Ternary complexes and cooperative interplay between NCoA-62/Ski-interacting protein and steroid receptor coactivators in vitamin D receptor-mediated transcription

The vitamin D receptor (VDR) is a ligand-dependent transcriptional factor that binds to vitamin D-responsive elements as a heterodimer with retinoid X receptor (RXR) to regulate target gene transcription. The steroid receptor coactivator (Src) proteins are coactivators that interact with the AF-2 domain of VDR to augment 1,25-dihydroxyvitamin D3-dependent transcription. In contrast, NCoA-62/Ski-interacting protein (SKIP) is a distinct, activation function-2-independent coactivator for VDR. The current study examined whether these two distinct classes of coactivators impact functionally on VDR-mediated transcription. Using a ternary complex binding assay, we observed a marked preference for the direct interaction of NCoA-62/SKIP with the VDR-RXR heterodimer as compared with the VDR-VDR homodimer or VDR monomer. The liganded VDR also formed a ternary complex with NCoA-62/SKIP and Src proteins in vitro. Competition experiments using LXXLL Peptides showed that NCoA-62/SKIP and Src coactivators contact different domains of the VDR-RXR heterodimer. Synergistic interplays were observed between NCoA-62/SKIP and Src coactivators in VDR-mediated transcriptional assays, and protein interference assays indicated a requirement for both NCoA-62/SKIP and SRCs in VDR- mediated transcription. These studies suggest that the ligand-dependent and simultaneous interaction of NCoA-62/SKIP and Src coactivators with distinct interaction domains within the VDR-RXR heterodimer results in cooperative interplays between coactivators in VDR-mediated transcription.