Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate

Authors

Kathleen M. Mulvaney, University of North Carolina at Chapel Hill
Jacob P. Matson, University of North Carolina at Chapel Hill
Priscila F. Siesser, University of North Carolina at Chapel Hill
Tigist Y. Tamir, University of North Carolina at Chapel Hill
Dennis Goldfarb, University of North Carolina at Chapel Hill
Timothy M. Jacobs, University of North Carolina at Chapel Hill
Erica W. Cloer, University of North Carolina at Chapel Hill
Joseph S. Harrison, University of the PacificFollow
Cyrus Vaziri, University of North Carolina at Chapel Hill
Jeanette G. Cook, University of North Carolina at Chapel Hill
Michael B. Major, University of North Carolina at Chapel Hill

ORCID

Joseph Harrison: 0000-0002-2118-6524

Document Type

Article

Publication Title

The Journal of Biological Chemistry

Department

Chemistry

ISSN

1083-351X

Volume

291

Issue

45

DOI

10.1074/jbc.M116.729418

First Page

23719

Last Page

23733

Publication Date

11-4-2016

Abstract

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.

Recommended Citation

Mulvaney, K. M., Matson, J. P., Siesser, P. F., Tamir, T. Y., Goldfarb, D., Jacobs, T. M., Cloer, E. W., Harrison, J. S., Vaziri, C., Cook, J. G., & Major, M. B. (2016). Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate. The Journal of Biological Chemistry, 291(45), 23719–23733. DOI: 10.1074/jbc.M116.729418
https://scholarlycommons.pacific.edu/cop-facarticles/569