Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Aug 8;32(32):3765-81.
doi: 10.1038/onc.2012.388. Epub 2012 Sep 10.

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

S Chowdhry  1 , Y Zhang, M McMahon, C Sutherland, A Cuadrado, J D Hayes
Affiliations

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

S Chowdhry et al. Oncogene. .

Abstract

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 β-transducin repeat-containing protein (β-TrCP), which acts as a substrate receptor for the Skp1-Cul1-Rbx1/Roc1 ubiquitin ligase complex. Deletion of either binding site in Nrf2 decreased β-TrCP-mediated ubiquitylation of the transcription factor. The ability of one of the two β-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 β-TrCP-binding motifs in Nrf2. Significantly, our pull-down assays indicated that β-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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The Neh6 domain of Nrf2 comprises two conserved regions that include putative β-TrCP binding sites and a potential PEST sequence. A) Amino acid sequences corresponding to the Neh6 domain of Nrf2 from mouse (m), human (h), rat (r), frog (f), and zebrafish (z), along with that of ECH (i.e. chicken Nrf2) have been aligned using the T-Coffee tool (at http://www.ebi.ac.uk/Tools/msa/tcoffee/). White letters on a black background represent residues that are identical across at least half of the species studied, and black letters on a grey background are conserved residues. The two boxes designated SDS1 and SDS2 contain sequences enriched with Ser and Asp residues. The solid horizontal bar over residues corresponding to 347 and 385 in mouse Nrf2 depicts a potential PEST sequence that is enriched with Pro, Glu, Ser and Thr residues (6). Within the SDS1 and SDS2 boxes, a solid horizonal bar is shown above sequences that represent putative β-TrCP binding sites. The residues that are predicted to be phosphorylated by GSK-3, based on the Scansite program (at http://scansite.mit.edu), are shown at the bottom as vertical arrows. B) Amino acid sequences of the SDS1 region in the Neh6 domain of mNrf2, hNrf2 and rNrf2 have been aligned with a similar region in the acidic domain-2 of mNrf1, hNrf1, rNrf1 and TCF11, a splice variant of Nrf1 (the protein shown is the human factor). C) Amino acid sequences of the SDS2 region in the Neh6 domain of mNrf2, hNrf2 and rNrf2 have been aligned with a similar region in the Neh6-like domain of mRn1, hNrf1, rNrf1 and TCF11.
Figure 2
Figure 2
Repression of Nrf2 by β-TrCP is abolished by deletion of two separate regions within its Neh6 domain. A) Keap1−/− MEFs were transfected for 24 h with pcDNA3.1 expression vectors encoding V5-tagged mouse Nrf2ΔNeh2 or related mutants that lack SDS1, PEST or the entire Neh6 domain, along with either an empty pcDNA4-FLAG plasmid or a pcDNA4-βTrCP1-FLAG plasmid. Twenty-four h later, the cells were serum-depleted by transfer to Delbecco’s modified Eagle’s medium (DMEM) containing 0.5% fetal bovine serum (FBS) for 16 h, after which whole cell lysates were prepared and ectopic Nrf2 measured by Western blotting using mouse anti-V5 antibodies. B) Keap1−/− MEFs were transfected with the same Nrf2ΔNeh2-V5, Nrf2ΔNeh2,SDS1-V5, Nrf2ΔNeh2,PEST-V5 and Nrf2ΔNeh2,Neh6-V5 expression vectors used in panel A. Following transfection, the MEFs were serum-depleted for 16 h before they were treated with CHX for various periods of time and the relative amounts of ectopic mutant Nrf2 measured by Western blotting. Results that were significantly higher than the Nrf2ΔNeh2 control with P values of 0.01-0.001 or <0.001 are indicated with double (**) or triple (***) asterisk signs, respectively. C) COS1 cells were co-transfected with an empty pcDNA3.1 expression vector or one encoding various mutant mouse Nrf2 proteins (as indicated at the bottom of the figure) along with the murine quinone reductase-based P-1016/nqo1-Luc reporter construct (2) and the pRL-TK Renilla control plasmid. At the same time, the cells were also transfected with combinations of empty vectors and pcDNA4-βTrCP1-FLAG and pCGN/GSK-3βΔ9 as indicated. Equal amounts of DNA were transfected into COS1 cells in the different experimental groups. After overnight transfection, the cells were serum-depleted for 16 h, before ARE-driven luciferase activity was measured. All results were normalized to Renilla luciferase activity. The data are presented as follows: cells transfected with empty pcDNA4 expression vector in open bars; cells transfected with pcDNA4-FLAG and pcDNA4-βTrCP1-FLAG in grey bars; cells transfected with pcDNA4-βTrCP1-FLAG and pCGN/GSK-3βΔ9 solid black bars. Results in which ectopic expression of compound mutant forms of Nrf2 stimulated an increase in ARE-driven luciferase activity with a P value of 0.05-0.01, relative to that produced by Nrf2ΔNeh2-V5, are indicated by a single asterisk (*). Results in which the stimulation of ARE-driven luciferase activity produced by Nrf2 or its mutants was reduced through forced expression of β-TrCP1 or forced co-expression of β-TrCP and GSK-3βΔ9 with a P value of 0.05-0.01 or <0.005 are indicated by $ or $$$, respectively.
