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Molecular and Cellular Biology, August 1999, p. 5363-5372, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Purification and Identification of p68 RNA Helicase Acting as a Transcriptional Coactivator Specific for the Activation Function 1 of Human Estrogen Receptor alpha

Hideki Endoh,1,2 Kazunori Maruyama,1 Yoshikazu Masuhiro,2 Yoko Kobayashi,2 Masahide Goto,1 Hitoshi Tai,2 Junn Yanagisawa,2 Daniel Metzger,3 Seiichi Hashimoto,1 and Shigeaki Kato2,4,*

Molecular Medicine Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical, Tsukuba, Ibaraki 305-8585,1 Institute for Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032,2 and CREST, Japan Science and Technology, Kawaguchi, Saitama 332-0012,4 Japan, and Institut de Genetique et de Biologie Moleculaire et Cellulaire, (IGBMC)/CNRS/INSERM/ULP College de France, 67404 Illkirch Cedex, C.U. de Strasbourg, France3

Received 19 January 1999/Returned for modification 1 March 1999/Accepted 5 May 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ERalpha (hERalpha ) is enhanced through phosphorylation of the Ser118 residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein (p68) which interacted with the A/B domain but not with the LBD of hERalpha . Phosphorylation of hERalpha Ser118 potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ERalpha in a cell-type-specific manner. However, it did not potentiate AF-1 or AF-2 of ERbeta , androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERalpha AF-1 coactivator activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERalpha A/B domain was essential for the full activity of hERalpha AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the ERalpha AF-1 and strongly suggest that the interaction between p68 and the hERalpha A/B domain is regulated by MAPK-induced phosphorylation of Ser118.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Fat-soluble ligands such as steroid and thyroid hormones, retinoids, and vitamin D regulate many biological processes, including cell proliferation and differentiation, through transcriptional control of target gene expression mediated by their cognate receptors (5, 10, 32). These nuclear receptors belong to a large family of ligand-inducible transcription factors, and studies of their molecular structure have identified five or six functional domains (designated A to F). The C domain, located in the middle of the protein, is responsible for specific binding to DNA-responsive elements. The ligand binding domain (LBD) is localized in the C-terminal E/F domain and contains a ligand-dependent transcriptional activation function (AF-2) (30, 46), whereas another activation function (AF-1), which is constitutive in the absence of the LBD, is located in the A/B region (46). The activities of both AF-1 and AF-2 are promoter context and cell type specific and can synergize (45). The role of AF-1 in the ligand-induced transactivation function of estrogen receptor alpha  (ERalpha ) is prominent in the actions of estrogen (17beta -estradiol [E2]) and partial agonists such as tamoxifen. Tamoxifen blocks the AF-2 but not the AF-1 activity of ERalpha (6), exerting agonistic or antagonistic activities in a tissue-specific way. Moreover, we have shown that the human ERalpha (hERalpha ) AF-1 activity is potentiated through phosphorylation of the Ser118 residue by mitogen-activated protein kinase (MAPK), which is activated by growth factors such as insulin, insulin-like growth factors and tumor necrosis factor alpha (26). To achieve such complex AF activity, the presence of common transcriptional mediators or cofactors mediating AF activity to basic transcription machinery was suggested. The observations that AF-1 and AF-2 of steroid receptors are transcriptionally squelched or interfere with each other further supported this idea (37, 45). Some components of the basic transcription machinery such as TATA-associated factors (22, 35, 36) and TFIIB (4) associate with nuclear receptors in a ligand-independent manner. Indeed, putative transcriptional mediators and cofactors interacting with and activating the AF-2 activities of nuclear receptors in a ligand-dependent fashion have been recently identified (17). They include the TIF2/SRC-1 (2, 9, 11, 39, 47) and CBP/p300 families (24), TIF1 (29), ARA70 (52), and many others (40). Some of these factors (CBP/p300 and SRC-1) exhibit autonomous histone acetyltransferase activity (11, 42) and appear to associate with P/CAF (51), and therefore some of the effects at the chromatin level might be mediated by histone acetylation. Despite the information on coactivators for AF-2, little is known about possible coactivator(s) for AF-1.

