title: Cancer-derived mutations in KEAP1 impair NRF2 degradation but not ubiquitination.
abstract: NRF2 is a transcription factor that mediates stress responses. Oncogenic mutations in NRF2 localize to one of its two binding interfaces with KEAP1, an E3 ubiquitin ligase that promotes proteasome-dependent degradation of NRF2. Somatic mutations in KEAP1 occur commonly in human cancer, where KEAP1 may function as a tumor suppressor. These mutations distribute throughout the KEAP1 protein but little is known about their functional impact. In this study, we characterized 18 KEAP1 mutations defined in a lung squamous cell carcinoma tumor set. Four mutations behaved as wild-type KEAP1, thus are likely passenger events. R554Q, W544C, N469fs, P318fs, and G333C mutations attenuated binding and suppression of NRF2 activity. The remaining mutations exhibited hypomorphic suppression of NRF2, binding both NRF2 and CUL3. Proteomic analysis revealed that the R320Q, R470C, G423V, D422N, G186R, S243C, and V155F mutations augmented the binding of KEAP1 and NRF2. Intriguingly, these "super-binder" mutants exhibited reduced degradation of NRF2. Cell-based and in vitro biochemical analyses demonstrated that despite its inability to suppress NRF2 activity, the R320Q "superbinder" mutant maintained the ability to ubiquitinate NRF2. These data strengthen the genetic interactions between KEAP1 and NRF2 in cancer and provide new insight into KEAP1 mechanics.
authors: Authors' Affiliations: Department of Cell Biology and Physiology; Lineberger Comprehensive Cancer Center; and Division of Medical Oncology, Department of Internal Medicine and Otolaryngology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine; Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and Department of Pharmacology, Howard Hughes Medical Institute, University of Washington, Seattle, Washington.
authors: Bridgid E
authors: BE
authors: Hast
authors: Erica W
authors: EW
authors: Cloer
authors: Dennis
authors: D
authors: Goldfarb
authors: Heng
authors: H
authors: Li
authors: Priscila F
authors: PF
authors: Siesser
authors: Feng
authors: F
authors: Yan
authors: Vonn
authors: V
authors: Walter
authors: Ning
authors: N
authors: Zheng
authors: D Neil
authors: DN
authors: Hayes
authors: Michael B
authors: MB
authors: Major
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1; D008958:Models, Molecular; D009154:Mutation; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D059748:Proteolysis; D054875:Ubiquitination
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2