Phosphorylation of Nrf2 at Ser-40 by Protein Kinase C Regulates Antioxidant Response Element-mediated Transcription

Abstract

Nrf2, a basic leucine zipper transcription factor, is an essential activator of the coordinated transcription of genes encoding antioxidant enzymes and phase II detoxifying enzymes through the regulatory sequence termed antioxidant response element (ARE). Recently we reported evidence for the involvement of protein kinase C (PKC) in phosphorylating Nrf2 and triggering its nuclear translocation in response to oxidative stress. We show here that phosphorylation of purified rat Nrf2 by the catalytic subunit of PKC was blocked by a synthetic peptide mimicking one of the potential PKC sites. Accordingly, Nrf2 bearing a Ser to Ala mutation at amino acid 40 (S40A) could not be phosphorylated by PKC. The S40A mutation did not affect in vitro binding of Nrf2/MafK to the ARE. However, it partially impaired Nrf2 activation of ARE-driven transcription in a reporter gene assay when Keap1 was overexpressed. In vitro transcribed/translated Keap1 could be coimmunoprecipitated with Nrf2. Phosphorylation of wild-type Nrf2 by PKC promoted its dissociation from Keap1, whereas the Nrf2-S40A mutant remained associated. These findings together with our prior studies suggest that the PKC-catalyzed phosphorylation of Nrf2 at Ser-40 is a critical signaling event leading to ARE-mediated cellular antioxidant response. Nrf2, a basic leucine zipper transcription factor, is an essential activator of the coordinated transcription of genes encoding antioxidant enzymes and phase II detoxifying enzymes through the regulatory sequence termed antioxidant response element (ARE). Recently we reported evidence for the involvement of protein kinase C (PKC) in phosphorylating Nrf2 and triggering its nuclear translocation in response to oxidative stress. We show here that phosphorylation of purified rat Nrf2 by the catalytic subunit of PKC was blocked by a synthetic peptide mimicking one of the potential PKC sites. Accordingly, Nrf2 bearing a Ser to Ala mutation at amino acid 40 (S40A) could not be phosphorylated by PKC. The S40A mutation did not affect in vitro binding of Nrf2/MafK to the ARE. However, it partially impaired Nrf2 activation of ARE-driven transcription in a reporter gene assay when Keap1 was overexpressed. In vitro transcribed/translated Keap1 could be coimmunoprecipitated with Nrf2. Phosphorylation of wild-type Nrf2 by PKC promoted its dissociation from Keap1, whereas the Nrf2-S40A mutant remained associated. These findings together with our prior studies suggest that the PKC-catalyzed phosphorylation of Nrf2 at Ser-40 is a critical signaling event leading to ARE-mediated cellular antioxidant response. antioxidant response element NF-E2-related factor 2 protein kinase C mitogen-activated protein kinase extracellular signal-regulated kinase phosphatidylinositol 3-kinase phorbol 12-myristate 13-acetate tert-butylhydroquinone NAD(P)H:quinone oxidoreductase chloramphenicol acetyltransferase electrophoretic mobility shift assay nickel-nitrilotriacetic acid wild type The antioxidant response element (ARE)1 is a regulatory sequence involved in the coordinated transcriptional activation of genes coding for a number of antioxidant enzymes and phase II detoxifying enzymes (1Rushmore T.H. Pickett C.B. J. Biol. Chem. 1990; 265: 14648-14653Abstract Full Text PDF PubMed Google Scholar, 2Friling R.S. Bensimon A. Tichauer Y. Daniel V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6258-6262Crossref PubMed Scopus (426) Google Scholar, 3Favreau L.V. Pickett C.B. J. Biol. Chem. 1991; 266: 4556-4561Abstract Full Text PDF PubMed Google Scholar, 4Li Y. Jaiswal A.K. J. Biol. Chem. 1992; 267: 15097-15104Abstract Full Text PDF PubMed Google Scholar, 5Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 6Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar). Reactive oxygen species and electrophiles are potent activators of genes containing an ARE, mediated by the basic leucine zipper (bZIP) transcription factor Nrf2 (NF-E2-related factor 2) (7Moi P Chan K. Asunis I. Cao A. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9926-9930Crossref PubMed Scopus (1238) Google Scholar, 8Igarashi K. Kataoka K. Itoh K. Hayashi N. Nishizawa M. Yamamoto M. Nature (Lond.). 1994; 367: 568-572Crossref PubMed Scopus (398) Google Scholar, 9Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3191) Google Scholar). Accumulated evidence from studies ofnrf2-null mice has established that Nrf2 is an essential ARE-binding factor involved in both constitutive and inducible gene expression via the ARE (9Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3191) Google Scholar, 10Kwak M.K. Itoh K. Yamamoto M. Sutter T.R. Kensler T.W. Mol. Med. 2001; 7: 135-145Crossref PubMed Google Scholar, 11McMahon M. Itoh K. Yamamoto M. Chanas S.A. Henderson C.J. McLellan L.I. Wolf C.R. Cavin C. Hayes J.D. Cancer Res. 2001; 61: 3299-3307PubMed Google Scholar). An important regulatory step leading to ARE activation is the oxidative stress-induced nuclear translocation of Nrf2, which normally appears to be sequestered in the cytoplasm by the cytoskeleton-binding Keap1 protein (12Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2792) Google Scholar, 13Dhakshinamoorthy S. Jaiswal A.K. Oncogene. 2001; 20: 3906-3917Crossref PubMed Scopus (255) Google Scholar, 14Sekhar K.R. Spitz D.R. Harris S. Nguyen T.T. Meredith M.J. Holt J.T. Guis D. Marnett L.J. Summar M.L. Freeman M.L. Free Rad. Biol. Med. 2002; 32: 650-662Crossref PubMed Scopus (65) Google Scholar). However, the precise mechanism by which ARE-activating signals reach Nrf2 and cause dissociation of the putative inhibitory Nrf2-Keap1 complex remains unclear. Several protein kinase pathways have been implicated in transducing oxidative stress signals to gene expression mediated through the ARE. A number of reports have addressed a possible role for extracellular signal-regulated kinase (ERK1/2) in ARE activation. The findings have however remained controversial: ERK1/2 has been found to regulate the ARE positively in certain hepatoma cells (15Yu R. Lei W. Mandlekar S. Weber M.J. Der C.J., Wu, J. Kong A.-N.T. J. Biol. Chem. 1999; 274: 27545-27552Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 16Yu R. Chen C., Mo, Y.Y. Hebbar V. Owuor E.D. Tan T.-H. Kong A.-N.T. J. Biol. Chem. 2000; 275: 39907-39913Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 17Zipper L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (218) Google Scholar) but negatively in others (18Kang K.W. Cho M.K. Lee C.H. Kim S.G. Mol. Pharmacol. 2001; 59: 1147-1156Crossref PubMed Scopus (102) Google Scholar). Similarly, p38 MAP (mitogen-activated protein) kinase has also been shown to affect ARE activity, either positively (17Zipper L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (218) Google Scholar, 19Kang K.W. Ryu J.H. Kim S.G. Mol. Pharmacol. 2000; 58: 1017-1025Crossref PubMed Scopus (105) Google Scholar,20Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) or negatively (16Yu R. Chen C., Mo, Y.Y. Hebbar V. Owuor E.D. Tan T.-H. Kong A.-N.T. J. Biol. Chem. 2000; 275: 39907-39913Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 21Yu R. Mandlekar S. Lei W. Fahl W.E. Tan T.-H. Kong A.-N.T. J. Biol. Chem. 2000; 275: 2322-2327Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). More recently, phosphatidylinositol 3-kinase and its downstream target Akt/PKB (protein kinase B) have been linked to activation of the ARE in hepatoma (18Kang K.W. Cho M.K. Lee C.H. Kim S.G. Mol. Pharmacol. 2001; 59: 1147-1156Crossref PubMed Scopus (102) Google Scholar, 19Kang K.W. Ryu J.H. Kim S.G. Mol. Pharmacol. 2000; 58: 1017-1025Crossref PubMed Scopus (105) Google Scholar) and neuroblastoma (22Lee J.