Figure 2
Figure 2
Repression of Nrf2 by β-TrCP is abolished by deletion of two separate regions within its Neh6 domain. A) Keap1−/− MEFs were transfected for 24 h with pcDNA3.1 expression vectors encoding V5-tagged mouse Nrf2ΔNeh2 or related mutants that lack SDS1, PEST or the entire Neh6 domain, along with either an empty pcDNA4-FLAG plasmid or a pcDNA4-βTrCP1-FLAG plasmid. Twenty-four h later, the cells were serum-depleted by transfer to Delbecco’s modified Eagle’s medium (DMEM) containing 0.5% fetal bovine serum (FBS) for 16 h, after which whole cell lysates were prepared and ectopic Nrf2 measured by Western blotting using mouse anti-V5 antibodies. B) Keap1−/− MEFs were transfected with the same Nrf2ΔNeh2-V5, Nrf2ΔNeh2,SDS1-V5, Nrf2ΔNeh2,PEST-V5 and Nrf2ΔNeh2,Neh6-V5 expression vectors used in panel A. Following transfection, the MEFs were serum-depleted for 16 h before they were treated with CHX for various periods of time and the relative amounts of ectopic mutant Nrf2 measured by Western blotting. Results that were significantly higher than the Nrf2ΔNeh2 control with P values of 0.01-0.001 or <0.001 are indicated with double (**) or triple (***) asterisk signs, respectively. C) COS1 cells were co-transfected with an empty pcDNA3.1 expression vector or one encoding various mutant mouse Nrf2 proteins (as indicated at the bottom of the figure) along with the murine quinone reductase-based P-1016/nqo1-Luc reporter construct (2) and the pRL-TK Renilla control plasmid. At the same time, the cells were also transfected with combinations of empty vectors and pcDNA4-βTrCP1-FLAG and pCGN/GSK-3βΔ9 as indicated. Equal amounts of DNA were transfected into COS1 cells in the different experimental groups. After overnight transfection, the cells were serum-depleted for 16 h, before ARE-driven luciferase activity was measured. All results were normalized to Renilla luciferase activity. The data are presented as follows: cells transfected with empty pcDNA4 expression vector in open bars; cells transfected with pcDNA4-FLAG and pcDNA4-βTrCP1-FLAG in grey bars; cells transfected with pcDNA4-βTrCP1-FLAG and pCGN/GSK-3βΔ9 solid black bars. Results in which ectopic expression of compound mutant forms of Nrf2 stimulated an increase in ARE-driven luciferase activity with a P value of 0.05-0.01, relative to that produced by Nrf2ΔNeh2-V5, are indicated by a single asterisk (*). Results in which the stimulation of ARE-driven luciferase activity produced by Nrf2 or its mutants was reduced through forced expression of β-TrCP1 or forced co-expression of β-TrCP and GSK-3βΔ9 with a P value of 0.05-0.01 or <0.005 are indicated by $ or $$$, respectively.
Figure 3
Figure 3
Transcription factor Nrf2 contains two separate sequences in its Neh6 domain to which β-TrCP can bind. A) COS1 cells were co-transfected with pcDNA3.1 expression plasmids encoding V5-tagged mouse Nrf2Δ17-32 or mutants lacking SDS1, SDS2, or SDS1 and SDS2, along with pcDNA4-βTrCP1-FLAG. Empty pcDNA3.1 vector was included in the transfection mixture to normalize the amount of DNA to which cells were exposed. Following overnight transfection, the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS before whole cell lysates were prepared. An aliquot (10%) of the lysate was withdrawn as the input sample, and the remainder was used for the pull-down assay that employed an antibody against FLAG as described in Materials and Methods. B) COS1 cells were co-transfected for 24 h with an expression vector for mouse Nrf2Δ17-32-V5, or its mutants lacking SDSGIS338, SDSEME370 and DSAPGS378, either individually or as double deletion mutants, along with an expression plasmid for FLAG-tagged β-TrCP1. As in panel A, β-TrCP1 was pulled-down after the cells had been subjected to 16 h serum-depletion using antibodies against FLAG, and the Nrf2 mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against the V5 epitope. C) COS1 cells were co-transfected with expression vectors for a YFP-Neh6 fusion protein, or YFP-Neh6 protein lacking SDSGIS338, SDSEME370 or DSAPGS378, or combinations thereof, along with an expression plasmid for FLAG-tagged β-TrCP1. The Neh6 domain mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against GFP.
Figure 3
Figure 3
Transcription factor Nrf2 contains two separate sequences in its Neh6 domain to which β-TrCP can bind. A) COS1 cells were co-transfected with pcDNA3.1 expression plasmids encoding V5-tagged mouse Nrf2Δ17-32 or mutants lacking SDS1, SDS2, or SDS1 and SDS2, along with pcDNA4-βTrCP1-FLAG. Empty pcDNA3.1 vector was included in the transfection mixture to normalize the amount of DNA to which cells were exposed. Following overnight transfection, the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS before whole cell lysates were prepared. An aliquot (10%) of the lysate was withdrawn as the input sample, and the remainder was used for the pull-down assay that employed an antibody against FLAG as described in Materials and Methods. B) COS1 cells were co-transfected for 24 h with an expression vector for mouse Nrf2Δ17-32-V5, or its mutants lacking SDSGIS338, SDSEME370 and DSAPGS378, either individually or as double deletion mutants, along with an expression plasmid for FLAG-tagged β-TrCP1. As in panel A, β-TrCP1 was pulled-down after the cells had been subjected to 16 h serum-depletion using antibodies against FLAG, and the Nrf2 mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against the V5 epitope. C) COS1 cells were co-transfected with expression vectors for a YFP-Neh6 fusion protein, or YFP-Neh6 protein lacking SDSGIS338, SDSEME370 or DSAPGS378, or combinations thereof, along with an expression plasmid for FLAG-tagged β-TrCP1. The Neh6 domain mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against GFP.
Figure 4
Figure 4
A peptide sequence in the SDS1 region of the Neh6 domain allows Nrf2 to interact with β-TrCP in a GSK-3-dependent manner. A) COS1 cells were co-transfected with expression vectors encoding the Gal4 DNA-binding domain fused to the Neh6 domain (i.e. Gal4(DBD)-Neh6), or expression vectors for Gal4(DBD)-Neh6 containing individual or combined deletion of the SDSGIS338, SDSEME370 and DSAPGS378 hexapeptides along with an expression vector for the Gal4 activating domain fused to the substrate-binding WD40 domain of β-TrCP1 (Gal4(AD)-WD40) and the reporter plasmids PTKUAS-Luc and pRL-TK Renilla. Forty-eight h later the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS, after which time Gal4-driven luciferase activity was measured. Significant increases in Gal4-driven reporter gene activity, relative to that produced by pM-Neh6 alone, with P values of 0.01-0.001 or <0.001 are indicated above the histogram bars by ** or ***, respectively. Significant decreases in reporter gene activity relative to that produced by Neh6 + WD40 with P values <0.001 are indicated by $$$ on a horizontal line from Neh6 with a vertical line directed at the relevant data points. B) COS1 cells were co-transfected with expression vectors for Gal4(DBD)-Neh6, Gal4(DBD)-Neh6ΔSDSGIS, Gal4(DBD)-Neh6ΔSDSEME or Gal4(DBD)-Neh6ΔDSAPGS and the expression plasmid for Gal4(AD)-WD40. At the same time, the COS1 cells were transfected with the reporter plasmids P TKUAS-Luc and pRL-TK Renilla, along with either an empty expression vector or one encoding GSK-3βΔ9. Following 48 h transfection, the COS1 cells were serum-depleted for 16 h, during which time a portion was treated with 5 μM CT99021 for 2 h or with 0.1% DMSO vehicle control. The cells were then harvested, lysed and Gal4-driven luciferase activity measured. Differences in reporter gene activity produced by a given two-hybrid pair that were observed upon either expression of GSK-3βΔ9 or treatment with CT99021 with P values of 0.01-0.001 (increase **, or decrease $$) are indicated by the linked lines above the histogram bars. C) COS1 cells were transfected with expression vectors as described in panel B, but a plasmid encoding GSK-3βY216F substituted for that encoding GSK-3βΔ9.
