The novel Nrf2-interacting factor KAP1 regulates susceptibility to oxidative stress by promoting the Nrf2-mediated cytoprotective response

The transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) co-ordinately regulates ARE (antioxidant-response element)-mediated induction of cytoprotective genes in response to electrophiles and oxidative stress; however, the molecular mechanism controlling Nrf2-dependent gene expression is not fully understood. To identify factors that regulate Nrf2-dependent transcription, we searched for proteins that interact with the Nrf2-NT (N-terminal Nrf2 transactivation domain) by affinity purification from HeLa nuclear extracts. In the present study, we identified KAP1 [KRAB (Kruppel-associated box)-associated protein 1] as a novel Nrf2-NT-interacting protein. Pull-down analysis confirmed the interaction between KAP1 and Nrf2 in cultured cells and demonstrated that the N-terminal region of KAP1 binds to Nrf2-NT in vitro. Reporter assays showed that KAP1 facilitates Nrf2 transactivation activity in a dose-dependent manner. Furthermore, the induction of the Nrf2-dependent expression of HO-1 (haem oxygenase-1) and NQO1 [NAD(P)H quinone oxidoreductase 1] by DEM (diethyl maleate) was attenuated by KAP1 knockdown in NIH 3T3 fibroblasts. This finding established that KAP1 acts as a positive regulator of Nrf2. Although Nrf2 nuclear accumulation was unaffected by KAP1 knockdown, the ability of Nrf2 to bind to the regulatory region of HO-1 and NQO1 was reduced. Moreover, KAP1 knockdown enhanced the sensitivity of NIH 3T3 cells to tert-butylhydroquinone, H2O2 and diamide. These results support our contention that KAP1 participates in the oxidative stress response by maximizing Nrf2-dependent transcription.