A mechanism of ubiquitin-independent proteasomal degradation of the tumor suppressors p53 and p73

Abstract

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...

Antizyme1 mediates AURKAIP1-dependent degradation of Aurora-A

“…Studies carried out using the A-box mutant of Aurora-A and temperature-sensitive cell line defective in ubiquitination at the restrictive temperature, suggest that Az1-mediated degradation of Aurora-A is mechanistically different from the cdh1-dependent degradation of Aurora-A and is Ub-independent. Despite the dismal knowledge we have on the significance and the physiological relevance of this alternative pathway, the growing list of protein substrates (Jin et al, 2003;Asher et al, 2005;Sdek et al, 2005) targeted to the proteasome in the absence of ubiquitination suggests that the Ub-independent route to the proteasome is as important as the Ub-dependent pathway. Az-mediated degradation of ODC, the first demonstrated prototype example of Ub-independent degradation, is essential for the maintenance of Thirty-six hours post-transfection, cells were harvested for western blot analysis of the endogenous Aurora-A.…”

A 19S proteasomal subunit cooperates with an ERK MAPK-regulated degron to regulate accumulation of Fra-1 in tumour cells

“…Fra-1 is one such protein, undergoing properly regulated proteasomal degradation even when all its lysine residues are substituted with arginines and its N terminus is blocked by fusion to a myc-epitope tag (Basbous et al, 2007. Other examples of cancer-associated proteins that can undergo ubiquitin-independent proteasomal degradation include c-Fos , p21 (Chen et al, 2007b), Rb (Kalejta and Shenk, 2003), KLF5 (Chen et al, 2007a), p53 and p73 (Asher et al, 2005). Although not well understood at the molecular level, ubiquitin-independent proteolysis may involve specific protein-protein interactions and the presence of poorly structured protein domains that can be recognized by proteasomal components (JarielEncontre et al, 2008).…”

p73 and caspase-cleaved p73 fragments localize to mitochondria and augment TRAIL-induced apoptosis

“…Under normal physiological conditions p73 is degraded either by calpains or by the 26S or 20S proteasomes (Asher et al, 2005;Munarriz et al, 2005;Rossi et al, 2005). While inhibition of calpains increases the steady-state levels of p73 , the E3 ubiquitin ligase Itch promotes 26S proteasome-dependent p73 degradation .…”

“…While inhibition of calpains increases the steady-state levels of p73 , the E3 ubiquitin ligase Itch promotes 26S proteasome-dependent p73 degradation . p73 is also subjected to ubiquitinindependent 20S proteasomal degradation that is regulated by NAD(P)H quinone oxidoreductase 1 together with NADH (Asher et al, 2005). After DNA damage, TAp73 rapidly accumulates (Agami et al, 1999;Gong et al, 1999) predominantly via transcription-independent mechanisms.…”