Identification and characterization of MCM3 as a kelch-like ECH-associated protein 1 (KEAP1) substrate

Kathleen M. Mulvaney, Jacob P. Matson, Priscila F. Siesser, Tigist Y. Tamir, Dennis Goldfarb, Timothy M. Jacobs, Erica W. Cloer, Joseph S. Harrison, Cyrus Vaziri, Jeanette G. Cook, Michael B. Major

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63 Scopus citations


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 candidatebased 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 MCMhexamer. 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 newfunctions for KEAP1within the nucleusandidentifyMCM3as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.

Original languageEnglish
Pages (from-to)23719-23733
Number of pages15
JournalJournal of Biological Chemistry
Issue number45
StatePublished - Nov 4 2016


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