Protein degradation is an essential and highly regulated process. The proteasomal degradation of the tumor suppressors p53 and p73 is regulated by both polyubiquitination and by an ubiquitin-independent process. Here, we show that this ubiquitin-independent process is mediated by the 20S proteasomes and is regulated by NQO1. NQO1 physically interacts with p53 and p73 in an NADH-dependent manner and protects them from 20S proteasomal degradation. Remarkably, the vast majority of NQO1 in cells is found in physical association with the 20S proteasomes, suggesting that NQO1 functions as a gatekeeper of the 20S proteasomes. We further show that this pathway plays a role in p53 accumulation in response to ionizing radiation. Our findings provide the first evidence for in vivo degradation of p53 and p73 by the 20S proteasomes and its regulation by NQO1 and NADH level. Protein degradation determines the outcome of many cellular physiological processes (Coux et al. 1996). Degradation of proteins by the proteasomes occurs via various pathways (Verma and Deshaies 2000;Pickart and Cohen 2004). The most intensely studied one is the ubiquitin-26S proteasome pathway (Hershko 1996;Hershko and Ciechanover 1998;Goldberg 2003). The tumor suppressor p53 is a very labile protein that undergoes Mdm2 and ubiquitin-dependent 26S proteasomal degradation (Haupt et al. 1997;Kubbutat et al. 1997). Recently, we reported that degradation of p53 also occurs in an Mdm2 and ubiquitin-independent manner (Asher et al. 2002b). This pathway of p53 degradation is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1) (Asher et al. 2001(Asher et al. , 2002a(Asher et al. ,b, 2003(Asher et al. , 2004), yet the underlying molecular mechanisms that control p53 degradation remained elusive. Results and DiscussionTo investigate the role of NQO1 in proteasomal degradation, we followed NQO1 distribution in fractionated mouse liver extracts. Ammonium sulfate precipitation and gel-filtration chromatography of liver extracts revealed that the majority of NQO1 cofractionated with the 20S proteasomes (Fig. 1A). These fractions are devoid of the 26S proteasomes that were excluded by the differential ammonium sulfate precipitation (Fig. 1A, IB: 26S, anti TBP1 a subunit of the 19S). These results suggest that the vast majority of cellular NQO1 is found in a large protein complex that possibly includes the 20S proteasomes.To further study this possibility, the 20S-containing fractions were pooled and fractionated by anion exchange chromatography according to a standard 20S purification protocol (Friguet et al. 2002). Remarkably, NQO1 was detected in the 0.3 M NaCl fraction containing the 20S proteasomes (Fig. 1B). Electrophoresis of the 0.3 M NaCl fraction on a nondenaturing PAGE, followed by peptidase activity assay, showed that the purified 20S is functional (Fig. 1C, Activity panel). Immunoblot analysis with anti NQO1 antibody revealed that NQO1 comigrated with the 20S proteasomes, but not with the 26S proteasomes (Fig. 1C). Finally, a coimmunopercipitation experim...

Hypoxic stress induces the expression of genes associated with increased energy flux, including the glucose transporters Glut1 and Glut3, several glycolytic enzymes, nitric oxide synthase, tyrosine hydroxylase, erythropoietin and vascular endothelial growth factor (VEGF). Induction of these genes is mediated by a common basic helix-loop-helix-PAS transcription complex, the hypoxia-inducible factor-1α (HIF-1α)/ aryl hydrocarbon nuclear translocator (ARNT). Insulin also induces some of these genes; however, the underlying mechanism is unestablished. We report here that insulin shares with hypoxia the ability to induce the HIF-1α/ARNT transcription complex in various cell types. This induction was demonstrated by electrophoretic mobility shift of the hypoxia response element (HRE), and abolished by specific antisera to HIF-1α and ARNT, and by transcription activation of HRE reporter vectors. Furthermore, basal and insulininduced expression of Glut1, Glut3, aldolase A, phosphoglycerate kinase and VEGF was reduced in cells having a defective ARNT. Similarly, the insulin-induced activation of HRE reporter vectors and VEGF was impaired in these cells and was rescued by re-introduction of ARNT. Finally, insulin-like growth factor-I (IGF-I) also induced the HIF-1α/ARNT transcription complex. These observations establish a novel signal transduction pathway of insulin and IGF-I and broaden considerably the scope of activity of HIF-1α/ARNT.

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 ...

We have previously shown that the degradation of c-myc and N-myc in vitro is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins in vivo and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins