Date of Graduation

2007

Document Type

Dissertation/Thesis

Abstract

Environmental pollutants and toxicants expose cells to oxidative and electrophilic challenges. Oxidative and xenobiotic stresses caused by environmental toxicants are known to cause human diseases including cancer and diabetes. To protect against the insults, cells have developed detection and response mechanisms. One such sensor-defense cellular mechanism involves the Nrf2 pathway. The transcription factor Nuclear factor erythriod 2 related factor 2 (Nrf2), a member of the cnc bZIP transcription factors, regulates the expression and induction of phase II antioxidant and detoxification genes via antioxidant response element (ARE). In the presence of an antioxidant, Nrf2 has been shown to be activated, translocate into the nucleus upon activation, heterodimerize with small Maf proteins like MafG and K, then bind to the ARE and activate transcription of phase II genes. Nrf2 has been shown to be important in neuronal function since ablation of Nrf2 induces leukoencephalopathy with wide spread gliosis in mouse brain. The signaling events leading to the activation of Nrf2 has not been clear; in particular, the role of phosphorylation in Nrf2 function remains controversial. We report that phenolic compounds, like antioxidant tert-butylhydroquinone (tBHQ) induced two forms of the Nrf2 protein in neuroblastoma cells (IMR-32), which migrated as distinctive bands on SDS-PAGE. The two bands were characterized to consist of the phosphorylated and the non phosphorylated forms of Nrf2. In vitro treatment with λ phosphatase eliminated the slower migrating form and increased the amount of the faster migrating form of Nrf2. While unphosphorylated Nrf2 predominated in the cytoplasm, the phosphorylated form preferentially localized in the nucleus. Deletional analyses from both the carboxyl- and the amino-ends revealed the transcription activation (TA) domains Neh4 and Neh5 as a major region necessary for the phosphorylation. The TA domains were characterized by the presence of multiple conserved phosphorylation sites of casein kinase 2 (CK2). Treatments with CK2 inhibitor DMAT blocked the induction of endogenous target genes of Nrf2 in cells and also significantly inhibited the TA activities of both the full length and the TA domains of Nrf2. Furthermore, phosphorylation of the TA domains correlated with the nuclear translocation of Nrf2 that was inhibited by DMAT in a concentration dependent manner. The findings demonstrated that phosphorylation of Nrf2 at the TA domains by CK2 is an integral component of Nrf2 activation necessary for the nuclear localization and transcription activation function of Nrf2 in neuroblastoma cells. Nrf2 activity is significantly affected by its interacting partners. I intended to identify new Nrf2 interacting partners. Using the yeast 2-hybrid assay, I identified N-Myc as a novel Nrf2-interacting protein. In addition, I identified MafG and ATF4, which have been known to interact with and affect Nrf2, and several other new proteins interacting with Nrf2. N-Myc is a known proto-oncogene required for embryonic development and organogenesis. Over expression of N-Myc leads to aggressive neuroblastomas. Further characterization revealed that N-Myc interacts with the C-terminal region of Nrf2. The C-terminal region of Nrf2 is involved in DNA binding and heterodimerization and therefore interaction of this region with N-Myc implicate the interaction in the transcriptional regulation of Nrf2 target gene and in cellular survival.

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