-M. Hanson J.M. Chu W.A. Johnson J.A. J. Biol. Chem. 2001; 276: 20011-20016Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) cell lines. However, none of the known cellular components involved in ARE regulation have been shown to be targets of any of these kinases. Recently, we reported several findings that indicate an important role for protein kinase C (PKC) in the ARE-mediated gene expression (23Huang H.-C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (440) Google Scholar). 1) Phorbol 12-myristate 13-acetate (PMA), a potent PKC-activating phorbol ester, stimulates ARE-driven transcription, which is blocked by selective PKC inhibitors. 2) Nuclear translocation of Nrf2 is induced by PMA but arrested by PKC inhibitors. 3) Both Nrf2 nuclear translocation and activation of the ARE bytert-butylhydroquinone (tBHQ) treatment are suppressed by PKC inhibitors. 4) Nrf2 is phosphorylated in vitro by purified PKC or immunoprecipitated PKC from tBHQ-induced cells. 5) Nrf2 phosphorylation in HepG2 cells is enhanced by PMA and tBHQ but abolished by PKC inhibitors. Together these results suggest that one critical step in the signaling cascade toward ARE activation may be the phosphorylation of Nrf2 by PKC, which promotes the nuclear translocation of this transcription factor in response to oxidative stress. The present study is a continuation of our investigation into the involvement of PKC in regulating the ARE. We sought to identify the site of phosphorylation in Nrf2 by PKC and to characterize the mechanistic significance of Nrf2 phosphorylation. A high level expression plasmid of rat Nrf2 gene linked at its N terminus to a His6tag was obtained by cloning the rat Nrf2 cDNA (GenBankTM accession number AF037350) into the pQE-30 vector (Qiagen). S40A mutant (AGT → GCT) was obtained by the PCR mutagenesis method and cloned into the same expression plasmid. Nrf2 wild-type or S40A mutant expression was induced by the addition of 0.5 mmisopropyl-β-d-thiogalactopyranoside for 6 h at 30 °C in Escherichia coli M15 cells. Cell pellets were suspended and sonicated in a buffer containing 100 mmNaH2PO4, 10 mm Tris-HCl (pH 8.0), 300 mm NaCl, 0.1 mm EDTA, 5 mmβ-mercaptoethanol, 10 mm imidazole, 0.5% Tween 20, and 15% glycerol. After centrifugation at 14,000 × g for 20 min, soluble lysates were loaded onto a Ni-NTA column. A 40 mm imidazole wash was followed by elution at 250 mm imidazole in the same buffer. Eluted fractions were pooled and dialyzed against 20 mm HEPES (pH 7.5), 200 mm NaCl, 0.1 mm EDTA, 1 mmdithiothreitol, and 20% glycerol and stored at −80 °C. Purified His6-tagged rat Nrf2 wild-type and S40A mutant proteins were used as substrates in in vitro kinase assays with catalytic subunits of rat brain PKC (Calbiochem). 50-μl reactions were carried out at 30 °C in a buffer containing 25 mm HEPES (pH 7.5), 10 mm MgCl2, 200 μm ATP, and 2 μCi of [γ-33P]ATP and stopped at the indicated times by the addition of sample buffer for SDS-PAGE analysis. The level of [33P]ATP incorporation into Nrf2 was determined by autoradiography or by a PhosphorImager (Fujifilm FLA-2000). [γ-33P]ATP was omitted from kinase reactions whose products were subsequently used in electrophoretic mobility shift assays (EMSA) or in immunoprecipitation studies as described below. EMSAs were performed essentially as described previously (24Nguyen T. Huang H.-C. Pickett C.B. J. Biol. Chem. 2000; 275: 15466-15473Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). A double-stranded oligonucleotide containing the ratQR gene ARE was used as probe after end-labeling with [γ-32P]ATP by T4 polynucleotide kinase. The sequence of the DNA probe was 5′-GATTTCAGTCTAGAGTCACAGTGACTTGGCAAAATCTGAGCCG-3′ (ARE core sequence highlighted in bold). Purified rat Nrf2 wild-type or S40A mutant protein was preincubated with rat MafK (unless otherwise indicated) for 20 min at 25 °C before the addition of DNA probe for another 20 min at 30 °C. MafK proteins were produced in vitro by the TnT coupled transcription/translation wheat germ extract system (Promega) (24Nguyen T. Huang H.