Original Contribution
Keap1 modification and nuclear accumulation in response to S-nitrosocysteine

https://doi.org/10.1016/j.freeradbiomed.2007.10.055Get rights and content

Abstract

Keap1 is a key regulator of the Nrf2 transcription factor, which transactivates the antioxidant response element (ARE) and upregulates numerous proteins involved in antioxidant defense. Under basal conditions, Keap1 targets Nrf2 for ubiquitination and proteolytic degradation and as such is responsible for the rapid turnover of Nrf2. In response to oxidants and electrophiles, Nrf2 is stabilized and accumulates in the nucleus. The mechanism for this effect has been proposed to involve thiol-dependent modulation of Keap1 leading to loss of its ability to negatively regulate Nrf2. We have previously shown that nitric oxide and S-nitrosothiols cause nuclear accumulation of Nrf2 and upregulation of the ARE-regulated gene HO-1. Here we show that nitric oxide and S-nitrosocysteine (CSNO) cause time- and dose-dependent Keap1 thiol modification. These studies were carried out in HEK293 cells and in HEK293 cells overexpressing hemagglutinin-tagged Keap1. Furthermore we demonstrate that in response to CSNO Keap1 accumulates in the nucleus with a time course similar to that of Nrf2.

Section snippets

Materials

Fetal bovine serum was obtained from HyClone (Logan, UT, USA) and minimal essential medium was from Sigma (St. Louis, MO, USA). All other cell culture reagents were from Gibco BRL (Grand Island, NY, USA). Tissue culture plasticware was obtained from Nunclon (Fisher Scientific, Raleigh, NC, USA). Spermine NONOate was from Cayman (Ann Arbor, MI, USA). Tris-glycine SDS–PAGE gels, Lipofectamine reagent, N′-(3-maleimidylpropionyl)biocytin (MPB), and NeutrAvidin–HRP were from Invitrogen (Carlsbad,

Keap1 modification by spermine NONOate and CSNO

These experiments investigated Keap1 modification by NO-related species using an assay developed by our laboratory to measure modification of specific proteins [34]. After exposure to the agents, cells were lysed in the presence of NEM to block reduced thiols. After removal of unreacted NEM, lysates were treated with DTT to reduce disulfide bridges, S-nitrosothiols, sulfenic acid derivatives, and other reversible thiol modifications. Subsequently the unreacted DTT was removed and lysates were

Keap1 modification

Results from this study clearly demonstrate that Keap1 thiols were modified in response to NO and CSNO. This is the first report of Keap1 thiol modification by reactive nitrogen species in an intact cell model. Similar responses were observed in HEK293H cells overexpressing Keap1 and in HEK293H cells containing only endogenous Keap1. It should be noted that the response to NONOate was much less pronounced than the response to CSNO. There are several possible explanations for this observation.

Acknowledgments

This work was supported by NHLBI HL61377(A.R.W.) and NHLBI HL42444 (A.R.W.).

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