Inhibitory regulation of Nrf2 activation by RAC3/AIB1/SRC-3

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Nuclear Factor-Erythroid-related Factor 2 (Nrf2, NM 006164, 605 aa) is essential for the antioxidant responsive element (ARE)-mediated expression of a group of detoxifying and antioxidant genes, which detoxify carcinogens and protect against oxidative stress. So far several proteins have been identified for the functional interaction with Nrf2. In this study, we found that overexpression of RAC3/AIB1/SRC-3, a co-factor/oncogene frequently amplified in human breast cancers, inhibited the activation of Nrf2 in HeLa cells tested using ARE-luciferase assay. We also conducted the interaction study between the two proteins using Co-IP and Fluorescence Resonance Energy Transfer (FRET) analysis. From this study, we found that Nrf2 binds to RAC3 protein directly in the nucleus in HeLa cells. Furthermore, we also verified the tentative regions of Nrf2 and RAC3 for their interaction by using GST-pull down assay. The results show that pas B and R3B3 regions of RAC3 are tightly bound to N-terminal (N1 and N2) of Nrf2. These results were confirmed by Co-IP using YFP-Nrf2, N1, N2, and N2a plus pCDNA3.1-RAC3-His or CFP-pasA, pasB and R3B3 plus pCDNA3.1-Nrf2-His plasmids transfected into HeLa cells. ChIP assay also supports that Nrf2 tightly binds to RAC3 in ARE region of HO-1 promoter. Taken together, our data shows that Nrf2 activation can be controlled by RAC3 protein in HeLa cells. (Supported by NIH R01-CA-94828).