-C. Pickett C.B. J. Biol. Chem. 2000; 275: 15466-15473Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). DNA-protein interactions were detected by electrophoresis on non-denaturing 6% polyacrylamide gels in Tris borate-EDTA (TBE) buffer, followed by autoradiography. For competition experiments, a 200-fold molar excess of either unlabeled probe or a random 43-base oligonucleotide was included in the preincubation mixture at 25 °C before the addition of the labeled probe. For supershift assay, an anti-Nrf2 was after the binding for h at °C before the of PKC phosphorylation of Nrf2 on ARE Nrf2 wild-type or S40A mutant protein was in kinase assay buffer for 1 h at 30 °C in the or of PKC, before an was for with MafK and the labeled probe for HepG2 cells were obtained from the and as previously described (1Rushmore T.H. Pickett C.B. J. Biol. Chem. 1990; 265: 14648-14653Abstract Full Text PDF PubMed Google Scholar). was performed as before on cells in at 1 of an expression plasmid containing rat ARE linked to chloramphenicol acetyltransferase reporter gene was with of plasmid bearing wild-type or S40A mutant Nrf2 with of plasmid containing rat Keap1 (GenBankTM accession number of DNA was at 2 by the addition of vector to the DNA were for h after from and in protein Cell lysates were for as described (23Huang H.-C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (440) Google and the results were by a In of the bearing Nrf2 and Keap1 was carried out the TnT coupled wheat germ extract system (Promega) in the of to the The products were together for min at 30 °C before immunoprecipitation by an anti-Nrf2 in an immunoprecipitation buffer containing mm Tris (pH 8.0), mm NaCl, 0.5% for h at followed by the addition of protein The mixture was at °C for 1 h and in immunoprecipitation buffer containing were by SDS-PAGE and detected by autoradiography. of was performed on a the of PKC phosphorylation on Nrf2-Keap1 an of labeled Nrf2 wild-type or S40A mutant was with PKC and Keap1 in kinase assay buffer for 30 min at 30 °C before In prior studies we that Nrf2 is phosphorylated in HepG2 cells (23Huang H.-C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (440) Google Scholar). Nrf2 phosphorylation was by the phorbol PMA but was blocked by the PKC that Nrf2 may be a PKC Accordingly, in vitro PKC assays that purified rat Nrf2 was phosphorylated by the catalytic subunit of rat brain PKC or by PKC immunoprecipitated from HepG2 identify the of phosphorylation in Nrf2 by PKC, a synthetic mimicking potential Nrf2 PKC were used as for in vitro phosphorylation of Nrf2 by PKC. Nrf2 is a acid protein containing potential PKC phosphorylation to the is any were to potential PKC target at and 1 were soluble in 20% peptide whose was when it was at both by one to a In vitro kinase assays were performed as described previously (23Huang H.-C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (440) Google Scholar) obtained catalytic subunits of rat brain PKC and purified rat Nrf2 as Nrf2 phosphorylation by PKC or enhanced by several of the it was by in the of 5 mm peptide 1 PKC against a was not suppressed by this peptide that its was not on the These findings suggest that Ser-40 of Nrf2 is an site of phosphorylation by PKC. these a mutagenesis was Nrf2 gene bearing a → to mutation at amino acid 40 was cloned into a high level expression plasmid containing a to the Nrf2-S40A protein was purified to by The at was to be Nrf2 by an against Nrf2 Nrf2-S40A was used in with wild-type Nrf2 as substrates in in vitro PKC shown in the amino acid from Ser to Ala at 40 abolished PKC phosphorylation of Nrf2. The of phosphorylation in this mutant that Ser-40 is the PKC with the peptide competition Nrf2 at PKC was as in 1 2 μm purified wild-type Nrf2 or Nrf2-S40A mutant protein as for the indicated times at 30 °C. an anti-Nrf2 was performed for an of the same as shown in the be that Ser-40 is also one of potential PKC in Nrf2 (GenBankTM accession number which is to the rat Nrf2. is that PKC Nrf2 at the same site and that the phosphorylation of Nrf2 in hepatoma HepG2 cells (23Huang H.