ABSTRACT
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 (Krüppel-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.
Subject(s)
Cytoprotection/physiology , NF-E2-Related Factor 2/physiology , Oxidative Stress/physiology , Repressor Proteins/physiology , Animals , Cells, Cultured , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Knockout , NIH 3T3 Cells , Protein Binding/physiology , Tripartite Motif-Containing Protein 28ABSTRACT
Fission yeast has two TOR kinases, Tor1 and Tor2. Recent studies have indicated that this microbe has a TSC/Rheb/TOR pathway like higher eukaryotes. Two TOR complexes, namely TORC1 and TORC2, have been identified in this yeast, as in budding yeast and mammals. Fission yeast TORC1, which contains Tor2, and TORC2, which contains Tor1, apparently have opposite functions with regard to the promotion of G1 arrest and sexual development. Rapamycin does not inhibit growth of wild-type fission yeast cells, unlike other eukaryotic cells, but precise analyses have revealed that rapamycin affects certain cellular functions involving TOR in this yeast. It appears that fission yeast has a potential to be an ideal model system to investigate the TOR signaling pathways.