Figure 4
Figure 4
A peptide sequence in the SDS1 region of the Neh6 domain allows Nrf2 to interact with β-TrCP in a GSK-3-dependent manner. A) COS1 cells were co-transfected with expression vectors encoding the Gal4 DNA-binding domain fused to the Neh6 domain (i.e. Gal4(DBD)-Neh6), or expression vectors for Gal4(DBD)-Neh6 containing individual or combined deletion of the SDSGIS338, SDSEME370 and DSAPGS378 hexapeptides along with an expression vector for the Gal4 activating domain fused to the substrate-binding WD40 domain of β-TrCP1 (Gal4(AD)-WD40) and the reporter plasmids PTKUAS-Luc and pRL-TK Renilla. Forty-eight h later the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS, after which time Gal4-driven luciferase activity was measured. Significant increases in Gal4-driven reporter gene activity, relative to that produced by pM-Neh6 alone, with P values of 0.01-0.001 or <0.001 are indicated above the histogram bars by ** or ***, respectively. Significant decreases in reporter gene activity relative to that produced by Neh6 + WD40 with P values <0.001 are indicated by $$$ on a horizontal line from Neh6 with a vertical line directed at the relevant data points. B) COS1 cells were co-transfected with expression vectors for Gal4(DBD)-Neh6, Gal4(DBD)-Neh6ΔSDSGIS, Gal4(DBD)-Neh6ΔSDSEME or Gal4(DBD)-Neh6ΔDSAPGS and the expression plasmid for Gal4(AD)-WD40. At the same time, the COS1 cells were transfected with the reporter plasmids P TKUAS-Luc and pRL-TK Renilla, along with either an empty expression vector or one encoding GSK-3βΔ9. Following 48 h transfection, the COS1 cells were serum-depleted for 16 h, during which time a portion was treated with 5 μM CT99021 for 2 h or with 0.1% DMSO vehicle control. The cells were then harvested, lysed and Gal4-driven luciferase activity measured. Differences in reporter gene activity produced by a given two-hybrid pair that were observed upon either expression of GSK-3βΔ9 or treatment with CT99021 with P values of 0.01-0.001 (increase **, or decrease $$) are indicated by the linked lines above the histogram bars. C) COS1 cells were transfected with expression vectors as described in panel B, but a plasmid encoding GSK-3βY216F substituted for that encoding GSK-3βΔ9.
Figure 5
Figure 5
The abundance of a Neh6-LacZ fusion protein is controlled by two peptide sequences and by GSK-3 A) COS1 cells were co-transfected with a pcDNA3.1 expression vector for a V5-tagged fusion protein comprising Neh6 coupled at its C-terminus to LacZ (i.e. Neh6(LacZ)-V5), or expression vectors for Neh6(LacZ)-V5 bearing individual or combined deletion of the SDSGIS338, SDSEME370 and DSAPGS378 hexapeptides from the Neh6 domain along with an expression plasmid for βTrCP1-FLAG and pRL-TK Renilla as a transfection control. As further controls, COS1 cells were transfected with the expression plasmid for Neh6(LacZ)-V5 alone, or were co-transfected with expression plasmids for LacZ-V5 and β-TrCP1-FLAG; all of these included the Renilla transfection control plasmid. After 24 h transfection, the cells were serum-depleted for 16 h before β-gal activity was measured and results normalised against Renilla. Enzyme activity results that are significantly higher than that produced by Neh6-LacZ with a P value of <0.001 are indicated by ***. B) COS1 cells were co-transfected with the expression plasmids for Neh6(LacZ)-V5, or its mutants, and β-TrCP1-FLAG as above along with the expression vector for GSK-3βΔ9. Thereafter, the cells were transferred to medium containing 0.5% FBS for 16 h, and were then treated with 5 μM CT99021 or 0.1% DMSO vehicle control for 2 h before being harvested and β-gal activity measured. The β-galactosidase activity was normalised against Renilla. The significance of changes in β-gal activity following treatment with CT99021 and/or ectopic expression of GSK-3βΔ9 for an individual Neh6-LacZ fusion protein is indicated above the histogram bars as outlined in Materials and Methods.
Figure 6
Figure 6
β-TrCP-mediated ubiquitylation of Nrf2 involves two separate peptide motifs in the Neh6 domain A) COS1 cells were co-transfected for 24 h with a pcDNA3.1 expression vector encoding V5-tagged mouse Nrf2Δ17-32, or mutants lacking SDSGIS338, SDSEME370 or DSAPGS378, along with expression plasmids for HisUb and β-TrCP1-FLAG. As controls, the cells were transfected with empty expression vectors, pHisUb alone or pcDNA3.1-Nrf2Δ17-32-V5 without pcDNA4-βTrCP1-FLAG. Following transfection, the cells were serum-depleted for 16 h, after which whole cell lysates were prepared in phosphate-buffered saline. To allow loading to be assessed, a 10% portion of the lysate was retained as input. The remainder of each sample was used to purify His-tagged protein separately using Ni2+-agarose beads, and the total amount of ubiquitylated Nrf2 protein in each sample was determined by Western blotting with anti-V5 antibodies. The input samples were also immunoblotted with anti-V5 and anti-FLAG antibodies to confirm equal loading of Nrf2 and β-TrCP1. B) The same ubiquitylation assay was performed for Nrf2Δ17-32-V5, Nrf2Δ17-32,SDSGIS-V5 and Nrf2Δ17-32,DSAPGS-V5 as in panel A, but on this occasion the Nrf2 expression constructs were co-transfected into COS1 cells with an expression vector for either GSK-3βΔ9 or an empty vector. C) The same ubiquitylation assay was performed for Nrf2Δ17-32-V5, Nrf2Δ17-32,SDSGIS-V5 and Nrf2Δ17-32,DSAPGS-V5 as in panel A, but in this case the COS1 cells were treated with 5 μM CT99021 to inhibit GSK-3.
