Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from the ubiquitin ligase CUL3.
The regulation of cellular stress responses to electrophiles and oxidants is mediated by the transcription factor NF-E2-related factor 2 (Nrf2), which, in turn, is regulated by CUL-E3 (CUL3) ligase-mediated ubiquitylation. The Kelch-like ECH-associated protein 1 (Keap1) serves as an adapter between CUL3 and Nrf2. We used the model electrophile N-iodoacetyl- N-biotinylhexylenediamine (IAB) to define the relationship among the adduction of Keap1 cysteine residues, structure, and function. Exposure of Keap1 to IAB in vitro was accompanied by progressive loss of protein secondary structure, as monitored by CD spectroscopy and a loss of the ability to associate with recombinant CUL3. Dissociation of Keap1 from CUL3 in vitro was dependent upon C151 in Keap1. A quantitative mass spectrometry-based kinetic analysis of adduction in HEK293 cells expressing FLAG-Keap1 revealed that Cys151 was one of the most reactive residues in vivo and that it was required for IAB-mediated dissociation of the Keap1-CUL3 interaction. These results demonstrate that Cys151 adduction confers a critical alkylation sensor function upon Keap1, making Keap1 unique among BTB CUL3 adapter proteins.
Rachakonda
Girish
G
Department of Radiation Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Xiong
Ying
Y
Sekhar
Konjeti R
KR
Stamer
Sheryl L
SL
Liebler
Daniel C
DC
Freeman
Michael L
ML
D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000737:chemistry*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D044842:Cullin Proteins / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004274:DNA, Recombinant / Q000138:chemical synthesis / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry*; D000072019:Kelch-Like ECH-Associated Protein 1; D007700:Kinetics; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000737:chemistry
C503645:CUL3 protein, human; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D004274:DNA, Recombinant; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D011994:Recombinant Proteins; D003545:Cysteine