Soo Kyung Lee

Steroid Receptor Coactivator-1 Interacts with the p50 Subunit and Coactivates Nuclear Factor κB-mediated Transactivations

Steroid receptor coactivator-1 (SRC-1) specifically bound to the transcription factor NFκB subunit p50 but not to p65 as demonstrated by the yeast two hybrid tests and glutathione S-transferase pull down assays. The p50-binding site was localized to a subregion of SRC-1 (amino acids 759–1141) that encompasses the previously described CBP-p300-binding domain. In mammalian cells, SRC-1 potentiated the NFκB-mediated transactivations in a dose-dependent manner. Coexpression of p300 further enhanced this SRC-1-potentiated level of transactivations, consistent with the recent findings in which CBP and p300 were shown to be transcription coactivators of the p65 subunit (Perkins, N. D., Felzien, L. K., Betts, J. C., Leung, K., Beach, D. H., and Nabel, G. J. (1997) Science 275, 523–527; Gerritsen, M. E., Williams, A. J., Neish, A. S., Moore, S., Shi, Y., and Collins, T. (1997) Proc. Acad. Natl. Sci. U.u2009S.u2009A. 94, 2927–2932). These results suggest that at least two distinct coactivator molecules may cooperate to regulate the NFκB-dependent transactivations in vivo and SRC-1, originally identified as a coactivator for the nuclear receptors, may constitute a more widely used coactivation complex.

A Nuclear Factor, ASC-2, as a Cancer-amplified Transcriptional Coactivator Essential for Ligand-dependent Transactivation by Nuclear Receptors in Vivo

Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. We isolated a nuclear factor (designated ASC-2) with such properties by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. ASC-2 also interacted with other nuclear receptors, including retinoic acid receptor, thyroid hormone receptor, estrogen receptor α, and glucocorticoid receptor, basal factors TFIIA and TBP, and transcription integrators CBP/p300 and SRC-1. In transient cotransfections, ASC-2, either alone or in conjunction with CBP/p300 and SRC-1, stimulated ligand-dependent transactivation by wild type nuclear receptors but not mutant receptors lacking the AF2 domain. Consistent with an idea that ASC-2 is essential for the nuclear receptor function in vivo, microinjection of anti-ASC-2 antibody abrogated the ligand-dependent transactivation of retinoic acid receptor, and this repression was fully relieved by coinjection of ASC-2-expression vector. Surprisingly, ASC-2 was identical to a gene previously identified during a search for genes amplified and overexpressed in breast and other human cancers. From these results, we concluded that ASC-2 is a bona fidetranscription coactivator molecule of nuclear receptors, and its altered expression may contribute to the development of cancers.

Ca2+/Calmodulin-dependent Protein Kinase IV Stimulates Nuclear Factor-κB Transactivation via Phosphorylation of the p65 Subunit

Calmodulin-dependent protein kinase IV (CaMKIV) is a key mediator of Ca2+-induced gene expression. In this study, CaMKIV was found to directly associate with and phosphorylate the nuclear factor-κB (NFκB) component p65 both in vitro and in vivo. The phosphorylation of p65 by CaMKIV resulted in recruitment of transcription coactivator cAMP-response element-binding protein-binding protein and concomitant release of corepressor silencing mediator for retinoid and thyroid hormone receptors, as demonstrated by the glutathioneS-transferase pull down and mammalian two hybrid assays. In addition, cotransfection of CaMKIV resulted in cytosolic translocation of the silencing mediator for retinoid and thyroid hormone receptors. Consistent with these results, cotransfected CaMKIV dramatically stimulated the NFκB transactivation in mammalian cells. From these results, NFκB is suggested to be a novel downstream effector molecule of CaMKIV.

Mutations in Retinoid X Receptor That Impair Heterodimerization with Specific Nuclear Hormone Receptor

Retinoid X receptor (RXR) serves as a promiscuous heterodimerization partner for many nuclear receptors through the identity box, a 40-amino acid subregion within the ligand binding domain. In this study, we randomly mutated two specific residues within the human RXRα identity box region previously identified as important determinants in heterodimerization (i.e. Ala416 and Arg421). Interestingly, most of these mutants still retained wild type interactions with thyroid hormone receptor (TR), retinoic acid receptor, peroxisome proliferator-activated receptor α, small heterodimer partner, and constitutive androstane receptor. However, RXR-A416D and R421L were specifically impaired for interactions with TR, whereas RXR-A416K lost both TR and retinoic acid receptor interactions. Accordingly, RXR-A416D did not support T3 transactivation in mammalian cells, whereas RXR-A416K was not supportive of transactivation by retinoids or T3. These results provide a basis upon which to further design mutant RXRs highly selective in heterodimerization, potentially useful tools to probe nuclear receptor function in vivo.

Dissecting the molecular mechanism of nuclear receptor action: transcription coactivators and corepressors.

Over 150 members that belong to the nuclear receptor superfamily have been discovered since glucocorticoid receptor was first reported in 1985, which primarily regulate, in a ligand-dependent manner, transcriptional initiation of the target genes by directly binding to specific DNA sequences named hormone response elements (HRE) (reviewed in Mangelsdorf et al., 1995). The Cterminus of the ligand binding domain (LBD) of these proteins harbors an essential ligand-dependent transactivation function (activation function 2, AF2), whereas the N-terminus of many nuclear receptors often includes AF1. Genetic studies implicated that transcription cofactors with no specific DNA-binding activity are essential components of transcriptional regulation, which ultimately led to identify a series of nuclear receptor-interacting coregulatory proteins (reviewed in Horwitz et al., 1996). Thus far, these proteins have been shown to have a few characteristic features, as summarized in Figure 1. First, they bind to target transcription factors in a liganddependent manner. Second, many of them are capable of directly interacting with the basal transcriptional machinery. Third, some of them exhibit enzymatic function intrinsically linked to gene regulation, such as the nucleosomal remodeling histone acetyl transferase (HAT) or deacetylase (HDAC) activities. Thus, these proteins appear to function by either remodeling chromatin structures and/or acting as adapter molecules between nuclear receptors and the components of the basal transcriptional apparatus. Herein, we discuss the recent progress in studies of these coactivators and corepressors of nuclear receptors.