To study the AF-1 of the ERalpha with respect to the MAPK-induced potentiation of this activity, we sought to identify a coactivator for AF-1 of the hERalpha . Here we report the purification and cloning of a 68-kDa protein (p68) and show that it interacts with the hERalpha A/B domain but not with its LBD and that this interaction is enhanced by the MAPK-mediated phosphorylation of the hERalpha at Ser118. Furthermore, the transcriptional activity of ERalpha AF-1, but not AF-2, was potentiated by p68, whereas overexpression of p68 had no effect on either AF-1 or AF-2 of ERbeta , androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. The RNA helicase activity previously ascribed to p68 (12, 16) was dispensable for the coactivator activity. p68 bound to CBP in vitro. Thus, these findings demonstrate that p68 acts as a coactivator specific for the ERalpha AF-1 and indicate that phosphorylation of the hERalpha at Ser118 by MAPK potentiates this interaction.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Vectors. The GAL4 chimeric proteins with hERalpha deletion mutants [pM-A/B(1-180), pM-AB-Ms, and pM-LBD(295-595)], with human ERbeta deletion mutants [pM-A/B(1-95) and pM-E/F(213-477)], human androgen receptor A/B domain [pM-AR(1-557)], human mineralocorticoid receptor A/B domain [pM-MR(1-279)], and human retinoic acid receptor alpha A/B domain [pM-RARalpha (1-99)] were generated by subcloning cDNA fragments into the appropriate sites of the multicloning region of the pM vector (Clontech). The regions of the nuclear receptors subcloned are given as the numbers of the amino acid sequences in parentheses. VP16-hERalpha and glutathione S-transferase-tagged hERalpha (GST-hERalpha ) fusion protein expression vectors were prepared in the same way, by subcloning into pVP16 (Clontech) and pGEX-2T/4T (Amersham Pharmacia Biotech), respectively. p68 cDNA was isolated by reverse transcription-PCR from an MCF-7 cDNA library, and the PCR-amplified cDNA was subcloned into the appropriate multicloning sites of the expression vectors pET-17b (Novagen), pM, and pSG5. SRC-1 and human ERbeta cDNAs were isolated from the HeLa cDNA library, and the expression vectors were constructed by introducing the cDNAs, which were verified (27, 39), into pcDNA3 (Invitrogen) (44, 50). Amino acid substitution of p68 (p68K144R) was performed by PCR-based mutagenesis with primers 5'-CAGACTGGATCTGGGAGAACATTGT-CTTATTTGC-3' and 5'-GGAAGCAAA-TAAGACAATGTTCTCCCAGATCCAG-3'.

GST pull-down and in vitro binding assays. GST-ER/p68 fusion proteins were expressed in bacteria and bound to glutathione-Sepharose 4B (Pharmacia) beads as described previously (20). Metabolic labeling of MCF-7 was performed by culturing the cells for 4 h in medium containing [35S]methionine (Amersham Pharmacia Biotech) (9). In vitro translation for the linearized expression vectors was performed with [35S]methionine by using the TNT kit (Promega). For the GST pull-down assay, a 50% suspension of GST-protein beads (50 µl), which contained up to 1.0 µg of protein, was resuspended in the same volume of binding buffer (20 mM Tris-HCl [pH 7.5], 0.12 M NaCl, 10% [vol/vol] glycerol, 0.055% 2-mercaptoethanol, 1 mM EDTA, 0.1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40) (50). The nuclear extract of 35S-labelled MCF-7 (3 × 106 cells) in 300 µl of binding buffer or an aliquot (15 µl) of the in vitro translation reaction mixture was mixed with GST-protein beads and suspended for 1 h at 4°C. The beads were then washed four times with washing buffer (replacing 0.12 M NaCl in the binding buffer with 0.1 M NaCl) and resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. After electrophoresis, radiolabeled proteins were visualized with an image analyzer (BAS2000; Fuji Film, Tokyo, Japan).

