-dependent transcription and induction of p21 in cells, consequently leading to G1 arrest. Inversely, the ΔN-p63γ isoform as well as p300AT2 inhibited the induction of p21 by p63γ. These results suggest that p300 regulates p63-dependent transcription of p21.

AMP-activated protein kinase induces p53 by phosphorylating MDMX and inhibiting its activity.

ABSTRACT AMP-activated protein kinase (AMPK) has been shown to activate p53 in response to metabolic stress. However, the underlying mechanisms remain unclear. Here we show that metabolic stresses induce AMPK-mediated phosphorylation of human MDMX on Ser342 in vitro and in cells, leading to enhanced association between MDMX and 14-3-3. This markedly inhibits p53 ubiquitylation and significantly stabilizes and activates p53. By striking contrast, no phosphorylation of MDM2 by AMPK was noted. AMPK-mediated MDMX phosphorylation, MDMX–14-3-3 binding, and p53 activation were drastically reduced in mouse embryo fibroblasts harboring endogenous MDMX with S341A (mouse homologue of human serine 342), S367A, and S402A (mouse homologue of human serine 403) mutations. Moreover, deficiency of AMPK prevented MDMX–14-3-3 interaction and p53 activation. The activation of p53 through AMPK-mediated MDMX phosphorylation and inactivation was further confirmed by using cell and animal model systems with two AMPK activators, metformin and salicylate (the active form of aspirin). Together, the results unveil a mechanism by which metabolic stresses activate AMPK, which, in turn, phosphorylates and inactivates MDMX, resulting in p53 stabilization and activation.

Ribosomopathies: Mechanisms of Disease

Ribosomopathies are diseases caused by alterations in the structure or function of ribosomal components. Progress in our understanding of the role of the ribosome in translational and transcriptional regulation has clarified the mechanisms of the ribosomopathies and the relationship between ribosomal dysfunction and other diseases, especially cancer. This review aims to discuss these topics with updated information.