Subcellular localization and cytoplasmic complex status of endogenous Keap1

Yoriko Watai, Akira Kobayashi, Hiroko Nagase, Mio Mizukami, Justina Dolorita McEvoy, Jeffrey D. Singer, Ken Itoh, Masayuki Yamamoto

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf2 activity through the interaction with Nrf2 Neh2 domain. However, the mechanism(s) of Nrf2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule. Here, we report the generation of a new monoclonal antibody for the detection of endogenous Keap1 molecules. Immunocytochemical analysis of mouse embryonic fibroblasts with the antibody revealed that under normal, unstressed condition, Keap1 is localized primarily in the cytoplasm with minimal amount in the nucleus and endoplasmic reticulum. This subcellular localization profile of Keap1 appears unchanged after treatment of cells with diethyl maleate, an electrophile, and/or Leptomycin B, a nuclear export inhibitor. Subcellular fractionation analysis of mouse liver cells showed similar results. No substantial change in the subcellular distribution profile could be observed in cells isolated from butylated hydroxyanisole-treated mice. Analyses of sucrose density gradient centrifugation of mouse liver cells indicated that Keap1 appears to form multiprotein complexes in the cytoplasm. These results demonstrate that endogenous Keap1 remains mostly in the cytoplasm, and electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of Keap1.

Original languageEnglish (US)
Pages (from-to)1163-1178
Number of pages16
JournalGenes to Cells
Volume12
Issue number10
DOIs
StatePublished - Oct 2007
Externally publishedYes

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Cytoplasm
diethyl maleate
Cullin Proteins
Butylated Hydroxyanisole
Multiprotein Complexes
Cell Nucleus Active Transport
Density Gradient Centrifugation
Antibodies
Liver
Ligases
Ubiquitin
Endoplasmic Reticulum
Sucrose
Oxidative Stress
Fibroblasts
Monoclonal Antibodies
leptomycin B

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Watai, Y., Kobayashi, A., Nagase, H., Mizukami, M., McEvoy, J. D., Singer, J. D., ... Yamamoto, M. (2007). Subcellular localization and cytoplasmic complex status of endogenous Keap1. Genes to Cells, 12(10), 1163-1178. https://doi.org/10.1111/j.1365-2443.2007.01118.x

Subcellular localization and cytoplasmic complex status of endogenous Keap1. / Watai, Yoriko; Kobayashi, Akira; Nagase, Hiroko; Mizukami, Mio; McEvoy, Justina Dolorita; Singer, Jeffrey D.; Itoh, Ken; Yamamoto, Masayuki.

In: Genes to Cells, Vol. 12, No. 10, 10.2007, p. 1163-1178.

Research output: Contribution to journalArticle

Watai, Y, Kobayashi, A, Nagase, H, Mizukami, M, McEvoy, JD, Singer, JD, Itoh, K & Yamamoto, M 2007, 'Subcellular localization and cytoplasmic complex status of endogenous Keap1', Genes to Cells, vol. 12, no. 10, pp. 1163-1178. https://doi.org/10.1111/j.1365-2443.2007.01118.x
Watai, Yoriko ; Kobayashi, Akira ; Nagase, Hiroko ; Mizukami, Mio ; McEvoy, Justina Dolorita ; Singer, Jeffrey D. ; Itoh, Ken ; Yamamoto, Masayuki. / Subcellular localization and cytoplasmic complex status of endogenous Keap1. In: Genes to Cells. 2007 ; Vol. 12, No. 10. pp. 1163-1178.
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