Figure 6
Figure 6
β-TrCP-mediated ubiquitylation of Nrf2 involves two separate peptide motifs in the Neh6 domain A) COS1 cells were co-transfected for 24 h with a pcDNA3.1 expression vector encoding V5-tagged mouse Nrf2Δ17-32, or mutants lacking SDSGIS338, SDSEME370 or DSAPGS378, along with expression plasmids for HisUb and β-TrCP1-FLAG. As controls, the cells were transfected with empty expression vectors, pHisUb alone or pcDNA3.1-Nrf2Δ17-32-V5 without pcDNA4-βTrCP1-FLAG. Following transfection, the cells were serum-depleted for 16 h, after which whole cell lysates were prepared in phosphate-buffered saline. To allow loading to be assessed, a 10% portion of the lysate was retained as input. The remainder of each sample was used to purify His-tagged protein separately using Ni2+-agarose beads, and the total amount of ubiquitylated Nrf2 protein in each sample was determined by Western blotting with anti-V5 antibodies. The input samples were also immunoblotted with anti-V5 and anti-FLAG antibodies to confirm equal loading of Nrf2 and β-TrCP1. B) The same ubiquitylation assay was performed for Nrf2Δ17-32-V5, Nrf2Δ17-32,SDSGIS-V5 and Nrf2Δ17-32,DSAPGS-V5 as in panel A, but on this occasion the Nrf2 expression constructs were co-transfected into COS1 cells with an expression vector for either GSK-3βΔ9 or an empty vector. C) The same ubiquitylation assay was performed for Nrf2Δ17-32-V5, Nrf2Δ17-32,SDSGIS-V5 and Nrf2Δ17-32,DSAPGS-V5 as in panel A, but in this case the COS1 cells were treated with 5 μM CT99021 to inhibit GSK-3.
Figure 6
Figure 6
β-TrCP-mediated ubiquitylation of Nrf2 involves two separate peptide motifs in the Neh6 domain A) COS1 cells were co-transfected for 24 h with a pcDNA3.1 expression vector encoding V5-tagged mouse Nrf2Δ17-32, or mutants lacking SDSGIS338, SDSEME370 or DSAPGS378, along with expression plasmids for HisUb and β-TrCP1-FLAG. As controls, the cells were transfected with empty expression vectors, pHisUb alone or pcDNA3.1-Nrf2Δ17-32-V5 without pcDNA4-βTrCP1-FLAG. Following transfection, the cells were serum-depleted for 16 h, after which whole cell lysates were prepared in phosphate-buffered saline. To allow loading to be assessed, a 10% portion of the lysate was retained as input. The remainder of each sample was used to purify His-tagged protein separately using Ni2+-agarose beads, and the total amount of ubiquitylated Nrf2 protein in each sample was determined by Western blotting with anti-V5 antibodies. The input samples were also immunoblotted with anti-V5 and anti-FLAG antibodies to confirm equal loading of Nrf2 and β-TrCP1. B) The same ubiquitylation assay was performed for Nrf2Δ17-32-V5, Nrf2Δ17-32,SDSGIS-V5 and Nrf2Δ17-32,DSAPGS-V5 as in panel A, but on this occasion the Nrf2 expression constructs were co-transfected into COS1 cells with an expression vector for either GSK-3βΔ9 or an empty vector. C) The same ubiquitylation assay was performed for Nrf2Δ17-32-V5, Nrf2Δ17-32,SDSGIS-V5 and Nrf2Δ17-32,DSAPGS-V5 as in panel A, but in this case the COS1 cells were treated with 5 μM CT99021 to inhibit GSK-3.
Figure 7
Figure 7
β-TrCP binds both phosphorylated and non-phosphorylated Nrf2-derived peptides containing the DSGIS and DSAPGS sequences. Biotinylated-peptides, designed around sequences in the SDS1 and SDS2 regions of the Neh6 domain in mouse Nrf2, were coupled with Streptavidin, which had first been immobilized on agarose beads, and used in pull-down assays to identify those that were bound by in vitro translated [35S]methionine-labelled β-TrCP1. A biotinylated ETGE-containing peptide, representing residues 73-90 of mouse Nrf2, was used as a negative control. A) Ala-scanning substitutions were introduced into the (SGSG)MEFNDSDSGISLNTSPSR peptide between residues equivalent to Asp-332 and Asn-340 in the Neh6 domain (the residues changed are shown underlined). Each of the peptides was used in the pull-down assay, and autoradiography was used to identify β-TrCP1 that bound the peptides. As a control, lane 1 contained total [35S]methionine-labelled in vitro translated protein. Lane 2 shows [35S]methionine-labelled protein pulled-down by the ETGE peptide. Lane 3 shows [35S]methionine-labelled protein pulled-down by the wild-type 22-mer peptide, and lanes 4-12 show protein pulled down by the peptides with Ala substitutions across residues 332-340. B) Ala-scanning substitutions were introduced into the (SGSG)SEMEELDSAPGSVKQNGP peptide between residues equivalent to Glu-371 and Ser-374 and Pro-376 and Lys-380 in the Neh6 domain (the residues changed are shown underlined). As above, lane 1 represents total [35S]methionine-labelled in vitro translated protein, lane 2 shows protein pulled-down by the ETGE peptide, lane 3 shows protein pulled-down by the wild-type 22-mer peptide. Lanes 4-7 show [35S]methionine-labelled protein pulled down by the peptides with Ala substitutions across residues 371-374, and lanes 8-12 show protein pulled down by peptide with Ala substitutions across residues 376-380. C) The biotinylated-peptide pull-down assay was used to test whether double phosphorylation of the peptides increased their binding by β-TrCP; the phosphorylated residues are indicated in bold italics in the peptide shown above the gel. Lane 1 represents total [35S]methionine-labelled in vitro translated protein, and lane 2 shows protein pulled down by the ETGE-containing peptide based on the Neh2 domain. Lanes 3 and 4 show the protein pulled-down by DSGIS- and DpSGIpS-containing peptides. Lanes 5 and 6 show that the SDSEME- and pSDpSEME-containing peptides did not pull-down protein. Lanes 7 and 8 show the protein pulled-down by DSAPGS- and DpSAPGpS-containing peptides. D) The effect of individual phosphorylation of Ser-333, Ser-335, Ser-338 and Ser-342 across the DSGIS-containing peptide on binding by β-TrCP was examined by pull-down assay. Lane 1 shows total [35S]methionine-labelled in vitro translated protein, and lane 2 shows protein pulled-down by the ETGE-containing peptide based on the Neh2 domain. Lanes 3 and 9 show protein bound to the non-phosphorylated DSGIS-containing peptide. Lanes 4-8 show the protein bound to the DSGIS-containing peptide in which only Ser-335 is phosphorylated, only Ser-338 is phosphorylated, both Ser-335 and Ser-338 are phosphorylated, Ser-333, Ser-335 and Ser-338 are phosphorylated, and Ser-335, Ser-338 and Ser-342 had been phosphorylated, respectively.
