Jae Woon 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.

Retinoids Inhibit Interleukin-12 Production in Macrophages through Physical Associations of Retinoid X Receptor and NFκB

Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a κB site within the IL-12 p40 promoter. In this study, we found that retinoids inhibit this LPS-stimulated production of IL-12 in a dose-dependent manner. The NFκB components p50 and p65 bound retinoid X receptor (RXR) in a ligand-independent manner in vitro, and the interaction interfaces involved the p50 residues 1–245, the p65 residues 194–441, and the N-terminal A/B/C domains of RXR. Activation of macrophages by LPS resulted in markedly enhanced binding activities to the κB site, which significantly decreased upon addition of retinoids, as demonstrated by the electrophoretic mobility shift assays. In cotransfections of CV-1 and HeLa cells, RXR also inhibited the NFκB transactivation in a ligand-dependent manner, whereas a mutant RXR lacking the AF2 transactivation domain, which serves as ligand-dependent binding sites for transcription integrators SRC-1 and p300, was without any effect. In addition, coexpression of increasing amounts of SRC-1 or p300 relieved the retinoid-mediated inhibition of the NFκB transactivation. From these results, we propose that retinoid-mediated suppression of the IL-12 production from LPS-activated macrophages may involve both inhibition of the NFκB-DNA interactions and competitive recruitment of transcription integrators between NFκB and RXR.

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.

Steroid Receptor Coactivator-1 Interacts with Serum Response Factor and Coactivates Serum Response Element-mediated Transactivations

Steroid receptor coactivator-1 (SRC-1) specifically bound to serum response factor (SRF), as demonstrated by glutathione S-transferase pull down assays, and the yeast and mammalian two-hybrid tests. In mammalian cells, SRC-1 potentiated serum response element (SRE)-mediated transactivations in a dose-dependent manner. Coexpression of p300 synergistically enhanced this SRC-1-potentiated level of transactivations, consistent with the recent finding (Ramirez, S., Ali, S. A. S., Robin, P., Trouche, D., and Harel-Bellan, A. (1997) J. Biol. Chem. 272, 31016–31021) in which the p300 homologue CREB-binding protein was shown to be a transcription coactivator of SRF. Thus, we concluded that at least two distinct classes of coactivator molecules may cooperate to regulate SRF-dependent transactivationsin vivo.

Bcl3, an IκB Protein, Stimulates Activating Protein-1 Transactivation and Cellular Proliferation

Bcl3, an IκB protein, was originally isolated as a putative proto-oncogene in a subset of B cell chronic lymphocytic leukemias. Bcl3 was subsequently shown to associate tightly with and transactivate the NFκB p50 or p52 homodimer. Herein, we show that Bcl3 stimulates the activating protein-1 (AP-1) transactivation, either alone or in conjunction with transcription integrators steroid receptor coactivator-1 and CREB-binding protein/p300. The C-terminal 158 residues of Bcl3 exhibited an autonomous transactivation function and interacted with specific subregions of the AP-1 components c-Jun and c-Fos, CREB-binding protein/p300, and steroid receptor coactivator-1, as demonstrated by the yeast and mammalian two-hybrid tests as well as glutathione S-transferase pull-down assays. In addition, anti-HA antibody co-precipitated c-Jun from HeLa cells co-expressing c-Jun and HA-tagged Bcl3, consistent with the idea that Bcl3 directly associates with AP-1 in vivo. Furthermore, microinjection of Bcl3 expression vector into Rat-1 fibroblast cells significantly enhanced DNA synthesis and expression of c-jun, one of the cellular target genes of AP-1. These results suggest that Bcl3 may directly participate in the tumorigenesis processes as a novel transcription coactivator of the mitogenic transcription factor AP-1in vivo.

Activating Signal Cointegrator 1, a Novel Transcription Coactivator of Nuclear Receptors, and Its Cytosolic Localization under Conditions of Serum Deprivation

ABSTRACT Activating signal cointegrator 1 (ASC-1) harbors an autonomous transactivation domain that contains a putative zinc finger motif which provides binding sites for basal transcription factors TBP and TFIIA, transcription integrators steroid receptor coactivator 1 (SRC-1) and CBP-p300, and nuclear receptors, as demonstrated by the glutathioneS-transferase pull-down assays and the yeast two-hybrid tests. The ASC-1 binding sites involve the hinge domain but not the C-terminal AF2 core domain of nuclear receptors. Nonetheless, ASC-1 appears to require the AF2-dependent factors to function (i.e., CBP-p300 and SRC-1), as suggested by the ability of ASC-1 to coactivate nuclear receptors, either alone or in cooperation with SRC-1 and p300, as well as its inability to coactivate a mutant receptor lacking the AF2 core domain. By using indirect immunofluorescence, we further show that ASC-1, a nuclear protein, is localized to the cytoplasm under conditions of serum deprivation but is retained in the nucleus when it is serum starved in the presence of ligand or coexpressed CBP or SRC-1. These results suggest that ASC-1 is a novel coactivator molecule of nuclear receptors which functions in conjunction with CBP-p300 and SRC-1 and may play an important role in establishing distinct coactivator complexes under different cellular conditions.

Bcl3, an IκB Protein, as a Novel Transcription Coactivator of the Retinoid X Receptor

We have recently shown that the IκB protein IκBβ interacted with the retinoid X receptor (RXR) and inhibited the 9-cis-retinoic acid (RA)-dependent transactivations (Na, S.-Y., Kim, H.-J., Lee, S.-K., Choi, H.-S., Na, D. S., Lee, M.-O., Chung, M., Moore, D. D., and Lee, J. W. (1998) J. Biol. Chem. 6, 3212–3215). Herein, we show that a distinct IκB protein Bcl3 also interacts with RXR, as shown in the yeast two-hybrid tests and glutathioneS-transferase pull-down assays. The Bcl3 interaction involved two distinct subregions of RXR, i.e. constitutive interactions of the N-terminal ABC domains and 9-cis-RA-dependent interactions of the C-terminal DEF domains. In contrast to IκBβ, Bcl3 did not interact with the AF2 domain of RXR. Bcl3 specifically interacted with the general transcription factors TFIIB, TBP, and TFIIA but not with TFIIEα in the GST pull-down assays. TBP and TFIIA, however, were not able to interact with IκBβ. Accordingly, Bcl3 coactivated the 9-cis-RA-induced transactivations of RXR, in contrast to the inhibitory actions of IκBβ. In addition, coexpression of SRC-1 but not p300 further stimulated the Bcl3-mediated enhancement of the 9-cis-RA-induced transactivations of RXR. These results suggest that distinct IκB proteins differentially modulate the 9-cis-RA-induced transactivations of RXR in vivo.

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.