-C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (440) Google Scholar) is at in by PKC on We and others have previously shown vitro transcribed/translated Nrf2 with high to the ARE as of a complex with proteins (9Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3191) Google Scholar, T. Huang H.-C. Pickett C.B. J. Biol. Chem. 2000; 275: 15466-15473Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, S. Jaiswal A.K. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of Nrf2 with the ARE is by the mutation PKC in vitro EMSAs with and in vitro MafK were ARE sequence from the rat gene as probe. Nrf2 or or MafK could not to the ARE. In the of both and Nrf2-S40A with the ARE probe. These were with an against Nrf2 The and mobility of the wild-type and S40A mutant are of both of was blocked by excess unlabeled ARE but not by random the S40A mutation did not the high Nrf2/MafK and the ARE. Nrf2 to the ARE in a as wild-type Nrf2 or the S40A mutant for PKC phosphorylation the of ARE-binding transcriptional complex containing Nrf2 and proteins not to be by the phosphorylation of Nrf2 by PKC. the Nrf2-S40A mutant has an on Nrf2 of the ARE, we in HepG2 cells. of plasmid into a number of hepatoma cell has previously been shown to in activation of the ARE-mediated transcription (16Yu R. Chen C., Mo, Y.Y. Hebbar V. Owuor E.D. Tan T.-H. Kong A.-N.T. J. Biol. Chem. 2000; 275: 39907-39913Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, T. Huang H.-C. Pickett C.B. J. Biol. Chem. 2000; 275: 15466-15473Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). A ratQR reporter gene was with a high plasmid vector bearing wild-type Nrf2. An activation of ARE-driven was of Nrf2 plasmid with an of Nrf2-S40A in of activation Keap1 has been shown to Nrf2 by it in the cytoplasm (12Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2792) Google we any of the Nrf2-S40A mutant of Keap1 with wild-type Nrf2 ARE activation. of Keap1 in a of ARE activation by the Nrf2-S40A mutant that was not with of Keap1 in HepG2 cells. ARE activation by Nrf2-S40A was to a level of that with wild-type Nrf2, from an activation to In these Nrf2 wild-type and S40A proteins were to as by an anti-Nrf2 These findings indicate a role for Keap1 in the by the Nrf2-S40A from Itoh (12Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2792) Google Scholar) have shown that Nrf2 with Keap1 through a of 100 amino acid at its N terminus PKC Nrf2 at we the of the S40A mutant in ARE activation is to its with We in vitro transcribed/translated Nrf2 and Keap1 proteins could with Nrf2 and Keap1 were labeled in the of The products were followed by immunoprecipitation with an anti-Nrf2 and the products were to SDS-PAGE and autoradiography. Keap1 could be with Nrf2 by the anti-Nrf2 We the Keap1 and the Nrf2-S40A of Keap1 with both wild-type and S40A Nrf2 6 However, when immunoprecipitation was carried out after of these components in the of PKC, the of Keap1 with wild-type Nrf2 was by the S40A mutant with Keap1 to a with or PKC 6 the dissociation of Keap1 from wild-type Nrf2 was abolished when PKC was preincubated in the of 10 a potent of PKC We that phosphorylation of Nrf2 by PKC at Ser-40 a critical role in the of Nrf2 from the impaired of the Nrf2-S40A mutant to ARE-mediated transcription is to a in the dissociation of Nrf2 from its We reported that phosphorylation of Nrf2 by PKC nuclear translocation of this transcription factor and activation of the ARE in response to oxidative stress (23Huang H.