Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate.
KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Toward this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1-interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2-7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests that MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2-7 complex. KEAP1 is thus poised to affect MCM2-7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.
From the Departments of Cell Biology and Physiology.|Lineberger Comprehensive Cancer Center, and.
Kathleen M
KM
Mulvaney
Biochemistry and Biophysics.
Jacob P
JP
Matson
Lineberger Comprehensive Cancer Center, and.
Priscila F
PF
Siesser
Lineberger Comprehensive Cancer Center, and.|Pharmacology.
Tigist Y
TY
Tamir
Lineberger Comprehensive Cancer Center, and.|Computer Science, and.
Dennis
D
Goldfarb
Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599.
Timothy M
TM
Jacobs
From the Departments of Cell Biology and Physiology.|Lineberger Comprehensive Cancer Center, and.
Erica W
EW
Cloer
Lineberger Comprehensive Cancer Center, and.|Biochemistry and Biophysics.
Joseph S
JS
Harrison
Lineberger Comprehensive Cancer Center, and.|Pathology.
Cyrus
C
Vaziri
Lineberger Comprehensive Cancer Center, and jgcook@email.unc.edu.|Biochemistry and Biophysics.
Jeanette G
JG
Cook
From the Departments of Cell Biology and Physiology, benmajor@med.unc.edu.|Lineberger Comprehensive Cancer Center, and.|Pharmacology.|Computer Science, and.
Michael B
MB
Major
D000818:Animals; D001343:Autophagy; D002352:Carrier Proteins / Q000378:metabolism; D002453:Cell Cycle; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D044842:Cullin Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D000072019:Kelch-Like ECH-Associated Protein 1 / Q000378:metabolism*; D051379:Mice; D064126:Minichromosome Maintenance Complex Component 3 / Q000378:metabolism*; D008958:Models, Molecular; D060066:Protein Interaction Maps; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination
C503645:CUL3 protein, human; D002352:Carrier Proteins; D002843:Chromatin; D044842:Cullin Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; C066470:MCM3 protein, human; C479529:RBX1 protein, human; D025801:Ubiquitin; D064126:Minichromosome Maintenance Complex Component 3
DNA replication; E3 ubiquitin ligase; KEAP1; MCM3; NFE2L2; Nrf2; erythroid-derived 2-like factor; nuclear factor 2; oxidative stress; proteomics