Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate
| Autor: | Timothy M. Jacobs, Erica W. Cloer, Jacob Peter Matson, Jeanette Gowen Cook, Tigist Y. Tamir, Cyrus Vaziri, Priscila F. Siesser, Dennis Goldfarb, Michael B. Major, Joseph S. Harrison, Kathleen M. Mulvaney |
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| Rok vydání: | 2016 |
| Předmět: |
Models
Molecular 0301 basic medicine Protein subunit Biology Proteomics Biochemistry Cell Line Mice 03 medical and health sciences Ubiquitin Autophagy Animals Humans Protein Interaction Maps Molecular Biology Transcription factor Kelch-Like ECH-Associated Protein 1 Cell Cycle Ubiquitination Minichromosome Maintenance Complex Component 3 Signal transducing adaptor protein Cell Biology Cullin Proteins Molecular biology Chromatin Ubiquitin ligase Cell biology HEK293 Cells 030104 developmental biology biology.protein Carrier Proteins HeLa Cells Signal Transduction |
| Zdroj: | Journal of Biological Chemistry. 291:23719-23733 |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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