In vitro phosphorylation. The GST-HE15 and GST-HE15/457 proteins (5 µg) purified on glutathione-Sepharose 4B beads were phosphorylated in vitro with MAPK as described previously (26). The activated MAPK was a gift from E. Nishida (Kyoto University, Kyoto, Japan). Thus, phosphorylated GST fusion proteins were separated from the free ATP and used as probes in the GST pull-down experiment.

Purification and microsequencing of the 68-kDa protein. The nuclear extract of MCF-7 cells was incubated for 1 h at 4°C with 30 mg of GST-HE15 protein immobilized on 750 µl of glutathione-Sepharose. After the beads were washed three times in 1.5 ml of washing buffer (replacing 0.15 M NaCl in binding buffer with 0.1 M NaCl), proteins were eluted in 750 µl of elution buffer 1 (replacing 0.15 M NaCl in binding buffer with 0.2 M NaCl). After mixing for 5 min, the supernatant was removed again and the beads were resuspended in 750 µl of elution buffer 2 (replacing 0.15 M NaCl in binding buffer with 0.3 M NaCl). After mixing for 5 min, the supernatant was collected and concentrated by trichloroacetic acid precipitation. The precipitated proteins were resuspended in SDS-PAGE sample buffer, electrophoresed on a 10% polyacrylamide gel, and electroblotted on a polyvinylidene difluoride (PVDF) membrane (ProBlott; Perkin-Elmer, Norwalk, Conn.) at 7 V/cm at 4°C for 15 h in 10 mM cyclohexyl aminopropene sulfonic acid-NaOH (pH 11)-10% methanol. After blotting, protein bands were visualized by Coomassie brilliant blue staining, and the band corresponding to the p68 protein was excised. Cysteine residues of the protein on the PVDF membrane were carboxymethylated by reductive alkylation as described by Iwamatsu (21). S-carboxymethylated proteins were digested in situ with lysylendopeptidase, and the resulting peptides were separated by micro-fast-performance liquid chromatography (FPLC) with the SMART system (Amersham Pharmacia Biotech) (21). Microsequencing was performed with a 473A protein sequencer (Perkin-Elmer). Peptide fragment sequences were searched in the nucleotide/protein database by using the BLAST search method.

Northern analysis. MTN blots were obtained from Clontech. We also extracted and analyzed poly(A)+ RNA from various human cell lines derived from estrogen target tissues, as follows: MCF-7, breast cancer cell line; T47D, breast cancer cell line; LNCaP, prostate carcinoma cell line; HOS, osteosarcoma cell line; HTOA, ovarian cancer cell line; Ishikawa, uterine body cancer cell line. The cDNA probe of p68 RNA helicase was labeled with [alpha -32P]dCTP by the random-primer method. Prehybridizations and hybridizations were done as described previously (43). A human glyceraldehyde-3-phosphate dehydrogenase probe from Clontech was used for control experiments.

Transient transfection, reporter assay, and mammalian two-hybrid assay. For transfection, COS-1 cells were seeded in 100-mm dishes containing phenol red-free Dulbecco's minimal essential medium (GIBCO BRL) supplemented with 10% charcoal dextran-treated fetal bovine serum. At 40 to 60% confluency, the cells were transfected with 2 µg of ERE-G-CAT or 17M2G-CAT reporter plasmid, 0.4 µg of ER expression vector, 0.15 to 0.6 µg of p68 expression vector, and 3 µg of pCH110 beta -galactosidase reporter, and Bluescribe M13+ was used as the carrier DNA to adjust the total amount of DNA (26). After 20 to 24 h, the medium was replaced with fresh medium with or without 10-7 M E2, 10-7 M 4-hydroxytamoxifen (OHT) or 10-7 M ICI164,384 (ICI). After 24 to 48 h, the cells were lysed to determine the beta -galactosidase and chloramphenicol acetyltransferase activity (25).

