To gain insights into AF-1 and to understand the molecular mechanism of the ligand-dependent transactivation of FXR, we isolated
Nutlin3a associated with AF-1 by GST pull-down assay using the A–C domain of FXR and nuclear extracts from HeLa cells. Here, we have identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR-interacting proteins. These factors often compose a heterotrimeric complex, called DNA-PK. DNA-PK is a serine/threonine kinase and phosphorylates various substrates including p53, H2AX, nuclear receptors such as the glucocorticoid receptor (GR; NR3C1) [5] and
progesterone receptor (PR; NR3C3) [6] and [7], Ku80, Ku70, and DNA-PK itself [8] and [9]. DNA-PK is involved in multiple nuclear processes, such as DNA repair, telomere maintenance, DNA recombination and transcriptional regulation [8], [9] and [10]. We found that both Ku proteins interact with the DBD and the hinge region of FXR, and these factors suppress the FXR-mediated BSEP promoter activity and gene expression. Furthermore, Ku proteins associated with FXR on the BSEP promoter. Taken together, these findings suggest that Ku proteins function as corepressors for FXR.
