Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR‐32 neuroblastoma cells

Abstract

The antioxidant-activated transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the induction of cytoprotective genes against chemical toxicity and oxidative injuries. The role of phosphorylation in Nrf2 activation has been suggested but remains elusive. We report that phenolic antioxidant/pro-oxidant tert-butylhydroquinone (tBHQ) induced two forms of the Nrf2 protein in neuroblastoma cells (IMR-32), which migrated as distinctive bands on SDS-PAGE. In vitro treatment with λ λ phosphatase eliminated the slower migrating form and increased the amount of the faster migrating form of Nrf2. In vivo 32 Pi-phosphorylation resulted in 32 Pi-labeling of the Nrf2 protein in the presence of tBHQ that can be dephosphorylated by λ λ phosphotase, indicating that the slower migrating form is a phosphorylated Nrf2 protein and the faster form an unphosphorylated Nrf2. Unphosphorylated Nrf2 predominated in the cytoplasm, whereas the phosphorylated form preferentially localized in the nucleus. Nuclear Nrf2 can be dephosphorylated by λ λ phosphotase in vitro and be converted to the faster migrating form, implicating phosphorylation of Nrf2 in the cytoplasmic-nuclear translocation of the protein. Deletional analyses from both the carboxyl-and amino-ends revealed the transcription activation (TA) domains Neh4 (Nrf2-ECH homology 4) and Neh5 (Nrf2-ECH homology 5) as a major region necessary for the phosphorylation. The TA domains are characterized by the presence of multiple phosphorylation sites of casein kinase 2 (CK2). Moreover,

SCF/β-TrCP Promotes Glycogen Synthase Kinase 3-Dependent Degradation of the Nrf2 Transcription Factor in a Keap1-Independent Manner

“…Conventional cell signaling studies have suggested that Nrf2 might be regulated by protein phosphorylation (2,6,15,18,36,44). Previously, we presented data suggesting that GSK-3␤ (glycogen synthase kinase 3␤) influences the nuclear exclusion and inactivation of Nrf2 (37)(38)(39).…”

Acetylation-Deacetylation of the Transcription Factor Nrf2 (Nuclear Factor Erythroid 2-related Factor 2) Regulates Its Transcriptional Activity and Nucleocytoplasmic Localization

“…In HEK293T cells, Salazar et al (23) showed that Nrf2 is phosphorylated by GSK-3␤ and that this phosphorylation localizes it to the cytoplasmic compartment; according to their report, diminished entry of Nrf2 into the nucleus resulted in impaired Nrf2-induced activation of ARE-driven gene promoters. In contrast, in neuroblastoma cells phosphorylation of the Neh4 (Nrf2-ECH homology 4) and Neh5 domains of Nrf2 positively influenced its nuclear translocation as well as its transactivation activity (24). In another study, Fyn kinase-mediated phosphorylation of Tyr 568 in Nrf2 was reported to regulate its nuclear export and degradation (25).…”

Increased BTB-Kelch type substrate adaptor protein immunoreactivity associates with advanced stage and poor differentiation in renal cell carcinoma

“…Modification of cysteine residues by ROS-induced stress allows the dissociation of Nfr2 from Keap1 (22). There are, however, other regulatory mechanisms such as phosphorylation and proteosomal degradation which are also important in regulating Nfr2 activity (23,24). The significance of these mechanisms could not be assessed in this study, although they might explain some of the discrepancy between the detected immunohistochemical staining of Keap1 and Nfr2.…”