Phosphorylation of Nrf2 at Ser40 by protein kinase C in response to antioxidants leads to the release of Nrf2 from INrf2, but is not required for Nrf2 stabilization/accumulation in the nucleus and transcriptional activation of antioxidant response element-mediated NAD(P)H:quinone oxidoreductase-1 gene expression.
The antioxidant response element (ARE) and transcription factor Nrf2 regulate basal expression and antioxidant induction of NAD(P)H:quinone oxidoreductase-1 (NQO1) and other detoxifying genes. Under normal conditions, Nrf2 is targeted for proteasomal degradation by INrf2. Oxidative stress causes release of Nrf2 from INrf2. Nrf2 translocates to the nucleus, binds to the ARE, and activates gene expression. In this study, we demonstrate that protein kinase C (PKC) plays a significant role in the regulation of ARE-mediated NQO1 gene expression and induction in response to t-butylhydroquinone. Treatment of HepG2 cells with the PKC inhibitors staurosporine and calphostin C repressed ARE-mediated induction of a luciferase reporter as well as that of the endogenous NQO1 gene. Similar experiments with inhibitors of MEK/ERK, p38, phosphatidylinositol 3-kinase, and tyrosine kinases failed to repress ARE-mediated gene expression. The PKC inhibitor staurosporine blocked the nuclear translocation of Nrf2, suggesting that Nrf2 might be the target for PKC regulation. A Prosite search revealed the presence of seven putative PKC sites in mouse Nrf2. The PKC site at Ser40 is conserved among species and lies in the Neh2 domain, which interacts with INrf2. We demonstrate that phosphorylation of Ser40 is necessary for Nrf2 release from INrf2, but is not required for Nrf2 stabilization/accumulation in the nucleus and transcriptional activation of ARE-mediated NQO1 gene expression. A peptide that competes with endogenous Nrf2 for INrf2 binding was able to induce ARE activity more effectively than t-butylhydroquinone, and Nrf2 that accumulated in the nucleus as a result was not phosphorylated.
Bloom
David A
DA
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030, USA.
Jaiswal
Anil K
AK
D000975:Antioxidants / Q000494:pharmacology*; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007163:Immunosorbent Techniques; D016350:Leucine Zippers; D016297:Mutagenesis, Site-Directed; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000032:analysis / Q000235:genetics*; D051267:NF-E2-Related Factor 2; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020218:Response Elements / Q000502:physiology; D012694:Serine / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D000975:Antioxidants; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D009281:Naphthalenes; D015534:Trans-Activators; D012694:Serine; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C