Phosphorylation of the human retinoid X receptor α at serine 260 impairs coactivator(s) recruitment and induces hormone resistance to multiple ligands

The retinoid X receptor alpha(RXR alpha) is a member of the nuclear receptor superfamily that regulates transcription of target genes through heterodimerization with several partners, including peroxisome proliferator-activated receptor, retinoic acid receptor, thyroid receptor, and vitamin D receptor (VDR). We have shown previously that signaling through VDR center dot RXR alpha heterodimers was attenuated in ras-transformed keratinocytes due to phosphorylation of serine 260 of the RXR alpha via the activated Ras-Raf-MAPK cascade in these cells. In this study we demonstrate that phosphorylation at serine 260, a site located in the omega loop-AF-2 interacting domain of RXR alpha, inhibits signaling through several heterodimeric partners of the RXR alpha. The inhibition of signaling results in reduced transactivational response to ligand presentation and the reduced physiological response of growth inhibition not only of 1,25-dihydroxyvitamin D-3 but also of retinoic acid receptor alpha ligands and LG1069 (an RXR alpha ligand). This partial resistance to ligands could be reversed by inhibition of MAPK activity or by overexpression of a non-phosphorylable RXR alpha mutant at serine 260 (RXR alpha Ser-260 -> Ala). Importantly, phosphorylation of RXR alpha at serine 260 impaired the recruitment of DRIP205 and other coactivators to the VDR center dot RXR alpha complex. Chromatin immunoprecipitation and pulldown assays further demonstrated that coactivator recruitment to the VDR center dot RXR complex could be restored by treatment with a MAPK inhibitor. Our data suggest that phosphorylation at serine 260 plays a critical role in inducing hormone resistance of RXR alpha-mediated signaling likely through structural changes in the H-1-H-3 omega loop-AF2 coactivator(s) interacting domain.