Figure 7
Figure 7
β-TrCP binds both phosphorylated and non-phosphorylated Nrf2-derived peptides containing the DSGIS and DSAPGS sequences. Biotinylated-peptides, designed around sequences in the SDS1 and SDS2 regions of the Neh6 domain in mouse Nrf2, were coupled with Streptavidin, which had first been immobilized on agarose beads, and used in pull-down assays to identify those that were bound by in vitro translated [35S]methionine-labelled β-TrCP1. A biotinylated ETGE-containing peptide, representing residues 73-90 of mouse Nrf2, was used as a negative control. A) Ala-scanning substitutions were introduced into the (SGSG)MEFNDSDSGISLNTSPSR peptide between residues equivalent to Asp-332 and Asn-340 in the Neh6 domain (the residues changed are shown underlined). Each of the peptides was used in the pull-down assay, and autoradiography was used to identify β-TrCP1 that bound the peptides. As a control, lane 1 contained total [35S]methionine-labelled in vitro translated protein. Lane 2 shows [35S]methionine-labelled protein pulled-down by the ETGE peptide. Lane 3 shows [35S]methionine-labelled protein pulled-down by the wild-type 22-mer peptide, and lanes 4-12 show protein pulled down by the peptides with Ala substitutions across residues 332-340. B) Ala-scanning substitutions were introduced into the (SGSG)SEMEELDSAPGSVKQNGP peptide between residues equivalent to Glu-371 and Ser-374 and Pro-376 and Lys-380 in the Neh6 domain (the residues changed are shown underlined). As above, lane 1 represents total [35S]methionine-labelled in vitro translated protein, lane 2 shows protein pulled-down by the ETGE peptide, lane 3 shows protein pulled-down by the wild-type 22-mer peptide. Lanes 4-7 show [35S]methionine-labelled protein pulled down by the peptides with Ala substitutions across residues 371-374, and lanes 8-12 show protein pulled down by peptide with Ala substitutions across residues 376-380. C) The biotinylated-peptide pull-down assay was used to test whether double phosphorylation of the peptides increased their binding by β-TrCP; the phosphorylated residues are indicated in bold italics in the peptide shown above the gel. Lane 1 represents total [35S]methionine-labelled in vitro translated protein, and lane 2 shows protein pulled down by the ETGE-containing peptide based on the Neh2 domain. Lanes 3 and 4 show the protein pulled-down by DSGIS- and DpSGIpS-containing peptides. Lanes 5 and 6 show that the SDSEME- and pSDpSEME-containing peptides did not pull-down protein. Lanes 7 and 8 show the protein pulled-down by DSAPGS- and DpSAPGpS-containing peptides. D) The effect of individual phosphorylation of Ser-333, Ser-335, Ser-338 and Ser-342 across the DSGIS-containing peptide on binding by β-TrCP was examined by pull-down assay. Lane 1 shows total [35S]methionine-labelled in vitro translated protein, and lane 2 shows protein pulled-down by the ETGE-containing peptide based on the Neh2 domain. Lanes 3 and 9 show protein bound to the non-phosphorylated DSGIS-containing peptide. Lanes 4-8 show the protein bound to the DSGIS-containing peptide in which only Ser-335 is phosphorylated, only Ser-338 is phosphorylated, both Ser-335 and Ser-338 are phosphorylated, Ser-333, Ser-335 and Ser-338 are phosphorylated, and Ser-335, Ser-338 and Ser-342 had been phosphorylated, respectively.
Figure 8
Figure 8
Nrf2 is down-regulated by prevention of the inhibitory phosphorylation of GSK-3 A) COS1 cells were transfected with a pcDNA3.1 expression vector for a V5-tagged fusion protein comprising Neh6(LacZ)-V5 or Neh6(LacZ)-V5 bearing individual deletion of the SDSGIS338, SDSEME370 and DSAPGS378 from the Neh6 domain for 24 h. Twenty-four h later the cells were serum depleted by transfer to DMEM containing 0.1% (v/v) FBS for a further 16 h before the cells were treated with either 10 μM LY294002, 5 μM MK-2206 or with vehicle (0.1% (v/v) DMSO) in media containing 0.1% (w/v) FBS for 8 h. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immuno-blotted with the indicated antibodies. The antibody that recognised phospho-GSK-3β (Ser-9) was from Abcam (ab30619). Gapdh was used as an internal control. B) Keap1−/− MEFs were seeded in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for a further 16 h. Thereafter, the cells were treated for 8 h with 1.0, 2.5, 10 or 40 μM LY294002 or 0.25, 1.0, 5.0 or 10 μM MK-2206, all of which were dissolved in DMSO to a final concentration of 0.1% (by vol), in media containing 0.1% (w/v) FBS; 0.1% (v/v) DMSO was used as vehicle control. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immunoblotted with the indicated antibodies. Gapdh was used as a sample loading control. C) Keap1−/− MEFs were grown in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for 16 h, as described in panel B above. The fibroblasts were then treated with LY294002 or MK-2206 in medium containing 0.1% FBS, at the doses indicated, for 2 h before they were transferred to fresh medium containing 0.1% FBS for 6 h. Thereafter, the fibroblasts were harvested, total RNA extracted, and mRNA for Nqo1, Hmox1, Gclc, Gclm, Gsta1 and Gstm1 measured by TaqMan chemistry as described by Higgins et al (61). The solid horizontal bar indicates that mRNA levels in MEFs treated with kinase inhibitors in medium containing 0.1% FBS were compared with MEFs treated with DMSO vehicle control in medium containing 0.1% FBS.
