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.
Highlights
NRF2 degradation has long been thought to be the primary function of Kelch-like ECH-associated protein 1 (KEAP1), we have shown that KEAP1 associates with a number of interesting and diverse proteins, suggesting previously unknown roles for KEAP1 [19, 20]
HEK293T cells engineered for stable expression of KEAP1 fused with streptavidin binding peptide (SBP) and HA epitope were grown in stable isotope labeling with amino acids in cell culture (SILAC)-light medium or SILAC-heavy medium before addition of vehicle or MG132 proteasome inhibitor (Fig. 1A)
In addition to NRF2 signaling and the coordinated antioxidant response, KEAP1 has been shown to regulate NF-B signaling through its degradation of IKBKB to target the mitochondrial membrane phosphatase PGAM5 for proteasome-mediated degradation and to regulate DNA break repair through degradation-independent ubiquitylation of PALB2 [21,22,23]
Summary
Identification of MCM3 as a KEAP1 Substrate—We used two complementary strategies to identify proteins ubiquitylated by the KEAP1-CUL3-RBX1 complex. With the exception of NRF2 and NRF1, PAC-based analysis of the KEAP1 protein complex did not reveal new putative substrates. The class 1 proteins NRF1 and NRF2 increased rapidly in whole cell lysates and within the purified KEAP1 protein complex (ϳ5-fold and ϳ18-fold, respectively) (Fig. 1, C and D). Both NRF1 and NRF2 responded rapidly to proteasome inhibition, NRF2 is the only KEAP1 substrate that robustly accumulated in response to treatment with a ROS mimetic (sulforaphane) or KEAP1-CUL3 antagonist (MLN4924, bardoxolone methyl (CDDO-me)) (Fig. 1E) These results suggest two distinct classes of putative KEAP1 substrates: NRF1 and NRF2, which are short-lived, stress-responsive proteins that are rapidly turned over by the proteasome, and a second, more stable and higher abundance class of KEAP1 substrates comprised of PGAM5, MCM3, SLK, and MAD2L1.
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