-C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (440) Google Scholar). In the present we the of Nrf2 phosphorylation and its significance in ARE activation. that PKC Nrf2 at Ser-40 and its from the Together with our these results suggest a mechanistic of ARE-mediated cellular antioxidant response the PKC-catalyzed phosphorylation of Nrf2 at Ser-40 as a for the nuclear translocation of this transcription studies from Itoh (12Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2792) Google Scholar) that Nrf2 is normally in the cytoplasm by its with the cytoskeleton-binding protein Nrf2 from the Nrf2-Keap1 it to into the to ARE-driven gene indicated that Keap1 through the of Nrf2, amino (12Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2792) Google Scholar). in vitro results here a mechanistic for the of this in Nrf2-Keap1 A critical this appears to be whose phosphorylation by PKC oxidative stress promotes the dissociation of Nrf2 from remains to be in cells Ser-40 phosphorylation is or and components also be involved for Nrf2 to from its and into the is also not known Keap1 is a PKC Keap1 potential PKC the and which have been as important for binding to Nrf2 S. Jaiswal A.K. Oncogene. 2001; 20: 3906-3917Crossref PubMed Scopus (255) Google Scholar). However, we did not a in the of Keap1 with the Nrf2-S40A mutant with PKC, on Keap1 that the in The present findings suggest that the significance of PKC phosphorylation of Nrf2 is to the nuclear The by the Nrf2-S40A mutant in ARE-driven reporter gene assay appears to with the of the the of ARE-mediated gene expression by of Keap1 in the of to the dissociation of this However, the of ARE-binding complex with Nrf2/MafK in the shift assay was not by the of PKC-catalyzed phosphorylation of Nrf2 or when the S40A mutant was PKC phosphorylation not to a role in either the of Nrf2/MafK with the ARE sequence or of Nrf2 with PKC has also been implicated in the transcriptional regulation J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Chen J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). PKC in that appears to a nuclear it not the of the transcription factor J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). be to the Nrf2 proteins present in the are of the phosphorylated phosphorylation by PKC not to be for high binding of Nrf2 to the ARE. The our results suggest that after from Keap1, is into the for binding to the ARE However, it is possible that protein before the binding of Nrf2 to its target sequence and the of this transcription factor into the cytoplasm as of a regulatory of nuclear of Nrf2. In studies of have been shown to and in T. Mol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). prior studies the involvement of PKC in ARE activation by Nrf2 were performed with of PKC as In a mutant (S40A) that PKC-catalyzed phosphorylation of Nrf2 and its dissociation from Keap1, we have an essential role for PKC phosphorylation of Nrf2 in the signaling leading to ARE activation. The present results a role for PKC, its downstream in the regulation of Nrf2 An important is PKC may be a for oxidative stress. An that and which are potent of the ARE, also PKC R. Chen U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). PKC that suggest it as an target for R. S. Free Rad. Biol. Med. 2000; PubMed Scopus Google Scholar). have been several reports of in both its regulatory and catalytic that to regulate PKC R. S. Free Rad. Biol. Med. 2000; PubMed Scopus Google Scholar). it is at present of PKC Nrf2, a may be which has been implicated in a number of cellular oxidative stress N. T. W. Cao J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus (183) Google Scholar, Wu, R. S. D. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). from several have indicated the involvement of kinase pathways in ARE-mediated transcription (17Zipper L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (218) Google Scholar, K.W. Cho M.K. Lee C.H. Kim S.G. Mol. Pharmacol. 2001; 59: 1147-1156Crossref PubMed Scopus (102) Google Scholar, 19Kang K.W. Ryu J.H. Kim S.G. Mol. Pharmacol. 2000; 58: 1017-1025Crossref PubMed Scopus (105) Google Scholar, J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 21Yu R. Mandlekar S. Lei W. Fahl W.E. Tan T.