Figure 8
Figure 8
Nrf2 is down-regulated by prevention of the inhibitory phosphorylation of GSK-3 A) COS1 cells were transfected with a pcDNA3.1 expression vector for a V5-tagged fusion protein comprising Neh6(LacZ)-V5 or Neh6(LacZ)-V5 bearing individual deletion of the SDSGIS338, SDSEME370 and DSAPGS378 from the Neh6 domain for 24 h. Twenty-four h later the cells were serum depleted by transfer to DMEM containing 0.1% (v/v) FBS for a further 16 h before the cells were treated with either 10 μM LY294002, 5 μM MK-2206 or with vehicle (0.1% (v/v) DMSO) in media containing 0.1% (w/v) FBS for 8 h. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immuno-blotted with the indicated antibodies. The antibody that recognised phospho-GSK-3β (Ser-9) was from Abcam (ab30619). Gapdh was used as an internal control. B) Keap1−/− MEFs were seeded in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for a further 16 h. Thereafter, the cells were treated for 8 h with 1.0, 2.5, 10 or 40 μM LY294002 or 0.25, 1.0, 5.0 or 10 μM MK-2206, all of which were dissolved in DMSO to a final concentration of 0.1% (by vol), in media containing 0.1% (w/v) FBS; 0.1% (v/v) DMSO was used as vehicle control. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immunoblotted with the indicated antibodies. Gapdh was used as a sample loading control. C) Keap1−/− MEFs were grown in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for 16 h, as described in panel B above. The fibroblasts were then treated with LY294002 or MK-2206 in medium containing 0.1% FBS, at the doses indicated, for 2 h before they were transferred to fresh medium containing 0.1% FBS for 6 h. Thereafter, the fibroblasts were harvested, total RNA extracted, and mRNA for Nqo1, Hmox1, Gclc, Gclm, Gsta1 and Gstm1 measured by TaqMan chemistry as described by Higgins et al (61). The solid horizontal bar indicates that mRNA levels in MEFs treated with kinase inhibitors in medium containing 0.1% FBS were compared with MEFs treated with DMSO vehicle control in medium containing 0.1% FBS.
Figure 8
Figure 8
Nrf2 is down-regulated by prevention of the inhibitory phosphorylation of GSK-3 A) COS1 cells were transfected with a pcDNA3.1 expression vector for a V5-tagged fusion protein comprising Neh6(LacZ)-V5 or Neh6(LacZ)-V5 bearing individual deletion of the SDSGIS338, SDSEME370 and DSAPGS378 from the Neh6 domain for 24 h. Twenty-four h later the cells were serum depleted by transfer to DMEM containing 0.1% (v/v) FBS for a further 16 h before the cells were treated with either 10 μM LY294002, 5 μM MK-2206 or with vehicle (0.1% (v/v) DMSO) in media containing 0.1% (w/v) FBS for 8 h. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immuno-blotted with the indicated antibodies. The antibody that recognised phospho-GSK-3β (Ser-9) was from Abcam (ab30619). Gapdh was used as an internal control. B) Keap1−/− MEFs were seeded in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for a further 16 h. Thereafter, the cells were treated for 8 h with 1.0, 2.5, 10 or 40 μM LY294002 or 0.25, 1.0, 5.0 or 10 μM MK-2206, all of which were dissolved in DMSO to a final concentration of 0.1% (by vol), in media containing 0.1% (w/v) FBS; 0.1% (v/v) DMSO was used as vehicle control. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immunoblotted with the indicated antibodies. Gapdh was used as a sample loading control. C) Keap1−/− MEFs were grown in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for 16 h, as described in panel B above. The fibroblasts were then treated with LY294002 or MK-2206 in medium containing 0.1% FBS, at the doses indicated, for 2 h before they were transferred to fresh medium containing 0.1% FBS for 6 h. Thereafter, the fibroblasts were harvested, total RNA extracted, and mRNA for Nqo1, Hmox1, Gclc, Gclm, Gsta1 and Gstm1 measured by TaqMan chemistry as described by Higgins et al (61). The solid horizontal bar indicates that mRNA levels in MEFs treated with kinase inhibitors in medium containing 0.1% FBS were compared with MEFs treated with DMSO vehicle control in medium containing 0.1% FBS.
Figure 8
Figure 8
Nrf2 is down-regulated by prevention of the inhibitory phosphorylation of GSK-3 A) COS1 cells were transfected with a pcDNA3.1 expression vector for a V5-tagged fusion protein comprising Neh6(LacZ)-V5 or Neh6(LacZ)-V5 bearing individual deletion of the SDSGIS338, SDSEME370 and DSAPGS378 from the Neh6 domain for 24 h. Twenty-four h later the cells were serum depleted by transfer to DMEM containing 0.1% (v/v) FBS for a further 16 h before the cells were treated with either 10 μM LY294002, 5 μM MK-2206 or with vehicle (0.1% (v/v) DMSO) in media containing 0.1% (w/v) FBS for 8 h. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immuno-blotted with the indicated antibodies. The antibody that recognised phospho-GSK-3β (Ser-9) was from Abcam (ab30619). Gapdh was used as an internal control. B) Keap1−/− MEFs were seeded in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for a further 16 h. Thereafter, the cells were treated for 8 h with 1.0, 2.5, 10 or 40 μM LY294002 or 0.25, 1.0, 5.0 or 10 μM MK-2206, all of which were dissolved in DMSO to a final concentration of 0.1% (by vol), in media containing 0.1% (w/v) FBS; 0.1% (v/v) DMSO was used as vehicle control. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immunoblotted with the indicated antibodies. Gapdh was used as a sample loading control. C) Keap1−/− MEFs were grown in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for 16 h, as described in panel B above. The fibroblasts were then treated with LY294002 or MK-2206 in medium containing 0.1% FBS, at the doses indicated, for 2 h before they were transferred to fresh medium containing 0.1% FBS for 6 h. Thereafter, the fibroblasts were harvested, total RNA extracted, and mRNA for Nqo1, Hmox1, Gclc, Gclm, Gsta1 and Gstm1 measured by TaqMan chemistry as described by Higgins et al (61). The solid horizontal bar indicates that mRNA levels in MEFs treated with kinase inhibitors in medium containing 0.1% FBS were compared with MEFs treated with DMSO vehicle control in medium containing 0.1% FBS.
