P53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1.
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.
Asher
Gad
G
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Lotem
Joseph
J
Tsvetkov
Peter
P
Reiss
Veronica
V
Sachs
Leo
L
Shaul
Yosef
Y
D000818:Animals; D017209:Apoptosis; D001120:Arginine / Q000737:chemistry; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004252:DNA Mutational Analysis; D001728:Dicumarol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D016158:Genes, p53; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry* / Q000378:metabolism; D009687:Nuclear Proteins; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism*
D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D001728:Dicumarol; D001120:Arginine; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex