Two Distinct Nuclear Receptor- Interaction Domains and CREB ...

Two Distinct Nuclear ReceptorInteraction Domains and CREBBinding Protein-Dependent Transactivation Function of Activating Signal Cointegrator-2

Soo-Kyung Lee*, Sung-Yun Jung, Youn-Sung Kim, Soon-Young Na†, Young Chul Lee, and Jae Woon Lee Center for Ligand and Transcription (S.-K.L., S.-Y.J., Y.-S.K., S.Y.N. Y.C.L., J.W.L.) Department of Biology (S.-K.L.,, S.-Y.N.) and Hormone Research Center (Y.C.L., J.W.L.) Chonnam National University Kwangju 500–757, Korea

ASC-2 is a recently isolated transcriptional cointegrator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors, AP-1, nuclear factor ␬B (NF␬B), serum response factor (SRF), and numerous other transcription factors. ASC-2 contained two nuclear receptor-interaction domains, both of which are dependent on the integrity of their core LXXLL sequences. Surprisingly, the C-terminal LXXLL motif specifically interacted with oxysterol receptor LXR␤, whereas the N-terminal motif bound a broad range of nuclear receptors. These interactions appeared to be essential because a specific subregion of ASC-2 including the N- or C-terminal LXXLL motif acted as a potent dominant negative mutant with transactivation by appropriate nuclear receptors. In addition, the autonomous transactivation domain (AD) of ASC-2 was found to consist of three separable subregions; i.e. AD1, AD2, and AD3. In particular, AD2 and AD3 were binding sites for CREB binding protein (CBP), and CBP-neutralizing E1A repressed the autonomous transactivation function of ASC-2. Furthermore, the receptor transactivation was not enhanced by ASC-2 in the presence of E1A and significantly impaired by overexpressed AD2. From these results, we concluded that ASC-2 directly binds to nuclear receptors and recruits CBP to mediate the nuclear receptor transactivation in vivo. (Molecular Endocrinology 15: 241–254, 2001)

INTRODUCTION The nuclear receptor superfamily is a group of liganddependent transcriptional regulatory proteins that function by binding to specific DNA sequences named hormone response elements in the promoters of target genes (1). The superfamily includes receptors for a variety of small hydrophobic ligands such as steroids, T3, and retinoids, as well as a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors. Functional analysis of nuclear receptors has shown that there are two major activation domains. The N-terminal domain (AF-1) contains a ligand-independent activation function, whereas the ligand-binding domain (LBD) (AF-2) exhibits ligand-dependent transactivation. The AF-2 core region, located at the extreme C terminus of the receptor LBDs, is conserved among nuclear receptors and undergoes a major conformational change upon ligand binding. Notably, deletion or point mutations in this region impair transcriptional activation without changing ligand and DNA binding affinities. This region has been shown to play a critical role in mediating transactivation by serving as a ligand-dependent interaction interface with many different coactivators (2, 3). Transcriptional coactivators either bridge transcription factors and the components of the basal transcriptional apparatus and/or remodel the chromatin structures (2, 3). In particular, CREB binding protein (CBP) and its functional homolog p300 were shown to be essential for the activation of transcription by a large number of regulated transcription factors (4). Similarly, steroid receptor coactivator-1 (SRC-1) and its family members were recently found to stimulate transactivation by many different transcription factors, including CREB and signal transducers and activators

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of transcription (STATs) (5), AP-1 (6), nuclear factor ␬B (NF␬B) (7, 8), p53 (9), and serum response factor (SRF) (10). SRC-1 (11) and its homolog ACTR (12), along with CBP and p300 (13, 14), were recently shown to contain histone acetyltransferase (HAT) activities and associate with yet another HAT protein P/CAF (15). In contrast, SMRT (16) and N-CoR (17), nuclear receptor corepressors, form complexes with Sin3 and histone deacetylase proteins (18, 19). These results are consistent with the notion that acetylation of histones destabilizes nucleosomes and relieves transcriptional repression by allowing transcription factors to access to recognition elements, whereas deacetylation of the histones stabilizes the repressed state (2, 3). We have recently described an AF-2 and liganddependent coactivator molecule of nuclear receptors that we named activating signal cointegrator-2 (ASC-2) (20, 21). Similar or identical molecules were subsequently reported by other groups and variously named TRBP (for thyroid hormone receptor-binding protein) (22), PRIP (for peroxisome proliferator-activated receptor interacting protein) (23), and RAP250 (for nuclear receptor-activating protein 250) (24). Like SRC-1 and CBP/p300, ASC-2 also enhanced transactivation by a large number of other transcription factors, including CREB, SRF, NF␬B, and AP-1 (21, 22). In particular, microinjection of anti-ASC-2 antibody abrogated the ligand-dependent transactivation of retinoic acid receptor (RAR) and the TPA-inducible AP-1 transactivation, suggesting that ASC-2 is essential for the nuclear receptor and AP-1 functions in vivo (20, 21). Northern blot and in situ hybridization analyses revealed that ASC-2 is widely expressed with the highest expression in reproductive organs and brain (24). Interestingly, ASC-2 was found to be highly amplified and overexpressed in human colon, breast, and lung cancers (20). However, it still remains an important, unresolved issue whether or not the altered expression of ASC-2 directly contributes to the development of cancers. In this report, we analyzed various functional domains of ASC-2 in an effort to understand its role in mediating transactivation of nuclear receptors. Surprisingly, ASC-2 contained two LXXLL-type nuclear receptor-interaction domains (5, 25), each of which was differentially used by different member(s) of the nuclear receptor superfamily. ASC-2 also appeared to directly bind nuclear receptors and subsequently recruit CBP to exert the receptor transactivation function in vivo.

RESULTS Identification of Two Nuclear ReceptorInteraction Domains We originally proposed the ASC-2 residues 586–860 to be a minimum receptor-interacting domain, based on its interaction with thyroid hormone receptors (TRs)

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in the yeast two-hybrid assays (20). However, this region was subsequently found to bind TR␣ very poorly in the glutathione S-transferase (GST) pulldown assays (results not shown), which prompted us to reexamine the nuclear receptor-interaction domains of ASC-2. Two potentially ␣-helical LXXLL motifs and a region distantly related to this motif (i.e. IXXIM) as indicated in Fig. 1A were suspected to be responsible for the nuclear receptor interactions. This LXXLL motif, in which L and X denote leucine and any amino acid, respectively, was recently shown to be responsible for the AF-2 and ligand-dependent interactions of nuclear receptors with coactivators (5, 25). However, the ASC2–2b fragment (i.e. the ASC-2 residues 622–849) containing the wild-type IXXIM motif or its mutated version (m1 in Fig. 1B) bound none of the nuclear receptors tested in yeast, as shown in Table 1. However, the ASC2–2c fragment (i.e. the ASC-2 residues 849–1,057), which includes the first LXXLL motif, showed a broad range of target specificity in yeast, binding retinoid X receptor (RXR), RAR, TR␣, TR␤, estrogen receptor ␣ (ER␣), ER␤, peroxisome proliferator-activated receptor ␣ (PPAR␣), and PPAR␥ (Table 1 and results not shown). Notably, this fragment did not interact with oxysterol receptor LXR␤/RIP15 (26, 27). As suspected, the LXXLL motif (5, 25) was essential in these interactions, as demonstrated by the complete loss of the receptor interactions by mutations in this region (i.e. m2 in Fig. 1B) (Table 1). Consistent with these results, similar conclusions were independently drawn from other laboratories (22–24). Finally, the second LXXLL motif contained in the ASC-2 residues 1,172–1,511 (i.e. ASC2–4C), which previously showed a very weak ligand-dependent interaction with RXR in the GST pull-down assays (20), did not interact with any of the receptors tested in yeast, as also shown by other groups (22–24). Surprisingly, however, this region strongly bound LXR␤ (26, 27), and mutation of the LXXLL motif to LXXAA (i.e. m3 in Fig. 1B) resulted in a complete loss of the ASC-2-LXR␤ interactions (Table 1). Similarly, ASC2–4 (i.e. the ASC-2 residues 1,172– 1,729), ASC2–4a (i.e. the ASC-2 residues 1,429–1,729) and ASC2–4LR (i.e. the ASC-2 residues 1,429–1,511) showed a relatively strong basal interaction with LXR␤ in yeast, which was further stimulated by ligand (Table 1 and results not shown). As expected, ASC2–4b (i.e. the ASC-2 residues 1559–1729) that does not contain the second LXXLL motif did not interact with LXR␤ (results not shown). Further corroborating with these results, we have independently isolated a partial cDNA clone encoding the LBD of LXR␤ by using ASC2–4 as bait in a separate yeast two-hybrid screening of a mouse liver cDNA library (results not shown). These interaction results were further confirmed in the mammalian two-hybrid tests as well as the GST pull-down assays. In the mammalian two-hybrid tests, ASC2–2c and ASC2–4a showed a ligand-stimulated interaction with LXR␤ and RAR, respectively (Fig. 2, A and C). In the GST pull-down assays, radiolabeled

Multiple Functional Domains of ASC-2

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Fig. 1. Schematic Representation of ASC-2 A, The full-length human ASC-2 and a series of 10 ASC-2 fragments are as depicted. The glutamic acid/aspartic acid-rich (D/E), glutamine-rich (Q-rich), glutamine/proline-rich (Q/P-rich), and serine/threonine-rich (S/T-rich), as well as three LXXLL-like motifs (5, 25) that are known to be important for the ligand-dependent interactions between coactivators and nuclear receptors, are as indicated. The identified interaction domains for nuclear receptors, along with the amino acid numbers for each construct, are also shown. B, The sequences of three LXXLL-like motifs are as indicated, along with their mutated sequences.

ASC2–2c but not ASC2–2c/m2, the LXXAA mutant version, specifically bound GST-TR␤ in a T3-dependent manner but neither GST alone nor GST-LXR␤ (Fig. 2B). In contrast, GST-LXR␤ specifically associated with radiolabeled ASC2–4a, but not the LXXAA mutant form ASC2–4a/m3, only in the presence of its ligand 22-OH-cholesterol (Fig. 2D). Interestingly, the basal interactions observed in yeast were not visible in this in vitro assay. Neither GST alone nor GST fusion to RAR or TR␤ was able to bind this region of ASC-2. Overall, these results led us to conclude that ASC-2 contains at least two LXXLL-dependent receptorinteraction domains, the N-terminal LXXLL motif associating with a broad range of nuclear receptors and the C-terminal motif specifically binding LXR␤. The N-Terminal LXXLL Motif Binds Similar TR Helices Recognized by SRC-1 and SMRT Surprisingly, similar LXXLL-type motifs have recently been shown to mediate the receptor interactions of not only coactivators but also corepressors, in which corepressors associated with specific residues in the

same TR pocket required for coactivator binding (28, 29). By using the identical set of TR␤-LBD mutants described in these works (28, 29), we searched for any mutation of TR that might distinguish ASC-2 from other receptor cofactors such as SRC-1 and SMRT, which would be of great value in elucidating the ASC-2 function. As shown in Table 2, the TR-ASC2–2c interactions were abolished by specific mutations in the TR␤ helices 3, 5, and 6, which were known to form the binding pocket for SRC-1 and SMRT (28, 29). Notably, these mutant TRs were shown to be nonfunctional in mediating the basal repression (28, 29) as well as the T3-dependent transactivation (results not shown). Interestingly, mut2 in the TR␤ helix 1 did not interact with SMRT in yeast, although it was previously shown to bind N-CoR in the GST pull-down assays (29). Overall, these results indicate that the ASC-2-receptor interactions are very similar to those of other coactivators and corepressors. However, a more extensive mutagenesis study is warranted to find TR mutants that specifically lack its ability to bind ASC-2 but not other cofactors.

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Table 1. Interactions of ASC-2 with Nuclear Receptors in Yeast LexA fusions B42 fusions

ASC2-2b ASC2-2b/m1 ASC2-2c ASC2-2c/m2 ASC2-4c ASC2-4c/m3 ASC2-4LR

(⫺)

RXR

RAR

TR␤

ER␤

LXR␤

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺(⫺) nd ⫹(⫹⫹⫹) ⫺(⫺) ⫺(⫺) ⫺(⫺) ⫺(⫹)

⫺(⫺) ⫺(⫺) ⫹⫹(⫹⫹⫹) ⫺(⫺) ⫺(⫺) ⫺(⫺) nd

⫺(⫺) nd ⫺(⫹⫹⫹) ⫺(⫺) ⫺(⫺) nd ⫺(⫺)

⫺(⫺) nd ⫹(⫹⫹⫹) ⫺(⫺) ⫺(⫺) nd nd

⫺(⫺) nd ⫺(⫺) nd ⫹⫹(⫹⫹⫹) ⫺(⫺) ⫹⫹(⫹⫹⫹)

The indicated B42- and LexA-plasmids were transformed into a yeast strain containing an appropriate LacZ reporter gene, as described previously (40). At least six separate transformants from each transformation were transferred to indicator plates containing X-gal, and reproducible results were obtained using colonies from a separate transformation. ⫹⫹⫹, Strongly blue colonies after 2 days of incubation; ⫹⫹, light blue colonies after 2 days of incubation; ⫹, light blue colonies after more than 2 days of incubation; ⫺, white colonies. The results within the parentheses were obtained in the presence of 100 nM of each cognate ligand.

Direct Bindings to Nuclear Receptors Required for the ASC-2 Function As we have previously shown (20), ASC-2 is an essential coactivator for the RAR transactivation. To assess the functional importance of the N-terminal LXXLL motif in the RAR transactivation, we cotransfected various ASC-2 fragments containing either the wild-type LXXLL or mutated LXXAA sequences. Interestingly, ASC2–2c but not ASC2–2c/m2 showed a dominant negative phenotype with the RAR or TR transactivation (Fig. 3 and results not shown). Similarly, expression of ASC-2 residues 786–1,132 was recently shown to specifically repress the 9-cis-RA and BRL 49653directed transactivation by RXR and PPAR␥, respectively (23). Consistent with the above interaction results, ASC-2 is also a bona fide transcription coactivator molecule of LXR␤ (26, 27). As reported previously (26), coexpression of increasing amounts of LXR␤ showed a constitutive activation of the LXRELuc reporter gene expression in a LXR␤-dose dependent manner (results not shown). This basal level of the reporter gene expression was further enhanced by coexpressed ASC-2 (Fig. 4A). 22-OH-cholesterol or 24, 25-epoxy-cholesterol, the recently described ligands for LXR␤ (26), stimulated the reporter gene expression approximately 2- to 4-fold, which was further enhanced by coexpressed ASC-2 (Fig. 4A and results not shown). As expected, the LXRE-Lucdependent transactivation was not observed either in the absence or presence of 22-OH-cholesterol when LXR␤ was not cotransfected (Fig. 4B). Interestingly, a full-length ASC-2 in which LXXAA sequences were incorporated into the second LXXLL motif (i.e. ASC-2/ m3) was completely inert, supporting the importance of the second LXXLL motif in the LXR␤ transactivation (Fig. 4A). In contrast, ASC-2/m3 was as functional as the wild-type ASC-2 with the RAR transactivation. Furthermore, overexpression of ASC2–4LR but not ASC2–4LR/m3 directed a potent dominant negative phenotype with the LXR␤ transactivation (Fig. 4B).

From these results, direct bindings to receptors through the LXXLL motifs appeared to be essential for the ASC-2 action with nuclear receptors. Dissection of the Autonomous Transactivation Domains of ASC-2 We have previously mapped at least two distinct autonomous transactivation domains of ASC-2 in yeast; i.e. a very strong transactivation domain contained within ASC-2 residues 1–557 and a rather modest transactivation domain within ASC-2 residues 3911057 (20). Surprisingly, these ASC-2 fragments showed reversed transactivation potency in mammalian cells, suggesting that their exact mode of actions is distinct between yeast and mammalian cells. In mammalian cells, the transactivation function within the ASC-2 residues 1–557 was localized to the ASC-2 residues 1–218 (as summarized in Fig. 5A). Notably, this transactivation function was modest in contrast to its strong activity in yeast (20), and we named this region autonomous transactivation domain 1 (AD1). As shown in Fig. 5B, ASC2-II (i.e. ASC-2 residues 391– 930) contained a potent transactivation function in mammalian cells, which was further localized to ASC2–2b (i.e. ASC-2 residues 622–849) (designated AD2) (Fig. 5B). In addition, ASC2–2c (i.e. ASC-2 residues 849-1057) also showed a significant activity. Thus, the modest autonomous transactivation function in yeast (20), included within ASC-2 residues 391– 1,057, was mapped to two separable subregions in mammalian cells; i.e. AD2 and AD3 (Fig. 5A). Notably, the activity of AD3 was relatively poor, whereas AD2 was much stronger than AD1 and AD3 (Fig. 5B). Consistent with the yeast results (20), the C-terminal region of ASC-2 (i.e. ASC2-III and ASC2–4.5) did not show any evidence of autonomous transactivation function. From these results, it was clear that ASC-2 contains three separable transactivation domains. Each of these multiple transactivation domains may represent just part of a consecutive, single transacti-


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Fig. 2. Interactions of Nuclear Receptors with ASC-2 A and C, HeLa cells were transfected with LacZ expression vector (100 ng) and VP16 fusion to the full-length LXR␤ or RAR (100 ng), along with expression vectors for Gal4 alone, Gal4/ASC2–2c and Gal4/ASC2–4a (100 ng), and a reporter gene Gal4-TK-Luc (100 ng), as indicated. All-trans-RA and 22-OH-cholesterol (at a concentration of 100 nM) were used as ligands for RAR and LXR␤, respectively. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions. The experiments were repeated at least three times and the representative results were expressed as fold-activation (n-fold) over the value obtained with Gal4 alone, with the error bars as indicated. Similar results were also obtained with CV-1 cells. B and D, The full-length ASC-2 and various ASC-2 fragments were labeled with [35S]methionine by in vitro translation and incubated with glutathione beads containing GST alone, GST-TR␤, GST-RAR, and GST-LXR␤, as indicated. ASC2–2c/m2 appears slightly larger than its wild-type version because it is hemagglutinin-tagged and contains the nuclear localization signal of SV40 T antigen. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-polyacrylamide gel electrophoresis. Approximately 5% of the total reaction mixture were loaded as input.– and ⫹ indicate the absence and presence of 100 nM of all-trans-RA, T3, or 22-OH-cholesterol, respectively.

vation domain, cooperating with each other to achieve the maximum transactivation potential of ASC-2. Alternatively, it is possible that each of these domains may represent an independent transactivation function, which differentially responds to various activating signals under certain conditions. AD2 and AD3 as CBP-Interaction Interfaces We have previously mapped the SRC-1 and CBPbinding sites to the N-terminal half of ASC-2 (20). In this report, we found that ASC2–2b encompassing the strongest transactivation domain AD2 was able to interact with the previously described CBP-E fragment (i.e. CBP residues 1,867–2,441) (Fig. 6A). The CBPAD2 interactions were further mapped to the CBP-E/C region (i.e. CBP residues 2,171–2,441) and the ASC2–

2b/C (i.e. ASC-2 residues 737–849) (Fig. 6, A and B). In addition, the ASC2–2b/AD2 region bound weakly to SRC1-D (i.e. SRC-1 residues 759–1,141) and very strongly to SRC-E (i.e. SRC-1 residues 1,101–1,441) (Fig. 6A). Interestingly, ASC2–2c encompassing the AD3 also bound CBP-E/C but not any of the SRC-1 fragments (Fig. 6C), whereas ASC2-I encompassing the AD1 did not bind any CBP fragment in yeast (results not shown). The LXXLL motifs similar to those involved with the receptor-coactivator interactions (5, 25) were also found within the SRC-1-CBP binding domains (2–4). Thus, we tested whether these motifs included within the ASC2–2b and ASC2–2c are involved with the observed ASC-2-CBP interactions. However, neither motif was involved with the CBP and SRC-1 bindings in yeast, as demonstrated by the lack of any effect by mutations in these motifs (Table 3).

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Table 2. Interactions of ASC-2 with TR␤ Mutants in Yeast B42 Fusions LexA Fusions

H1 H3 H5 H6 H11

Wild type mut1 (K206A, P209R) mut2 (E213R, L216R) mut4 (V279R, K283A) mut5 (C293R, I297R) mut6 (C304R, I307R) mut8 (M418R, D422R) mut9 (M425R, C429R) mut11 (F434R, L435R)

(⫺)

ASC2-2c

SRC-C

SMRT-D

⫺(⫺) ⫺(⫺) ⫺(⫺) ⫺(⫺) ⫺(⫺) ⫺(⫺) ⫺(⫺) ⫺(⫺) ⫺(⫺)

⫺(⫹⫹⫹) ⫺(⫹⫹⫹) ⫺(⫹⫹) ⫺(⫺) ⫺(⫺) ⫺(⫺) ⫺(⫹⫹⫹) ⫺(⫹⫹⫹) ⫺(⫹⫹⫹)

⫹⫹⫹(⫹⫹⫹) ⫹⫹⫹(⫹⫹⫹) ⫺(⫹⫹⫹) ⫺(⫺) ⫺(⫺) ⫺(⫺) ⫹⫹⫹(⫹⫹⫹) ⫹⫹⫹(⫹⫹⫹) ⫹⫹⫹(⫹⫹⫹)

⫹⫹⫹ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹

Same as Table 1 except that the results within the parentheses were obtained in the presence of 100 nM T3.

Fig. 3. The N-Terminal LXXLL Subregion as a Dominant Negative Mutant for the Receptor Transactivation HeLa cells were transfected with LacZ expression vector (100 ng) and ASC2–2c or ASC2–2c/m2-expression vector along with a reporter gene ␤RARE-Luc (100 ng), as indicated. For optimal RA response, 10 ng of RAR expression vector were also cotransfected. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions. The experiments were repeated at least three times, and the representative results were expressed as foldactivation (n-fold) over the value obtained with no ligand and ASC-2, with the error bars as indicated. Open and closed boxes indicate the absence and presence of 100 nM of alltrans-RA, respectively. Similar results were also obtained with TRE-Luc and CV-1 cells.

The specific ASC-2 residues involved with the CBP and SRC-1 bindings are currently being extensively mapped. Consistent with these results, the full-length ASC-2 interacted with GST fusions to either CBP4 (i.e. CBP residues 1,459–1,891) or CBP5 (i.e. CBP residues 1,891–2,441) in the GST pull-down assays, whereas ASC2–2bc and ASC2–2 interacted only with GSTCBP5 (Fig. 6D and results not shown). Overall, these studies identified AD2 and AD3 as CBP-interaction interfaces and AD2 as an SRC-1-binding domain. The Autonomous Transactivation Function of ASC-2 Requires CBP Interestingly, transactivation by Gal4 fusion to AD2 in the context of ASC2–2b or ASC2–2bc was signifi-

cantly stimulated by coexpressed CBP but neither SRC-1 nor P/CAF (Fig. 7A). Consistent with the lack of CBP interactions, however, transactivation by Gal4 fusion to ASC2-Ia (i.e. AD1) was not enhanced by coexpressed CBP (results not shown). Unexpectedly, however, transactivation by Gal4 fusion to ASC2–2c (i.e. AD3) was not stimulated by coexpressed CBP, despite the fact that this fragment also binds to CBP (Fig. 6C). Interestingly, a mutant CBP defective for its HAT activity (i.e. F1541A) (30) stimulated the AD2dependent transactivation (Fig. 7A). In addition, the AD2-dependent transactivation, either alone or in combination with AD1 or AD3, was specifically inhibited by CBP-neutralizing E1A but not E1A(⌬2–36), an E1A N-terminal deletion mutant that lacks the CBP bindings (30) (Fig. 7B and results not shown). Similarly, transactivation by Gal4 fusion to ASC-2 residues 1–929 that includes AD1, AD2, and part of AD3 was also inhibited by E1A (results not shown). In contrast, E1A or CBP alone had no effect on the significant basal activity directed by Gal4 alone (results not shown). Overall, these results clearly suggest that recruitment of CBP is essential for the autonomous transactivation function of ASC-2. The CBP Recruitment Is Essential for the ASC-2 Function with Nuclear Receptors The results presented in Figs. 6 and 7, along with the result in Figs. 3 and 4 in which direct recruitment of ASC-2 to receptors appears to be important, indicated that ASC-2 may act as an adaptor molecule between CBP and nuclear receptors, and these interactions could be essential for the coactivation function of ASC-2 with nuclear receptors. Consistent with this idea, ASC2–2bc but not ASC2–2bc/m2 led LexA/TR␤LBD to interact, in a strictly T3-dependent manner, with B42 fusion to CBP-E/C in yeast (Fig. 8). It should be noted that the receptor-interaction domain was previously mapped to the N-terminal region of CBP (4), and thus LexA/TR␤-LBD was incapable of directly interacting with B42/CBP-E/C in this yeast system. However, despite our extensive effort, we were not able to show formation of a ternary complex between


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Fig. 4. The C-Terminal LXXLL Motif Required for the LXR␤ Transactivation HeLa cells were transfected with LacZ expression vector (100 ng) and ASC-2, ASC-2/m3, ASC2–4LR, ASC2–4LR/m3, or LXR␤-expression vector along with a reporter gene ␤RARE-Luc (100 ng) or LXRE-Luc (100 ng), as indicated. For optimal RA-response with the ␤RARE-Luc construct, 10 ng of RAR-expression vector were also cotransfected. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions. The experiments were repeated at least three times, and the representative results were expressed as fold-activation (n-fold) over the value obtained with no ligand and ASC-2, with the error bars as indicated. Open and closed boxes indicate the absence and presence of 100 nM of all-trans-RA or 10 ␮M of 22-OH-cholestrerol, respectively. Similar results were also obtained with CV-1 cells.

ASC-2, receptor, and CBP in the GST pull-down assays (results not shown), suggesting that the ternary complex observed in yeast (Fig. 8) could have been mediated by other yeast protein(s). In addition, overexpressed ASC2–2b, which contains the potent transactivation function AD2 serving as a CBP-interaction interface, significantly repressed the 9-cis-RA-dependent RAR transactivation (Fig. 9A), indicating that the CBP-ASC-2 interactions are pivotal for the nuclear receptor transactivation. As we have previously shown for the RAR transactivation with p300 (20), CBP also synergized with ASC-2 to mediate the T3-dependent transactivation (Fig. 9B). It is noted that only 25 ng of CBP, which alone exerted negligible effects on the T3 transactivation, was used in this specific experiment, mainly because the endogenous level of CBP and ASC-2 within the cells interferes with readily observing such synergistic effects in standard cotransfections. Furthermore, the ASC-2-dependent enhancement of the RAR or TR transactivation was not observed in the presence of CBP-neutralizing E1A, whereas E1A(⌬2– 36) was inert (Fig. 9C and results not shown). From these results, along with the results in which CBP was shown to be required for the autonomous transactivation function of ASC-2 (Fig. 7), we concluded that ASC-2 should recruit CBP, either directly or indirectly, to mediate the nuclear receptor transactivation in vivo.

DISCUSSION ASC-2 is a novel transcription cointegrator molecule that mediates transactivation by a variety of nuclear

receptors, AP-1, CREB, NF␬B, SRF, and numerous other transcription factors (Refs. 20–24 and our unpublished results). The LXXLL-type motifs have recently been shown to mediate the receptor interactions of coactivators and corepressors (5, 25), although residues immediately adjacent to the LXXLL motif modulated the affinity of the interaction (31). Accordingly, a few different classes of the LXXLLcontaining peptides that showed a variety of distinct receptor-binding selectivity have been isolated by a combinatorial phage display approach (32). Notably, ASC-2 contains two LXXLL motifs (5, 25). In this report, we have shown that both of these motifs constitute the functional receptor interaction domains of ASC-2. The N-terminal LXXLL motif turned out to be responsible for binding many different nuclear receptors (Table 1 and Refs. 22–24). In contrast, the Cterminal LXXLL motif was capable of binding only LXR␤. These interactions were absolutely dependent on the integrity of the LXXLL motifs (Table 1 and Fig. 2). Interestingly, the C-terminal LXXLL motif resembled none of the LXXLL sequences of known receptor cofactors or the subclasses of LXXLL peptides isolated from a combinatorial phage display (32). Thus, we believe that this C-terminal LXXLL motif of ASC-2 may represent a distinct subtype that specifically recognizes LXR␤. In contrast, the N-terminal motif resembled the class III peptides (32), which contain the residues proline and leucine preceding the LXXLL motif (i.e. PLLXXLL, as shown in Fig. 1B). This class of peptides was shown to prefer RXR and the RXR partner receptors (32), consistent with our results.

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Fig. 5. Localization of the Autonomous Transactivation Domains of ASC-2 A, The autonomous transactivation domains of ASC-2 (AD1, AD2, and AD3) are schematically indicated, along with a series of nine ASC-2 fragments. B, HeLa cells were transfected with LacZ expression vector (100 ng), expression vectors for various Gal4 fusions (100 ng), and a reporter gene Gal4-TK-Luc (100 ng), as indicated. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions. The experiments were repeated at least three times, and the representative results were expressed as fold-activation (n-fold) over the value obtained with Gal4 alone, with the error bars as indicated. Similar results were also obtained with CV-1 cells.

The current model for the nuclear receptor-mediated transactivation involves two-step mechanisms (2, 3). According to this model, SRC-1 appears to be directly recruited to the liganded receptors first, which serves as a platform to recruit CBP. Consistent with this idea, the receptor-interacting LXXLL motif located at the N terminus of CBP could be deleted without significantly affecting transactivation by RAR-RXR heterodimers, whereas the SRC-1 LXXLL motifs were absolutely essential (33, 34). These factors and associated proteins such as P/CAF (15), by using their HAT activities (11–15), are known to remodel the nucleosomal structures so that the TRAP/DRIP complex (35, 36) can replace SRC-1/CBP and bind the liganded receptors. Subsequent recruitment of RNA polymerase II complex to TRAP/DRIP (35, 36) completes the second step in the nuclear receptor transactivation. The results from this and other reports (20, 23) are

consistent with an idea that ASC-2 may play a similar, essential role as SRC-1; i.e. direct bindings to nuclear receptors and recruitment of CBP to the receptorASC-2 complex, as summarized in Fig. 10. First, we have previously shown that ASC-2 is essential for the AP-1 and RAR transactivation in vivo by using antibody-blocking experiments (20, 21). In addition, ASC2–2c (but not its mutated version in which the LXXLL motif was converted to LXXAA) and a fragment slightly larger than this (i.e. the ASC-2 residues 786– 1,132) exhibited a potent dominant negative phenotype with the receptor transactivation (Fig. 3 and Ref. 23). Similarly, ASC2–4LR containing the LXR␤-interacting LXXLL motif acted as a dominant negative repressor of the LXR␤ transactivation in an LXXLLdependent manner (Fig. 4B). In addition, a full-length ASC-2 with the second LXXLL motif mutated to LXXAA was inert with the LXR␤ transactivation (Fig.


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Fig. 6. Localization of the CBP-Binding Domain of ASC-2 A, B, and C, The indicated B42- and LexA-plasmids were transformed into a yeast strain containing an appropriate LacZ reporter gene, as described (40). Closed and hatched boxes indicate the presence of LexA fusions to ASC2–2b (A) and ASC2–2c (C) or ASC2–2b/N (i.e. the ASC-2 residues 622–736) and ASC2–2b/C (i.e. the ASC-2 residues 737–849) (B), respectively. CBP-A, B, C, D, E, E/N, and E/C indicate the CBP residues 1–446, 452–721, 722–1,166, 1,161–1,752, 1,867–2,441, 1,867–2,170, and 2,171–2,441, respectively. SRC-A, B, C, D, and E indicate the SRC-1 residues 1–361, 361–568, 568–779, 759–1,141, and 1,101–1,441, respectively. The data are representative of at least two similar experiments, and the error bars are as indicated. D, The full-length ASC-2 and ASC2–2bc were labeled with [35S]methionine by in vitro translation and incubated with glutathione beads containing GST alone, GST-CBP4, and GST-CBP5, as indicated. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-PAGE. Approximately 5% of the total reaction mixture were loaded as input. Similarly, the full-length CBP labeled with [35S]methionine by in vitro translation specifically bound GST-ASC2–2bc (results not shown).

4A). Thus, direct bindings between receptors and ASC-2 appear to be required for the ASC-2 function with the nuclear receptor transactivation. Second, the autonomous transactivation function of ASC-2 was dependent on CBP (Fig. 7), in which AD2 and AD3 appear to play a pivotal role as CBP interaction interfaces (Fig. 6). It is also notable that the mutant CBP without apparent HAT activities (i.e. F1541A) (30) was functionally intact in promoting the autonomous transactivation function of AD2, suggesting that the autonomous transactivation function of ASC-2 does not require the nucleosomal remodeling activities of CBP. Third, ASC2–2b, which contains the CBP interaction domain, acted as a potent dominant negative mutant with the RAR transactivation (Fig. 9A), suggesting that the ASC-2-CBP interactions are critical components of the ASC-2-mediated receptor transactivation. Consistent with this idea, ASC-2-dependent enhancement of the RAR transactivation was not observed in the presence of CBP-neutralizing E1A, but not E1A(⌬2–36) (30), a mutated E1A incapable of associating with CBP

Table 3. Interactions of ASC-2 with CBP and SRC-1 in Yeast LexA Fusions

B42 Fusions

YTH Results

(⫺) CBP-E CBP-E CBP-E ASC2-2bc ASC2-2bc ASC2-2bc ASC2-2bc/m2 ASC2-2bc/m2 ASC2-2bc/m2

(⫺) (⫺) ASC2-2b ASC2-2b/m1 (⫺) CBP-E SRC-E (⫺) CBP-E SRC-E

⫺ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹

Same as Table 1 except that no ligand was utilized.

(Fig. 9B). Although the exact molecular mechanism by which E1A blocks the CBP-dependent ASC-2 function is not clearly understood, one interesting possibility is that E1A may impair the CBP-ASC-2 interactions, as was shown for the CBP-SRC-1 interactions (37). How-

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Fig. 7. CBP Required for the Autonomous Transactivation Function of ASC-2 HeLa cells were transfected with LacZ expression vector (100 ng) and a reporter gene Gal4-TK-Luc (100 ng), along with expression vectors for various Gal4 fusion proteins (100 ng) and increasing amounts of expression vectors (100 and 200 ng) for CBP, the HAT-negative mutant mCBP (F1541A) (30), SRC-1, P/CAF, E1A, and E1A(⌬2–36) (30), as indicated. Open, shaded, hatched, and closed boxes indicate the presence of Gal4 alone, Gal4/ASC2–2b, Gal4/ASC2–2c, and Gal4/ASC2–2bc, respectively. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions. The experiments were repeated at least three times and the representative results were expressed as fold-activation (n-fold) over the value obtained with Gal4 alone, with the error bars as indicated. Similar results were also obtained with CV-1 cells.

ever, our preliminary GST pull-down experiments indicated that E1A does not interfere with the CBPASC-2 interactions. More importantly, it still remains unresolved whether the interactions between ASC-2 and CBP are direct. In particular, the ternary interactions of ASC-2, receptor, and CBP observed in yeast (Fig. 8) were not detected in the GST pull- down assays (our unpublished results). These results suggest the requirement of other protein(s) in these ternary interactions, although we could not easily rule out the possibility of technical problems associated with the lack of the ternary interactions in the GST pull-down assays. Interestingly, the transcriptionally inert LexA/ TR␤-LBD became an excellent T3-dependent transactivator upon coexpression of ASC2–2bc in yeast (see the third column in Fig. 8), in which ASC2–2bc may have helped TR␤ to recruit yeast proteins that are essential for the receptor function in yeast, such as Swi/Snf proteins (38). These Swi/Snf proteins, conserved between yeast and mammals, could also be responsible for the putative, indirect ASC-2-CBP interactions in yeast (Fig. 8) and thus essential components in the ASC-2 function in mammalian cells. Considering the fact that ASC-2 is expressed in relatively low amounts in most cells but can be upregulated in certain cells by various cytokines and growth factors (our unpublished results), ASC-2 may serve as an inducible factor that represents an alter-

Fig. 8. The Ternary Interactions of ASC-2, Receptor, and CBP in Yeast Expression vectors for B42/CBP-E/C, ASC2–2bc, ASC2– 2bc/m2, and LexA/TR␤-LBD were transformed into a yeast strain containing an appropriate LacZ reporter gene, as described previously (40). Open and closed boxes indicate the absence and presence of 100 nM of T3, respectively. The data are representative of at least two similar experiments and the error bars are as indicated.

native functional homolog of SRC-1. Similarly, the ASC-2 overexpressing human cancer cells (20) could be potential sites where ASC-2 may play a functionally


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Fig. 9. CBP Is Essential for the Nuclear Receptor Transactivation HeLa cells were transfected with expression vectors for LacZ, ASC-2, ASC2–2b, CBP, E1A, and E1A(⌬2–36) along with a reporter gene ␤RARE-Luc (A and C) or TRE-Luc (B). For optimal RA- or T3-response, 10 ng of RAR- or TR-expression vector were also cotransfected. – and ⫹ indicate the absence and presence of 100 nM of all-trans-RA (A and C) or T3 (B), respectively. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions. The experiments were repeated at least three times, and the representative results were expressed as fold-activation (n-fold) over the value obtained with unstimulated cells, with the error bars as indicated. Similar results were also obtained with CV-1 cells.

dominant role over SRC-1. As indicated in the model (Fig. 10), however, it is not clear how ASC-2 functionally corroborates with other known coactivators of nuclear receptors such as SRC-1 and TRAP/DRIP (35, 36). In the previously shown antibody-blocking experiments (20), neutralization of the endogenous ASC-2 in rat-1 fibroblast cells significantly abolished the RAR transactivation, and coexpressed CBP but not SRC-1 was able to partly overcome these ␣ASC-2 antibodyblocking effects. These results indicate that either the RAR transactivation simply requires ASC-2 but not SRC-1 within this cell type (i.e. rat-1 fibroblast cells) or SRC-1 requires the presence of ASC-2 to function. The latter possibility is consistent with an idea that ASC-2 may function as a general platform to recruit other related coactivators such as SRC-1 and TRAP/ DRIP. In fact, ASC-2 can interact with SRC-1 (Fig. 6A and Ref. 20) although the functional role of these SRC1-ASC-2 interactions is not currently clear, and a component of the TRAP/DRIP complex and CBP/p300 were recently found to associate with the C-terminal region of ASC-2 (22). Interestingly, the latter results are in contrast to our yeast two-hybrid-based findings in which CBP appeared to exclusively bind to AD2 and AD3 located at the N-terminal region (Fig. 6). This

discrepancy is not clearly understood, although it is possible that the ASC-2 fragments used in our experiments may have impaired a cryptic CBP-binding site. Indeed, in our own GST pull-down assays, the fulllength ASC-2 interacted with multiple regions of CBP, as opposed to the yeast two-hybrid-based assays (Ref. 20 and Fig. 6D). Furthermore, ASC2–4a (i.e. the ASC-2 residues 1,429–1,729) but not ASC2–4a/m3 stimulated the LXR␤ transactivation (results not shown), for which the putative TRAP/CBP interaction(s) at the C terminus of ASC-2 could be responsible. Thus, more detailed analyses to localize the exact CBP interaction interfaces of ASC-2 are currently under way. Alternatively, the antibody used in the original antibody-blocking experiments may have crossreacted with other essential component(s) that are required for the SRC-1 and ASC-2 functions. We are currently reexamining this issue by using more defined monoclonal antibody against ASC-2, other cell-types, and various LacZ reporters responsive to different receptors. In conclusion, we have shown that ASC-2 interacted with oxysterol receptor LXR␤ in a manner specifically dependent on the C-terminal LXXLL motif, whereas the N-terminal LXXLL motif of ASC-2 bound a broad

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Fig. 10. The Model for the ASC-2 Action ASC-2 acts as a bridging molecule between nuclear receptors and CBP. Supporting this model, ASC2–2c (i.e. a subregion of ASC-2 containing the binding site for many different receptors) or ASC2–2b (i.e. a subregion of ASC-2 containing the binding site for CBP) acted as potent dominant negative mutants of the RAR transactivation. Similarly, ASC2–4LR (i.e. a subregion of ASC-2 containing the binding site for LXR␤) also acted as a dominant negative mutant of the LXR␤ transactivation. However, neither the exact relationship of ASC-2 to different coactivators such as SRC-1 and TRAP/DRIP (35, 36) nor whether or not the CBP-ASC-2 interactions are direct are clearly understood. HRE indicates hormone response elements, and CBP, ASC-2, TRAP, and SRC-1 are schematically shown as a single polypeptide, although each of them is known to exist as a steady state complex of multiple polypeptides in vivo (Refs. 35, 36, 44, and our unpublished results).

range of nuclear receptors. We have also demonstrated that the CBP-ASC-2 interactions are essential for the autonomous transactivation and the receptor coactivation functions of ASC-2, in which ASC-2 appears to serve as a bridging molecule between CBP and nuclear receptors. Finally, we have noted that ASC-2 contains a numerous number of putative phosphorylation sites (20) and, thus, the possible regulation of its inherent activity through various signal transduction pathways could play an important role. In this regard, it was interesting to note that DNA-dependent protein kinase complex (39) was recently copurified with GST-ASC-2 from HeLa nuclear extracts (22).

MATERIALS AND METHODS Plasmids PCR fragments encoding ASC2-I, ASC2-Ia, ASC2-Ib, ASC2II, ASC2-IIa, ASC2–2b/N, ASC2–2b/C, ASC2–2b/m1, ASC2– 2bc, ASC2–2c/m2, ASC2-III, ASC2–4a, ASC2–4b, ASC2–4c, ASC2–4c/m3, ASC2–4LR, ASC2–4LR/m3, ASC2–4.5, CBPE/N, CBP-E/C, and LXR␤ were inserted into EcoRI and XhoI/ SalI restriction sites of the LexA-fusion vector pEG202PL (40), the B42-fusion vector pJG4–5 (40), and the mammalian expression/in vitro translation vector pcDNA3, respectively. PCR fragments, obtained by using Gal4 fusion vectors to a series of TR␤ mutants (kind gift of Drs. M. G. Rosenfeld and Chris Glass at University of California, San Diego) as templates, were cloned into EcoRI and SalI restriction sites of pEG202PL to express appropriate LexA fusion proteins in

yeast. PCR fragments encoding ASC2–4a and ASC2–2c were cloned into BamHI and EcoRI restriction sites of pCMX1 to express Gal4-ASC2–4a and Gal4-ASC2–2c, respectively. Vectors encoding GST, LexA, and B42-fusions to ASC2–2, ASC2–2a, ASC2–2b, ASC2–2c, ASC2–4, CBP-A, CBP-B, CBP-C, CBP-D, CBP-E, CBP-4, CBP-5, SRC-A, SRC-B, SRC-C, SRC-D, SRC-E, RXR, RAR, TR␤, and ER␤, along with ␤RARE-Luc, TRE-Luc, LXRE-Luc, Gal4-TK-Luc, the transfection indicator construct pRSV-␤-gal, and expression vectors for ASC-2, E1A, E1A(⌬2–36), CBP, and mCBP (i.e. F1541A), were as previously described (6, 7, 9, 10, 20, 21, 26, 30). To express GST fusion to LXR␤, PCR fragment encoding the full-length LXR␤ was cloned into EcoRI and XhoI restriction sites of pGEX4T (Pharmacia Biotech, Piscataway, NJ). The site-directed mutagenesis kit QuickChange (Stratagene, San Diego, CA) was used to create a mutated, full-length ASC-2, in which the second LXXLL motif was converted to LXXAA. ASC2–2bc and its m2 mutant were subcloned into the HindIII and XhoI restriction sites of the yeast expression vector p425-Gal1 (41). The Yeast Two-Hybrid Screening and Yeast ␤-Galactosidase Assay The LexA-ASC2–4 was used as a bait to screen a mouse liver cDNA library in pJG4–5 (40) for ASC-2-interacting proteins, and the screening was executed essentially as previously described (42). The yeast ␤-galactosidase assay was done as described (40). For each experiment, at least three independently derived colonies expressing chimeric proteins were tested. GST Pull-Down Assays The GST fusions or GST alone was expressed in Escherichia coli, bound to glutathione-Sepahrose-4B beads (Pharmacia


Biotech) in binding buffer (25 mM HEPES, pH 7.8, 0.2 mM EDTA, 20% glycerol, 100 mM KCl, and 0.1% NP40), and incubated with labeled proteins expressed by in vitro translation by using the TNT-coupled transcription-translation system, with conditions as described by the manufacturer (Promega Corp., Madison, WI). Specifically bound proteins were eluted from beads with 40 mM reduced glutathione in 50 mM Tris (pH 8.0) and analyzed by SDS-PAGE and autoradiography as described (43). Cell Culture and Transfections HeLa and CV-1 cells were grown in 24-well plates with medium supplemented with 10% FCS for 24 h and transfected with 100 ng of LacZ expression vector pRSV-␤-gal and 100 ng of an indicated reporter gene, along with indicated amounts of various mammalian expression vectors. Total amounts of expression vectors were kept constant by adding pcDNA3. Transfections and luciferase assays were done as described previously (40), and the results were normalized to the LacZ expression. Similar results were obtained in more than two similar experiments.

Acknowledgments We thank Drs. M. G. Rosenfeld, Chris Glass, Joonho Choe, and David Mangelsdorf for various plasmids.

Received June 16, 2000. Re-revision received October 23, 2000. Accepted October 25, 2000. Address requests for reprints to: Jae Woon Lee, Ph.D., Center for Ligand and Transcription, Chonnam National University, Kwangju 500–757, Korea. E-mail: jlee@chonnam. chonnam.ac.kr. This work was supported by a grant from the National Creative Research Initiatives Program of the Korean Ministry of Science and Technology, Republic of Korea. *Present address: Howard Hughes Medical Institute, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0651. †Present address: Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114.

Note Added In Proof. While this manuscript was being revised, another paper describing hASC-2 (hNRC) as well as the rat homolog rNRC was published in which the CBP-ASC-2 associations in vivo were also suggested (Mahajan MA, Samulels HH 2000 A new family of nuclear receptor coregulators that integrate nuclear receptor signaling through CREBbinding protein. Mol Cell Biol 20:5048–5063).

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5th International Workshop on Resistance to Thyroid Hormone June 6–8, 2001, Verbania, Italy Further details: Prof. Paolo Beck-Peccoz, Institute of Endocrine Sciences, Ospedale Maggiore IRCCS, Via F. Sforza 35, 20122 Milan, Italy Fax: ⫹39-02-55195438 E-mail: [email protected] www.infinito.it/utenti/endocrinology