Figure 9
Figure 9
The sensitivity of Keap1−/− MEFs to anti-cancer drugs is increased by prevention of inhibitory phosphorylation of GSK-3 Keap1-null MEFs (1.32 × 104) were seeded in 96-well microtitre plates 24 h prior to serum depletion (0.1% FBS) for a further 16 h. The cells were then pre-treated for 8 h with (A) 10 μM LY29400 2 or 10 μM LY294002 plus 5 μM CT99021 (B) 5 μM MK-2206 or 5 μM MK-2206 plus 5 μM CT99021 in media containing 0.1% (w/v) FBS; in both (A) and (B) the kinase inhibitors were dissolved in DMSO to a final concentration of 0.1% (by volume). Thereafter, the MEFs were challenged for 48 h with increasing doses of acrolein (dissolved in ethanol), cisplatin (dissolved in media), chlorambucil (dissolved in DMSO) or a constant amount of vehicle control in media containing 0.1% (w/v) FBS. Finally, MTT was added to each of the wells and cell viability assessed by reduction of the dye (OD570), as described in the Materials and Methods section. The relative viability of Keap1−/− MEFs following exposure to different concentrations of acrolein, cisplatin or chlorambucil is shown as mean values ± SEM from three separate experiments. The EC50 dose represents the effective concentration of acrolein, cisplatin or chlorambucil required to reduce the viability of Keap1-null MEFs to 50% of maximum; the EC50 results shown in the table are mean values, with standard deviation as the 95% confidence interval presented in parenthesis. The statistical significance of differences in EC50 of Keap1-null MEFs for acrolein, cisplatin or chlorambucil with or without pre-treatment with LY294002 and/or CT99021, or MK-2206 and/or CT99021 were calculated by Two-Way ANOVA followed by Bonferroni post-tests. C) The MTT curve for Keap1-null MEFs treated for 48 h with different doses of LY294002 or MK-2206 in medium containing 0.1% FBS is shown as a control.
Figure 9
Figure 9
The sensitivity of Keap1−/− MEFs to anti-cancer drugs is increased by prevention of inhibitory phosphorylation of GSK-3 Keap1-null MEFs (1.32 × 104) were seeded in 96-well microtitre plates 24 h prior to serum depletion (0.1% FBS) for a further 16 h. The cells were then pre-treated for 8 h with (A) 10 μM LY29400 2 or 10 μM LY294002 plus 5 μM CT99021 (B) 5 μM MK-2206 or 5 μM MK-2206 plus 5 μM CT99021 in media containing 0.1% (w/v) FBS; in both (A) and (B) the kinase inhibitors were dissolved in DMSO to a final concentration of 0.1% (by volume). Thereafter, the MEFs were challenged for 48 h with increasing doses of acrolein (dissolved in ethanol), cisplatin (dissolved in media), chlorambucil (dissolved in DMSO) or a constant amount of vehicle control in media containing 0.1% (w/v) FBS. Finally, MTT was added to each of the wells and cell viability assessed by reduction of the dye (OD570), as described in the Materials and Methods section. The relative viability of Keap1−/− MEFs following exposure to different concentrations of acrolein, cisplatin or chlorambucil is shown as mean values ± SEM from three separate experiments. The EC50 dose represents the effective concentration of acrolein, cisplatin or chlorambucil required to reduce the viability of Keap1-null MEFs to 50% of maximum; the EC50 results shown in the table are mean values, with standard deviation as the 95% confidence interval presented in parenthesis. The statistical significance of differences in EC50 of Keap1-null MEFs for acrolein, cisplatin or chlorambucil with or without pre-treatment with LY294002 and/or CT99021, or MK-2206 and/or CT99021 were calculated by Two-Way ANOVA followed by Bonferroni post-tests. C) The MTT curve for Keap1-null MEFs treated for 48 h with different doses of LY294002 or MK-2206 in medium containing 0.1% FBS is shown as a control.
Figure 9
Figure 9
The sensitivity of Keap1−/− MEFs to anti-cancer drugs is increased by prevention of inhibitory phosphorylation of GSK-3 Keap1-null MEFs (1.32 × 104) were seeded in 96-well microtitre plates 24 h prior to serum depletion (0.1% FBS) for a further 16 h. The cells were then pre-treated for 8 h with (A) 10 μM LY29400 2 or 10 μM LY294002 plus 5 μM CT99021 (B) 5 μM MK-2206 or 5 μM MK-2206 plus 5 μM CT99021 in media containing 0.1% (w/v) FBS; in both (A) and (B) the kinase inhibitors were dissolved in DMSO to a final concentration of 0.1% (by volume). Thereafter, the MEFs were challenged for 48 h with increasing doses of acrolein (dissolved in ethanol), cisplatin (dissolved in media), chlorambucil (dissolved in DMSO) or a constant amount of vehicle control in media containing 0.1% (w/v) FBS. Finally, MTT was added to each of the wells and cell viability assessed by reduction of the dye (OD570), as described in the Materials and Methods section. The relative viability of Keap1−/− MEFs following exposure to different concentrations of acrolein, cisplatin or chlorambucil is shown as mean values ± SEM from three separate experiments. The EC50 dose represents the effective concentration of acrolein, cisplatin or chlorambucil required to reduce the viability of Keap1-null MEFs to 50% of maximum; the EC50 results shown in the table are mean values, with standard deviation as the 95% confidence interval presented in parenthesis. The statistical significance of differences in EC50 of Keap1-null MEFs for acrolein, cisplatin or chlorambucil with or without pre-treatment with LY294002 and/or CT99021, or MK-2206 and/or CT99021 were calculated by Two-Way ANOVA followed by Bonferroni post-tests. C) The MTT curve for Keap1-null MEFs treated for 48 h with different doses of LY294002 or MK-2206 in medium containing 0.1% FBS is shown as a control.
Figure 10
Figure 10
Down-regulation of Nrf2 in human lung A549 cells decreases expression of cytoprotective genes A) A549 cells were seeded and grown in DMEM that contained 10% FBS for about 24 h before transfer to DMEM containing 0.1% FBS for 16 h. The cells were then treated for 8 h with various doses of either LY294002 or MK-2206 in DMEM containing 0.1% FBS, as indicated, before lysates were prepared and the levels of individual proteins measured by Western blotting. The antibody that recognised both phospho-GSK-3α (Ser-21) and phospho-GSK-3β (Ser-9) was from Cell Signalling (#9331). B) A549 cells were grown as described above. After serum depletion for 16 h, they were treated for 2 h with various doses of LY294002 or MK-2206 and transferred to fresh DMEM containing 0.1% FBS for a further 6 h before being harvested. Messenger RNA levels were measured by TaqMan RT-PCR.
