title: BTB protein Keap1 targets antioxidant transcription factor Nrf2 for ubiquitination by the Cullin 3-Roc1 ligase.
abstract: The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (Cul3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. Here we report that the human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promoted NRF2 ubiquitination in vitro and knocking down Keap1 or CUL3 by short interfering RNA resulted in NRF2 protein accumulation in vivo. We suggest that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down Cul3, resulted in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic sequestration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF2 degradation.
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA.
authors.forename: Manabu
authors.initials: M
authors.lastname: Furukawa
authors.forename: Yue
authors.initials: Y
authors.lastname: Xiong
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000975:Antioxidants / Q000378:metabolism*; D050976:Basic-Leucine Zipper Transcription Factors; D002352:Carrier Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D044842:Cullin Proteins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation; D020868:Gene Silencing; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D000072019:Kelch-Like ECH-Associated Protein 1; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000502:physiology*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D000975:Antioxidants; D050976:Basic-Leucine Zipper Transcription Factors; C503645:CUL3 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; C117963:NFE2L3 protein, human; D011506:Proteins; C479529:RBX1 protein, human; D011994:Recombinant Proteins; D014157:Transcription Factors; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D046988:Proteasome Endopeptidase Complex