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Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity

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Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity. / Chowdhry, S; Zhang, Ying; McMahon, Michael; Sutherland, C; Cuadrado, A; Hayes, J D (Lead / Corresponding author).

In: Oncogene, Vol. 32, 2012, p. 3765-3781.

Research output: Contribution to journalArticle

Harvard

Chowdhry, S, Zhang, Y, McMahon, M, Sutherland, C, Cuadrado, A & Hayes, JD 2012, 'Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity' Oncogene, vol 32, pp. 3765-3781.

APA

Chowdhry, S., Zhang, Y., McMahon, M., Sutherland, C., Cuadrado, A., & Hayes, J. D. (2012). Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity. Oncogene, 32, 3765-3781doi: 10.1038/onc.2012.388

Vancouver

Chowdhry S, Zhang Y, McMahon M, Sutherland C, Cuadrado A, Hayes JD. Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity. Oncogene. 2012;32:3765-3781.

Author

Chowdhry, S; Zhang, Ying; McMahon, Michael; Sutherland, C; Cuadrado, A; Hayes, J D (Lead / Corresponding author) / Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity.

In: Oncogene, Vol. 32, 2012, p. 3765-3781.

Research output: Contribution to journalArticle

Bibtex - Download

@article{b14b0fe7164a4af2978d8fbbaeb60be5,
title = "Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity",
author = "S Chowdhry and Ying Zhang and Michael McMahon and C Sutherland and A Cuadrado and Hayes, {J D}",
year = "2012",
volume = "32",
pages = "3765--3781",
journal = "Oncogene",
issn = "0950-9232",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity

A1 - Chowdhry,S

A1 - Zhang,Ying

A1 - McMahon,Michael

A1 - Sutherland,C

A1 - Cuadrado,A

A1 - Hayes,J D

AU - Chowdhry,S

AU - Zhang,Ying

AU - McMahon,Michael

AU - Sutherland,C

AU - Cuadrado,A

AU - Hayes,J D

PY - 2012

Y1 - 2012

N2 - Identification of regulatable mechanisms by which transcription factor NF-E2 p45-related factor 2 (Nrf2) is repressed will allow strategies to be designed that counter drug resistance associated with its upregulation in tumours that harbour somatic mutations in Kelch-like ECH-associated protein-1 (Keap1), a gene that encodes a joint adaptor and substrate receptor for the Cul3-Rbx1/Roc1 ubiquitin ligase. We now show that mouse Nrf2 contains two binding sites for ß-transducin repeat-containing protein (ß-TrCP), which acts as a substrate receptor for the Skp1-Cul1-Rbx1/Roc1 ubiquitin ligase complex. Deletion of either binding site in Nrf2 decreased ß-TrCP-mediated ubiquitylation of the transcription factor. The ability of one of the two ß-TrCP-binding sites to serve as a degron could be both increased and decreased by manipulation of glycogen synthase kinase-3 (GSK-3) activity. Biotinylated-peptide pull-down assays identified DSGIS(338) and DSAPGS(378) as the two ß-TrCP-binding motifs in Nrf2. Significantly, our pull-down assays indicated that ß-TrCP binds a phosphorylated version of DSGIS more tightly than its non-phosphorylated counterpart, whereas this was not the case for DSAPGS. These data suggest that DSGIS, but not DSAPGS, contains a functional GSK-3 phosphorylation site. Activation of GSK-3 in Keap1-null mouse embryonic fibroblasts (MEFs), or in human lung A549 cells that contain mutant Keap1, by inhibition of the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB)/Akt pathway markedly reduced endogenous Nrf2 protein and decreased to 10-50% of normal the levels of mRNA for prototypic Nrf2-regulated enzymes, including the glutamate-cysteine ligase catalytic and modifier subunits, glutathione S-transferases Alpha-1 and Mu-1, haem oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Pre-treatment of Keap1(-/-) MEFs or A549 cells with the LY294002 PI3K inhibitor or the MK-2206 PKB/Akt inhibitor increased their sensitivity to acrolein, chlorambucil and cisplatin between 1.9-fold and 3.1-fold, and this was substantially attenuated by simultaneous pre-treatment with the GSK-3 inhibitor CT99021.Oncogene advance online publication, 10 September 2012; doi:10.1038/onc.2012.388.

AB - Identification of regulatable mechanisms by which transcription factor NF-E2 p45-related factor 2 (Nrf2) is repressed will allow strategies to be designed that counter drug resistance associated with its upregulation in tumours that harbour somatic mutations in Kelch-like ECH-associated protein-1 (Keap1), a gene that encodes a joint adaptor and substrate receptor for the Cul3-Rbx1/Roc1 ubiquitin ligase. We now show that mouse Nrf2 contains two binding sites for ß-transducin repeat-containing protein (ß-TrCP), which acts as a substrate receptor for the Skp1-Cul1-Rbx1/Roc1 ubiquitin ligase complex. Deletion of either binding site in Nrf2 decreased ß-TrCP-mediated ubiquitylation of the transcription factor. The ability of one of the two ß-TrCP-binding sites to serve as a degron could be both increased and decreased by manipulation of glycogen synthase kinase-3 (GSK-3) activity. Biotinylated-peptide pull-down assays identified DSGIS(338) and DSAPGS(378) as the two ß-TrCP-binding motifs in Nrf2. Significantly, our pull-down assays indicated that ß-TrCP binds a phosphorylated version of DSGIS more tightly than its non-phosphorylated counterpart, whereas this was not the case for DSAPGS. These data suggest that DSGIS, but not DSAPGS, contains a functional GSK-3 phosphorylation site. Activation of GSK-3 in Keap1-null mouse embryonic fibroblasts (MEFs), or in human lung A549 cells that contain mutant Keap1, by inhibition of the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB)/Akt pathway markedly reduced endogenous Nrf2 protein and decreased to 10-50% of normal the levels of mRNA for prototypic Nrf2-regulated enzymes, including the glutamate-cysteine ligase catalytic and modifier subunits, glutathione S-transferases Alpha-1 and Mu-1, haem oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Pre-treatment of Keap1(-/-) MEFs or A549 cells with the LY294002 PI3K inhibitor or the MK-2206 PKB/Akt inhibitor increased their sensitivity to acrolein, chlorambucil and cisplatin between 1.9-fold and 3.1-fold, and this was substantially attenuated by simultaneous pre-treatment with the GSK-3 inhibitor CT99021.Oncogene advance online publication, 10 September 2012; doi:10.1038/onc.2012.388.

U2 - 10.1038/onc.2012.388

DO - 10.1038/onc.2012.388

M1 - Article

JO - Oncogene

JF - Oncogene

SN - 0950-9232

VL - 32

SP - 3765

EP - 3781

ER -

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