CR6-interacting Factor 1 (CRIF1) Regulates NF-E2-related Factor 2 (NRF2) Protein Stability by Proteasome-mediated Degradation

Hyo Jin Kang,Young Bin Hong,Hee Jeong Kim,Insoo Bae

doi:10.1074/jbc.m109.084590

Hyo Jin Kang, Young Bin Hong + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.084590

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Abstract

Free radicals generated by oxidative stress cause damage that can contribute to numerous chronic diseases. Mammalian cells respond to this damage by increased transcription of cytoprotective phase II genes, which are regulated by NRF2. Previously, it has been shown that NRF2 protein levels increase after oxidative stress because its negative regulator, KEAP1, loses its ability to bind NRF2 and cause its proteasome-mediated degradation during oxidative stress. Here, we show that CRIF1, a protein previously known as cell cycle regulator and transcription cofactor, is also able to negatively regulate NRF2 protein stability. However, in contrast to KEAP1, which regulates NRF2 stability only under normal reducing conditions, CRIF1 regulates NRF2 stability and its target gene expression under both reducing and oxidative stress conditions. Thus, CRIF1-NRF2 interactions and their consequences are redox-independent. In addition, we found that CRIF1, unlike KEAP1 (which only interacts with N-terminal region of NRF2), physically interacts with both N- and C-terminal regions of NRF2 and promotes NRF2 ubiquitination and subsequent proteasome-mediated NRF2 protein degradation.

Highlights

Oxidative stress leads to the accelerated production and increased accumulation of reactive oxygen species (ROS),2 which contribute to both cancer and aging by causing oxidative damage to proteins and DNA [1]
Under conditions of oxidative stress, KEAP1 loses its ability to limit NF-E2-related factor 2 (NRF2) protein accumulation and to block NRF2 from transcribing its target genes. This occurs because oxidative stress/oxidative stress-generated ROS oxidize a Cys–Cys covalent bond, which causes a conformational change in KEAP1, enabling NRF2 to escape from the imposed cytoplasmic localization, ubiquitination, and degradation of KEAP1
By using an expression vector construct encoding a fusion protein between the GAL4 DNA binding domain and a bait protein, a cDNA encoding part of NRF2 was identified in a Matchmaker cDNA library. To confirm this result and to test for a direct NRF2-CRIF1 protein-protein interaction, we performed reciproantibodies against ␣/␤-tubulin and lamin B1 as indicators for cal co-immunoprecipitation experiments with total lysates prethe purity of the cytosol and nuclear fractions, respectively. pared from cells transfected with expression vectors for FLAG

Summary

Introduction

Oxidative stress leads to the accelerated production and increased accumulation of reactive oxygen species (ROS),2 which contribute to both cancer and aging by causing oxidative damage to proteins and DNA [1]. Overexpression of either CRIF1 or KEAP1 reduced exogenous wild-type NRF2 protein levels in total cell lysates (Fig. 4A, 2nd panel from the top, lanes 2 and 3).

Results

Conclusion