The Keap1-BTB Protein Is an Adaptor That Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 Ligase

ABSTRACT The Nrf2 transcription factor promotes survival following cellular insults that trigger oxidative damage. Nrf2 activity is opposed by the BTB/POZ domain protein Keap1. Keap1 is proposed to regulate Nrf2 activity strictly through its capacity to inhibit Nrf2 nuclear import. Recent work suggests that inhibition of Nrf2 may also depend upon ubiquitin-mediated proteolysis. To address the contribution of Keap1-dependent sequestration versus Nrf2 proteolysis, we identified the E3 ligase that regulates Nrf2 ubiquitination. We demonstrate that Keap1 is not solely a cytosolic anchor; rather, Keap1 is an adaptor that bridges Nrf2 to Cul3. We demonstrate that Cul3-Keap1 complexes regulate Nrf2 polyubiquitination both in vitro and in vivo. Inhibition of either Keap1 or Cul3 increases Nrf2 nuclear accumulation, leading to promiscuous activation of Nrf2-dependent gene expression. Our data demonstrate that Keap1 restrains Nrf2 activity via its capacity to target Nrf2 to a cytoplasmic Cul3-based E3 ligase and suggest a model in which Keap1 coordinately regulates both Nrf2 accumulation and access to target genes.

[1]  E. Gallagher,et al.  Induction of phase I and phase II drug-metabolizing enzyme mRNA, protein, and activity by BHA, ethoxyquin, and oltipraz. , 1995, Toxicology and applied pharmacology.

[2]  R. Vierstra,et al.  Identification of a Family of Closely Related Human Ubiquitin Conjugating Enzymes (*) , 1995, The Journal of Biological Chemistry.

[3]  K. Itoh,et al.  An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. , 1997, Biochemical and biophysical research communications.

[4]  M. Roussel,et al.  Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. , 1998, Genes & development.

[5]  A. Levine,et al.  Nuclear Export Is Required for Degradation of Endogenous p53 by MDM2 and Human Papillomavirus E6 , 1998, Molecular and Cellular Biology.

[6]  Wild Ac,et al.  Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. , 1999 .

[7]  Y. Kan,et al.  Nrf2 is essential for protection against acute pulmonary injury in mice. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. D. Engel,et al.  Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. , 1999, Genes & development.

[9]  R. Deshaies.  SCF and Cullin/Ring H2-based ubiquitin ligases. , 1999, Annual review of cell and developmental biology.

[10]  A. Choi,et al.  Nrf2, a Cap’n’Collar Transcription Factor, Regulates Induction of the Heme Oxygenase-1 Gene* , 1999, The Journal of Biological Chemistry.

[11]  C. Wolf,et al.  The Nrf2 transcription factor contributes both to the basal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin. , 2000, Biochemical Society transactions.

[12]  H. Huang,et al.  Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Ron,et al.  Perk is essential for translational regulation and cell survival during the unfolded protein response. , 2000, Molecular cell.

[14]  J. Chan,et al.  Impaired expression of glutathione synthetic enzyme genes in mice with targeted deletion of the Nrf2 basic-leucine zipper protein. , 2000, Biochimica et biophysica acta.

[15]  M. McMahon,et al.  Molecular basis for the contribution of the antioxidant responsive element to cancer chemoprevention. , 2001, Cancer letters.

[16]  L. Zipper,et al.  The Keap1 BTB/POZ Dimerization Function Is Required to Sequester Nrf2 in Cytoplasm* , 2002, The Journal of Biological Chemistry.

[17]  Peichuan Zhang,et al.  The PERK Eukaryotic Initiation Factor 2α Kinase Is Required for the Development of the Skeletal System, Postnatal Growth, and the Function and Viability of the Pancreas , 2002, Molecular and Cellular Biology.

[18]  M. Tyers,et al.  The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase , 2003, Nature.

[19]  J. D. Engel,et al.  Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation , 2003, Nature Genetics.

[20]  Mark Hannink,et al.  Distinct Cysteine Residues in Keap1 Are Required for Keap1-Dependent Ubiquitination of Nrf2 and for Stabilization of Nrf2 by Chemopreventive Agents and Oxidative Stress , 2003, Molecular and Cellular Biology.

[21]  J. Yates,et al.  BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases. , 2003, Molecular cell.

[22]  Y. Xiong,et al.  Targeting of protein ubiquitination by BTB–Cullin 3–Roc1 ubiquitin ligases , 2003, Nature Cell Biology.

[23]  J. Alam,et al.  Degradation of Transcription Factor Nrf2 via the Ubiquitin-Proteasome Pathway and Stabilization by Cadmium* , 2003, The Journal of Biological Chemistry.

[24]  K. Itoh,et al.  Keap1-dependent Proteasomal Degradation of Transcription Factor Nrf2 Contributes to the Negative Regulation of Antioxidant Response Element-driven Gene Expression* , 2003, Journal of Biological Chemistry.

[25]  Yanping Zhang,et al.  Nucleocytoplasmic Shuttling of p53 Is Essential for MDM2-Mediated Cytoplasmic Degradation but Not Ubiquitination , 2003, Molecular and Cellular Biology.

[26]  K. Itoh,et al.  Keap1 regulates both cytoplasmic‐nuclear shuttling and degradation of Nrf2 in response to electrophiles , 2003, Genes to cells : devoted to molecular & cellular mechanisms.

[27]  S. Elledge,et al.  BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3 , 2003, Nature.

[28]  Randal J. Kaufman,et al.  Nrf2 Is a Direct PERK Substrate and Effector of PERK-Dependent Cell Survival , 2003, Molecular and Cellular Biology.

[29]  H. Huang,et al.  Increased Protein Stability as a Mechanism That Enhances Nrf2-mediated Transcriptional Activation of the Antioxidant Response Element , 2003, The Journal of Biological Chemistry.

[30]  J. Alan Diehl,et al.  PERK-dependent Activation of Nrf2 Contributes to Redox Homeostasis and Cell Survival following Endoplasmic Reticulum Stress* , 2004, Journal of Biological Chemistry.