Ubiquitination of Keap1, a BTB-Kelch substrate adaptor protein for Cul3, targets Keap1 for degradation by a proteasome-independent pathway.
Keap1 is a BTB-Kelch protein that functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 targets its substrate, the Nrf2 transcription factor, for ubiquitination and subsequent degradation by the 26 S proteasome. Inhibition of Keap1-dependent ubiquitination of Nrf2 increases steady-state levels of Nrf2 and enables activation of cytoprotective Nrf2-dependent genes. In this report, we demonstrate that Keap1 and three other BTB-Kelch proteins, including GAN1, ENC1, and Sarcosin, are ubiquitinated by a Cul3-dependent complex. Ubiquitination of Keap1 is markedly increased in cells exposed to quinone-induced oxidative stress, occurs in parallel with inhibition of Keap1-dependent ubiquitination of Nrf2, and results in decreased steady-state levels of Keap1, particularly in cells that are unable to synthesize glutathione. Degradation of Keap1 is independent of the 26 S proteasome, because inhibitors of the 26 S proteasome do not prevent loss of Keap1 following exposure of cells to quinone-induced oxidative stress. Our results suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates.
Zhang
Donna D
DD
Department of Biochemistry, University of Missouri, Columbia, Missouri 65212, USA.
Lo
Shih-Ching
SC
Sun
Zheng
Z
Habib
Geetha M
GM
Lieberman
Michael W
MW
Hannink
Mark
M
D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002686:Chitin / Q000378:metabolism; D044842:Cullin Proteins / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005822:Genetic Vectors; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; D018384:Oxidative Stress; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
C503645:CUL3 protein, human; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; C586302:KLHL41 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D011506:Proteins; D011809:Quinones; D011994:Recombinant Proteins; D015534:Trans-Activators; D025801:Ubiquitin; D002686:Chitin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D005978:Glutathione