Interactions of SKIP/NCoA-62, TFIIB, and Retinoid X Receptor with Vitamin D Receptor Helix H10 Residues

Abstract

The vitamin D receptor (VDR) is a ligand-dependent transcription factor that heterodimerizes with retinoid X receptor (RXR) and interacts with the basal transcription machinery and transcriptional cofactors to regulate target gene activity. The p160 coactivator GRIP1 and the distinct coregulator Ski-interacting protein (SKIP)/NCoA-62 synergistically enhance ligand-dependent VDR transcriptional activity. Both coregulators bind directly to and form a ternary complex with VDR, with GRIP1 contacting the activation function-2 (AF-2) domain and SKIP/NCoA-62 interacting through an AF-2 independent interface. It was previously reported that SKIP/NCoA-62 interaction with VDR was independent of the heterodimerization interface (specifically, helices H10/H11). In contrast, the present study defines specific residues within a conserved and surface-exposed region of VDR helix H10 that are required for interaction with SKIP/NCoA-62 and for full ligand-dependent transactivation activity. SKIP/NCoA-62, the basal transcription factor TFIIB, and RXR all interacted with VDR helix H10 mutants at reduced levels compared with wild type in the absence of ligand and exhibited different degrees of increased interaction upon ligand addition. Thus, SKIP/ NCoA-62 interacts with VDR at a highly conserved region not previously associated with coregulator binding to regulate transactivation by a molecular mechanism distinct from that of p160 coactivators. The vitamin D receptor (VDR)1 is a ligand-dependent transcription factor important in the regulation of calcium homeostasis, development, cell growth, and differentiation. VDR belongs to the nuclear receptor superfamily, members of which share a common modular structure including a highly conserved DNA binding domain (DBD) and a conserved ligand binding domain (LBD). The LBD has a predominantly ␣-helical structure with an activation function-2 (AF-2) domain in the COOH-terminal helix (H12). The LBD is the main site of VDR interaction with its heterodimer partner retinoid X receptor (RXR) and basal transcription factors, and the AF-2 domain in combination with the hydrophobic cleft forms an interaction surface for transcriptional corepressors and coactivators (1, 2). In the absence of its ligand 1,25-dihydroxyvitamin D 3

Nuclear Coactivator-62 kDa/Ski-interacting Protein Is a Nuclear Matrix-associated Coactivator That May Couple Vitamin D Receptor-mediated Transcription and RNA Splicing

“…NCoA62/SKIP is a putative transcriptional coactivator for VDR that interacts selectively with the VDR-RXR heterodimer (20), with basal transcription factors such as TFIIB (41), and it augments VDR-mediated transcription in transient reporter gene expression studies (18,20). However, parallel studies also implicate NCoA62/SKIP in RNA splicing, since it was identified as a non-snRNP component of global spliceosome complexes as well as interchromatin granule complexes (21)(22)(23).…”

Prp8 protein: At the heart of the spliceosome

“…The transcriptional coactivator protein NCoA-62/ hSKIP(yPrp45) is a remodeling protein that is involved with vitamin D-regulated promoters (Baudino et al 1998;Zhang et al 2001Zhang et al , 2003Barry et al 2003). Adding GST-SKIP to HeLa cell nuclear extracts allowed the purification of a protein complex containing amongst others, the splicing factors hPrp8, PSF (polypyrimidine tract-binding protein-associated splicing factor), hPrp28, h200K (yBrr2p), and h116K (ySnu114p).…”

Nuclear Receptors and Their Selective Pharmacologic Modulators

“…Several coactivators have been identified that interact with VDR, including those of the steroid receptor coactivator family (SRC1, SRC2, and SRC3) as well as CBP [cAMP response element binding protein (CREB) binding protein]/p300, pCAF (p300/CBP-associated factor), and thyroid receptor interacting protein 1/Sug1, to name a few (Lee et al, 1995;Hermanson et al, 2002). VDR also directly interacts with certain components of the basal transcriptional machinery, including TF-IIB, TF-IIA, and TAF (Lavigne et al, 1999;Mengus et al, 1997Mengus et al, , 2000Barry et al, 2003).…”