Figure 10
Figure 10
Down-regulation of Nrf2 in human lung A549 cells decreases expression of cytoprotective genes A) A549 cells were seeded and grown in DMEM that contained 10% FBS for about 24 h before transfer to DMEM containing 0.1% FBS for 16 h. The cells were then treated for 8 h with various doses of either LY294002 or MK-2206 in DMEM containing 0.1% FBS, as indicated, before lysates were prepared and the levels of individual proteins measured by Western blotting. The antibody that recognised both phospho-GSK-3α (Ser-21) and phospho-GSK-3β (Ser-9) was from Cell Signalling (#9331). B) A549 cells were grown as described above. After serum depletion for 16 h, they were treated for 2 h with various doses of LY294002 or MK-2206 and transferred to fresh DMEM containing 0.1% FBS for a further 6 h before being harvested. Messenger RNA levels were measured by TaqMan RT-PCR.
Figure 10
Figure 10
Down-regulation of Nrf2 in human lung A549 cells decreases expression of cytoprotective genes A) A549 cells were seeded and grown in DMEM that contained 10% FBS for about 24 h before transfer to DMEM containing 0.1% FBS for 16 h. The cells were then treated for 8 h with various doses of either LY294002 or MK-2206 in DMEM containing 0.1% FBS, as indicated, before lysates were prepared and the levels of individual proteins measured by Western blotting. The antibody that recognised both phospho-GSK-3α (Ser-21) and phospho-GSK-3β (Ser-9) was from Cell Signalling (#9331). B) A549 cells were grown as described above. After serum depletion for 16 h, they were treated for 2 h with various doses of LY294002 or MK-2206 and transferred to fresh DMEM containing 0.1% FBS for a further 6 h before being harvested. Messenger RNA levels were measured by TaqMan RT-PCR.
Figure 11
Figure 11
The sensitivity of A549 cells to chemotherapeutic agents is increased by inhibition of the PI3K-PKB/Akt pathway A549 cells were seeded and grown in 96-well plates for 24 h before being subjected to serum depletion (in 0.1% FBS) for 16 h. A) The A549 cells were then pretreated for 8 h with LY294002, or LY294002 plus CT99021, before they were challenged with various doses of acrolein, cisplatin or chlorambucil for 48 h and cytotoxicity measured using the MTT assay. B) The A549 cells were pretreated with MK-2206, or MK-2206 plus CT99021, for 8 h before being exposed to acrolein, cisplatin or chlorambucil for 48 h and MTT cytotoxicity testing performed. C) An MTT curve for A549 cells treated for 48 h with different doses of LY294002, MK-2206 or CT99021 in medium containing 0.1% FBS is shown as a control.
Figure 11
Figure 11
The sensitivity of A549 cells to chemotherapeutic agents is increased by inhibition of the PI3K-PKB/Akt pathway A549 cells were seeded and grown in 96-well plates for 24 h before being subjected to serum depletion (in 0.1% FBS) for 16 h. A) The A549 cells were then pretreated for 8 h with LY294002, or LY294002 plus CT99021, before they were challenged with various doses of acrolein, cisplatin or chlorambucil for 48 h and cytotoxicity measured using the MTT assay. B) The A549 cells were pretreated with MK-2206, or MK-2206 plus CT99021, for 8 h before being exposed to acrolein, cisplatin or chlorambucil for 48 h and MTT cytotoxicity testing performed. C) An MTT curve for A549 cells treated for 48 h with different doses of LY294002, MK-2206 or CT99021 in medium containing 0.1% FBS is shown as a control.
Figure 11
Figure 11
The sensitivity of A549 cells to chemotherapeutic agents is increased by inhibition of the PI3K-PKB/Akt pathway A549 cells were seeded and grown in 96-well plates for 24 h before being subjected to serum depletion (in 0.1% FBS) for 16 h. A) The A549 cells were then pretreated for 8 h with LY294002, or LY294002 plus CT99021, before they were challenged with various doses of acrolein, cisplatin or chlorambucil for 48 h and cytotoxicity measured using the MTT assay. B) The A549 cells were pretreated with MK-2206, or MK-2206 plus CT99021, for 8 h before being exposed to acrolein, cisplatin or chlorambucil for 48 h and MTT cytotoxicity testing performed. C) An MTT curve for A549 cells treated for 48 h with different doses of LY294002, MK-2206 or CT99021 in medium containing 0.1% FBS is shown as a control.
Figure 12
Figure 12
Repression of Nrf2 by β-TrCP occurs in both a GSK-3-dependent and a GSK-3-independent manner Nrf2 is subject to dual regulation by Keap1 and β-TrCP. The cartoon shows that Nrf2 is repressed by Keap1 though DLG and ETGE motifs in its Neh2 domain, both of which are required for ubiquitylation of the CNC-bZIP protein by Cul3-Rbx1. By contrast, Nrf2 is repressed by β-TrCP though DSGIS and DSAPGS motifs in its Neh6 domain, each of which is sufficient for ubiquitylation of the CNC-bZIP protein by Cul1-Rbx1. Phosphorylation of the DSGIS motif increases its degron activity, and this is positively regulated by GSK-3. The GSK-3 inhibitor CT99021 decreases the degron activity of the DSGIS destruction motif whereas PI3K and PKB/Akt inhibitors increase the degron activity of the DSGIS motif. By contrast, the DSAPGS destruction motif is not influenced by GSK-3 activity.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol. 2007;47:89–116. - PubMed
    1. Nioi P, McMahon M, Itoh K, Yamamoto M, Hayes JD. Identification of a novel Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone oxidoreductase 1 gene: reassessment of the ARE consensus sequence. Biochem J. 2003;374:337–348. - PMC - PubMed
    1. Hayes JD, McMahon M, Chowdhry S, Dinkova-Kostova AT. Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway. Antioxid Redox Signal. 2010;13:1713–1748. - PubMed
    1. McMahon M, Itoh K, Yamamoto M, Hayes JD. Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J Biol Chem. 2003;278:21592–21600. - PubMed
    1. Nguyen T, Sherratt PJ, Huang HC, Yang CS, Pickett CB. Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element. Degradation of Nrf2 by the 26 S proteasome. J Biol Chem. 2003;278:4536–4541. - PubMed

Publication types

MeSH terms

Substances