-H. Kong A.-N.T. J. Biol. Chem. 2000; 275: 2322-2327Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, J.-M. Hanson J.M. Chu W.A. Johnson J.A. J. Biol. Chem. 2001; 276: 20011-20016Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). studies wild-type and and Nrf2 have a role for in ARE regulation in HepG2 cells (15Yu R. Lei W. Mandlekar S. Weber M.J. Der C.J., Wu, J. Kong A.-N.T. J. Biol. Chem. 1999; 274: 27545-27552Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 16Yu R. Chen C., Mo, Y.Y. Hebbar V. Owuor E.D. Tan T.-H. Kong A.-N.T. J. Biol. Chem. 2000; 275: 39907-39913Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). However, of in cells in expression of the a role for in ARE-mediated (18Kang K.W. Cho M.K. Lee C.H. Kim S.G. Mol. Pharmacol. 2001; 59: 1147-1156Crossref PubMed Scopus (102) Google Scholar). The role of the p38 kinase in ARE regulation is also from as several have reported a (17Zipper L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (218) Google Scholar, 19Kang K.W. Ryu J.H. Kim S.G. Mol. Pharmacol. 2000; 58: 1017-1025Crossref PubMed Scopus (105) Google Scholar, J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) whereas another that a p38 the activation of reporter gene R. Mandlekar S. Lei W. Fahl W.E. Tan T.-H. Kong A.-N.T. J. Biol. Chem. 2000; 275: 2322-2327Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Recently, 3-kinase and its downstream kinase have been shown by studies to be of ARE in hepatoma (18Kang K.W. Cho M.K. Lee C.H. Kim S.G. Mol. Pharmacol. 2001; 59: 1147-1156Crossref PubMed Scopus (102) Google Scholar) and neuroblastoma cells (22Lee J.-M. Hanson J.M. Chu W.A. Johnson J.A. J. Biol. Chem. 2001; 276: 20011-20016Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). of the precise of these be by the of cellular targets known to be involved in ARE be that in ARE-mediated transcription has been reported to be J.-M. J.D. Hanson J.M. Johnson J.A. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar). However, the same system was also shown to be oxidative in to the studies from oxidative stress-induced ARE-mediated gene expression has been the results indicate that Nrf2/MafK with ARE is of PKC it remains possible that binding of Nrf2 with a nuclear factor proteins to the ARE may be to regulation by PKC. studies have that may as nuclear extract from HepG2 and cells with the ARE that are from by in Nrf2/MafK proteins (24Nguyen T. Huang H.-C. Pickett C.B. J. Biol. Chem. 2000; 275: 15466-15473Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). have been several reports of Nrf2 binding to the ARE J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Fahl W.E. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar, Y. Itoh K. M. A. Yamamoto M. Genes 2001; PubMed Scopus Google Scholar, C.H. Gong Stewart D. Choi M.E. Choi A.M. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). protein) has been shown to Nrf2 via on Nrf2 in a to ARE transcription Y. Itoh K. M. A. Yamamoto M. Genes 2001; PubMed Scopus Google Scholar). has also been to with Nrf2 in regulating ARE-driven gene expression C.H. Gong Stewart D. Choi M.E. Choi A.M. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). be that the nuclear translocation of Nrf2 has been shown to be a mechanism for ARE activation in cell Nrf2 also has a role in constitutive ARE-mediated gene We have shown that Nrf2 is present in the tBHQ and its essential role in the of the ARE has been reported in a system (24Nguyen T. Huang H.-C. Pickett C.B. J. Biol. Chem. 2000; 275: 15466-15473Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) and in mice M. Itoh K. Yamamoto M. Chanas S.A. Henderson C.J. McLellan L.I. Wolf C.R. Cavin C. Hayes J.D. Cancer Res. 2001; 61: 3299-3307PubMed Google Scholar). that the coordinated transcriptional activation of antioxidant of which phosphorylation of Nrf2 at Ser-40 by PKC is but one of critical regulatory We for and