Mdm-2 and ubiquitin-independent p53 proteasomal degradation regulated by NQO1

Abstract

The tumor suppressor p53 is a labile protein whose level is known to be regulated by the Mdm-2-ubiquitin-proteasome degradation pathway. We have found another pathway for p53 proteasomal degradation regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). Inhibition of NQO1 activity by dicoumarol induces p53 and p73 proteasomal degradation. A mutant p53 (p53 [22,23] ), which is resistant to Mdm-2-mediated degradation, was susceptible to dicoumarol-induced degradation. This finding indicates that the NQO1-regulated proteasomal p53 degradation is Mdm-2-independent. The tumor suppressor p14 ARF and the viral oncogenes SV40 LT and adenovirus E1A that are known to stabilize p53 inhibited dicoumarol-induced p53 degradation. Unlike Mdm-2-mediated degradation, the NQO1-regulated p53 degradation pathway was not associated with accumulation of ubiquitin-conjugated p53. In vitro studies indicate that dicoumarol-induced p53 degradation was ubiquitin-independent and ATP-dependent. Inhibition of NQO1 activity in cells with a temperature-sensitive E1 ubiquitinactivating enzyme induced p53 degradation and inhibited apoptosis at the restrictive temperature without ubiquitination. Mdm-2 failed to induce p53 degradation under these conditions. Our results establish a Mdm-2-and ubiquitin-independent mechanism for proteasomal degradation of p53 that is regulated by NQO1. The lack of NQO1 activity that stabilizes a tumor suppressor such as p53 can explain why humans carrying a polymorphic inactive NQO1 are more susceptible to tumor development. Wild-type p53 is a tumor suppressor that accumulates in response to DNA damage and other types of stress and induces either growth arrest (1-4) or apoptosis (5-8). p53 level is regulated by the rate of its degradation, which is known to be mediated by the Mdm-2-ubiquitin-proteasome degradation pathway (9, 10). Mdm-2 is an E3 ubiquitin ligase that binds to the N terminus of p53 and ubiquitinates it. Stabilization of p53 can be enhanced by disruption of p53-Mdm-2 interaction, which prevents Mdm-2 from ubiquitinating p53. This stabilization occurs by posttranslational modifications of p53 and Mdm-2 (3, 4) or by their interaction with other proteins including simian virus 40 (SV40) large T antigen (LT; refs. 11 and 12), hsp90 (13-15), and p14 ARF (16,17). Ubiquitin-dependent degradation is a multistep process that plays an important role in the degradation of abnormal and short-lived proteins. Ubiquitin is first activated by an E1 ubiquitin-activating enzyme, transferred to one of several E2 ubiquitin-conjugating enzymes, and then ligated to lysine residues of target proteins by specific E3 ubiquitin ligases. Further ubiquitination leads to the formation of polyubiquitinated proteins that are recognized by proteasomes for degradation (18,19).We have previously reported that NAD(P)H quinone oxidoreductase 1 (NQO1) regulates p53 stability and that inhibition of NQO1 activity by dicoumarol induces proteasomal degradation of p53 and inhibits p53-mediated apoptosis (20). We have also shown that wild-type ...

Ubiquitin-independent p53 proteasomal degradation

“…Furthermore, the degradation takes place even under conditions whereby the pathway of protein ubiquitination is completely inhibited. 18 These findings and some others, as detailed below, argue for an alternative pathway of p53 degradation that takes place by default. Cells that are dicoumarol treated to induce p53 degradation by default escape DNA damage-induced apoptosis.…”

“…12 Accordingly, knockdown of NQO1 reduces the basal p53 level. 13 Furthermore, NQO1 null mice exhibit reduced p53 protein levels and decreased apoptosis in the bone marrow. 14 NQO1 binds to p53 in an NADH-dependent manner.…”

“…Consequently, the adenovirus E1A oncogene that stabilizes p53 by inducing p14 ARF also inhibits p53 ubiquitin-independent degradation. 13 Thus, p14 ARF exhibits a double lock activity by negating the two p53-degradation pathways, ensuring maximal p53 accumulation on demand. The viral oncogene SV40 LT also exhibits double lock activity by binding to p53 and protecting it from both Mdm2-mediated as well as ubiquitin-independent degradation.…”

“…68,69 The product of this NQO1 genotype is less active in stabilizing p53. 18 Several studies have found that the C609T allele is associated with increased risk of developing different types of tumors such as urothelial tumors, basal cell carcinoma, pediatric leukemia, colorectal cancer, esophageal squamous cell carcinoma, and gastric carcinoma. 70 A recent paper reports that this polymorphism is a strong prognostic and predictive factor in breast cancer.…”

Redox-regulated Turnover of Nrf2 Is Determined by at Least Two Separate Protein Domains, the Redox-sensitive Neh2 Degron and the Redox-insensitive Neh6 Degron

“…Cell Culture, Transfections, Chemical Challenge, and Reporter Assays-ts20TG R and H38.5 cells (26) [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] and the ETGE motif (amino acids 79 -82) of the Neh2 domain are highlighted, as are two highly conserved regions of the Neh6 domain (amino acids 329 -339 and 363-379). A complete sequence alignment of orthologous Nrf2 proteins from human to fish and a description of the known functions of the Neh domains can be found in the supplemental Fig.…”