p53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1

Abstract

Proteasomal degradation of p53 is mediated by two alternative pathways that are either dependent or independent of both Mdm2 and ubiquitin. The ubiquitin-independent pathway is regulated by NAD(P)H: quinone oxidoreductase 1 (NQO1) that stabilizes p53. The NQO1 inhibitor dicoumarol induces ubiquitin-independent p53 degradation. We now show that, like dicoumarol, several other coumarin and flavone inhibitors of NQO1 activity, which compete with NAD(P)H for binding to NQO1, induced ubiquitin-independent p53 degradation and inhibited wild-type p53-mediated apoptosis. Although wild-type p53 and several p53 mutants were sensitive to dicoumarol-induced degradation, the most frequent ''hot-spot'' p53 mutants in human cancer, R175H, R248H, and R273H, were resistant to dicoumarol-induced degradation, but remained sensitive to Mdm2-ubiquitin-mediated degradation. The two alternative pathways for p53 degradation thus have different p53 structural requirements. Further mutational analysis showed that arginines at positions 175 and 248 were essential for dicoumarol-induced p53 degradation. NQO1 bound to wild-type p53 and dicoumarol, which induced a conformational change in NQO1, inhibited this binding. Compared with wild-type p53, the hot-spot p53 mutants showed increased binding to NQO1, which can explain their resistance to dicoumarol-induced degradation. NQO1 thus has an important role in stabilizing hot-spot p53 mutant proteins in human cancer.W ild-type p53 is a labile protein and its cellular level is mainly regulated by the rate of its proteasomal degradation (reviewed in ref. 1). p53 degradation is mediated by two alternative pathways that either depend on Mdm2 and ubiquitin (2, 3) or are independent of both (4). The Mdm2-and ubiquitinindependent pathway is regulated by NAD(P)H: quinone oxidoreductase 1 (NQO1) (4-7). Our previous studies showed that NQO1 stabilizes p53 (5, 6) and that reducing the NQO1 level by small interfering RNA decreases the level of p53 (4). Dicoumarol is a competitive inhibitor of NQO1 activity that competes with NAD(P)H for binding to NQO1 (8) and induces ubiquitinindependent p53 degradation (4-7). We suggested that p53 stabilization requires the physical interaction of p53 with NQO1 (6), and recent studies have shown that NQO1 can bind to p53 (9). The tumor suppressor wild-type p53 is mutated in Ͼ50% of human cancers (1, 10, 11). We have now studied the control of ubiquitin-independent p53 degradation by using different NQO1 inhibitors, different p53 mutants, and binding of NQO1 to p53. Our results include the finding that the most frequent ''hot-spot'' p53 mutants in human cancer showed increased binding to NQO1 and resisted dicoumarol-induced degradation. These findings indicate that NQO1 plays a major role in stabilizing hot-spot p53 mutant proteins in human cancer cells. Materials and MethodsCells and Cell Culture. The cell lines used were: p53 null HCT116 human colon carcinoma cells (12), 293 human kidney cells, Huh 7 human hepatocellular carcinoma cells that carry the Y220P mutant 53 (1...

Ubiquitin-independent p53 proteasomal degradation

“…14 NQO1 binds to p53 in an NADH-dependent manner. [15][16][17] Interestingly, dicoumarol and other competitive inhibitors of NQO1 compete with NADH for the binding to NQO1, resulting in the dissociation of the NQO1-p53 complex. Remarkably, p53 becomes highly unstable and prone to proteasomal degradation in the presence of these inhibitors.…”

“…Strong alterations in the NAD þ / NADH ratio in the cells are expected to affect the degradation by default of p53 default in cells as they bind NQO1 with higher affinity. 16 Given the fact that some of the mutations strongly disrupt p53 conformation, they may fold into structures that resist degradation by the 20S proteasome. The loss of zinc binding to the DNA-binding domain of p53 has also been shown to increase thermodynamic instability, aggregation, and decrease specific DNA binding in vitro.…”

“…As described above, some of the p53 mutants are more resistant to the ubiquitin-independent pathway simply by binding NQO1 with higher affinity. 16 This provides another mechanism for relatively high steady-state expression levels of these mutant proteins in cancer cells. 65 By virtue of its ability to protect p53 from ubiquitinindependent degradation, NQO1 can be considered a tumor suppressor.…”

Mutant p53 in MDA-MB-231 breast cancer cells is stabilized by elevated phospholipase D activity and contributes to survival signals generated by phospholipase D

“…The observation that other tumor suppressor genes are commonly deleted in tumors leading to loss-of-function phenotypes suggests that the low incidence of deletion mutants for p53 in human cancer reflects a positive selection for the missence mutations in p53. Moreover, mutant p53 in human cancer is commonly expressed at high levels and is more stable than wild-type p53 (Midgley and Lane 1997;Peng et al, 2001a, b;Asher et al, 2003). These observations suggest that a positive selection for mutations to p53 that contribute to tumorigenesis.…”

Ets1 is an effector of protein kinase Cα in cancer cells

“…NQO1 plays a role in the degradation of the tumor suppressor protein p53. Intriguingly, it stabilizes predominantly mutant p53 (Asher et al, 2003). Mutant p53, which is expressed in MDA-MB-231 cells, cooperates with Ets1 to induce the multidrug-resistance protein MDR1 (Sampath et al, 2001).…”