Keap1 is a Redox-regulated Substrate Adaptor Protein for a Cul3-dependent Ubiquitin Ligase Complex

Overview

Journal Mol Cell Biol

Specialty Cell Biology

Date 2004 Dec 2

PMID 15572695

Citations 626

Authors

Donna D Zhang

Shih-Ching Lo

Janet V Cross

Dennis J Templeton

Mark Hannink

Affiliations

Soon will be listed here.

Abstract

The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.

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References

Chan K, Kan Y . Nrf2 is essential for protection against acute pulmonary injury in mice. Proc Natl Acad Sci U S A. 1999; 96(22):12731-6. PMC: 23072. DOI: 10.1073/pnas.96.22.12731. View

Cullinan S, Zhang D, Hannink M, Arvisais E, Kaufman R, Diehl J . Nrf2 is a direct PERK substrate and effector of PERK-dependent cell survival. Mol Cell Biol. 2003; 23(20):7198-209. PMC: 230321. DOI: 10.1128/MCB.23.20.7198-7209.2003. View

Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y . Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem. 2000; 275(21):16023-9. DOI: 10.1074/jbc.275.21.16023. View

Buschmann T, Fuchs S, Lee C, Pan Z, Ronai Z . SUMO-1 modification of Mdm2 prevents its self-ubiquitination and increases Mdm2 ability to ubiquitinate p53. Cell. 2000; 101(7):753-62. DOI: 10.1016/s0092-8674(00)80887-9. View

Bomont P, Cavalier L, Blondeau F, Ben Hamida C, Belal S, Tazir M . The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy. Nat Genet. 2000; 26(3):370-4. DOI: 10.1038/81701. View

Enomoto A, Itoh K, Nagayoshi E, Haruta J, Kimura T, OConnor T . High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicol Sci. 2001; 59(1):169-77. DOI: 10.1093/toxsci/59.1.169. View

Dinkova-Kostova A, Massiah M, Bozak R, Hicks R, Talalay P . Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc Natl Acad Sci U S A. 2001; 98(6):3404-9. PMC: 30666. DOI: 10.1073/pnas.051632198. View

Ramos-Gomez M, Kwak M, Dolan P, Itoh K, Yamamoto M, Talalay P . Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci U S A. 2001; 98(6):3410-5. PMC: 30667. DOI: 10.1073/pnas.051618798. View

McMahon M, Itoh K, Yamamoto M, Chanas S, Henderson C, McLellan L . The Cap'n'Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer Res. 2001; 61(8):3299-307. View

10.

Butterfield D, Howard B, LaFontaine M . Brain oxidative stress in animal models of accelerated aging and the age-related neurodegenerative disorders, Alzheimer's disease and Huntington's disease. Curr Med Chem. 2001; 8(7):815-28. DOI: 10.2174/0929867013373048. View

11.

Aoki Y, Sato H, Nishimura N, Takahashi S, Itoh K, Yamamoto M . Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust. Toxicol Appl Pharmacol. 2001; 173(3):154-60. DOI: 10.1006/taap.2001.9176. View

12.

Imlay J . Pathways of oxidative damage. Annu Rev Microbiol. 2003; 57:395-418. DOI: 10.1146/annurev.micro.57.030502.090938. View

13.

Geyer R, Wee S, Anderson S, Yates J, Wolf D . BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases. Mol Cell. 2003; 12(3):783-90. DOI: 10.1016/s1097-2765(03)00341-1. View

14.

Zhang D, Hannink M . Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress. Mol Cell Biol. 2003; 23(22):8137-51. PMC: 262403. DOI: 10.1128/MCB.23.22.8137-8151.2003. View

15.

Furukawa M, He Y, Borchers C, Xiong Y . Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases. Nat Cell Biol. 2003; 5(11):1001-7. DOI: 10.1038/ncb1056. View

16.

Wakabayashi N, Itoh K, Wakabayashi J, Motohashi H, Noda S, Takahashi S . Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation. Nat Genet. 2003; 35(3):238-45. DOI: 10.1038/ng1248. View

17.

Bloom D, Jaiswal A . Phosphorylation of Nrf2 at Ser40 by protein kinase C in response to antioxidants leads to the release of Nrf2 from INrf2, but is not required for Nrf2 stabilization/accumulation in the nucleus and transcriptional activation of antioxidant response.... J Biol Chem. 2003; 278(45):44675-82. DOI: 10.1074/jbc.M307633200. View

18.

Wolf D, Zhou C, Wee S . The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases?. Nat Cell Biol. 2003; 5(12):1029-33. DOI: 10.1038/ncb1203-1029. View

19.

Pickart C . Mechanisms underlying ubiquitination. Annu Rev Biochem. 2001; 70:503-33. DOI: 10.1146/annurev.biochem.70.1.503. View

20.

Cho H, Jedlicka A, Reddy S, Kensler T, Yamamoto M, Zhang L . Role of NRF2 in protection against hyperoxic lung injury in mice. Am J Respir Cell Mol Biol. 2002; 26(2):175-82. DOI: 10.1165/ajrcmb.26.2.4501. View