Immunoblot analysis. The cell lysates of COS-1 transfected both with hERalpha and the p68 expression vector were subjected to SDS-PAGE and transferred to a nitrocellulose membrane (Hybond-ECL; Amersham Pharmacia Biotech). The membrane was probed with the monoclonal anti-hERalpha antibody F3 (1) and then with the peroxidase-labeled second antibody. Antibody staining was visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotech).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

A 68-kDa protein interacts with the hERalpha A/B domain, but not the E/F domain. To isolate a hERalpha AF-1-specific coactivator, a 35S-labeled MCF-7 nuclear extract was loaded on a GST-tagged hERalpha N-terminal A-C region (GST-HE15) (Fig. 1A) column. After extensive washing, a 68-kDa protein (p68) interacting with GST-HE15 was detected (Fig 1B). Under similar conditions, no 68-kDa protein interacted with GST alone or with the ERalpha LBD (GST-LBD [Fig. 1]) in either the absence or presence of 17beta -estradiol (E2) whereas a 160-kDa protein (9, 14) specifically interacted with GST-LBD in the presence of E2 (Fig. 1B). Interestingly, the interaction of p68 with the hERalpha N-terminal region was enhanced by replacing the hERalpha Ser118 residue with a glutamate residue (S118E; GST-HE15/458 [Fig. 1B, right panel]). Since this amino acid substitution mimics the phosphorylation of the Ser118 residue in terms of transactivation properties (1), these results indicated that the interaction between p68 and the hERalpha A/B region might be increased by phosphorylation of Ser118.


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  		FIG. 1.   Detection of binding proteins to the hERalpha A/B domain in MCF-7 cells. (A) The hERalpha (with regions A to F shown) and the GST-ER fusion proteins used. (B) Binding proteins to the hERalpha A/B domain in MCF-7 cells. Aliquots of the 35S-labeled MCF-7 nuclear extract were incubated with glutathione-Sepharose beads loaded with GST alone, GST-HE15, GST-HE15/458, GST-HE15/457, or GST-LBD in the absence or presence of E2 at 1 and 10 µM. The bound proteins were subjected to SDS-PAGE (5 to 20% polyacrylamide gradient gel) followed by autoradiography. Open arrowheads indicate the position of a protein of 68 kDa. Size markers are indicated in kilodaltons. The solid arrowhead indicates the position of the SRC-1/TIF2 160-kDa family proteins (9, 14).

From these findings, we speculated that p68 is a putative coactivator specific for ERalpha AF-1 and is involved in the enhancement of AF-1 activity by the MAPK-mediated phosphorylation of the Ser118 residue in the hERalpha A/B region.

Peptide microsequencing identifies the purified 68-kDa protein as p68 RNA helicase. To purify p68 from the MCF-7 cell nuclear extracts, we used a GST pull-down assay to concentrate the protein. The p68 protein absorbed to a GST-chimeric hERalpha A/B fusion protein (GST-HE15) on the beads was dissociated in a 0.25 to 0.3 M NaCl fraction in a stepwise elution, subjected to SDS-PAGE, and electroblotted on a PVDF membrane. The 68-kDa band was cut out of the Coomassie brilliant blue (G)-stained membrane, S-carboxymethylated, and digested with lysylendopeptidase. The five digested peptides were isolated by reversed-phase semi-micro-high-performance liquid chromatography. The peptide sequences of five peptides perfectly matched the sequences of the known p68 RNA helicase (31), as underlined in Fig. 2. We cloned the p68 RNA helicase cDNA by reverse transcription-PCR from an MCF-7 cDNA library and verified the DNA sequence. With this cloned cDNA, we studied the tissue distribution of the p68 transcripts and their expression in cell lines by Northern blotting. Two transcripts of mouse p68 of 2.2 and 3.5 kb were found in human tissue (31). We detected human p68 transcripts of 2.4 and 4.2 kb (Fig. 3), both of which were expressed ubiquitously in all tissues except the colon. These findings reveal no significant tissue specificity in p68 gene expression. Relatively high-level expression of p68 was seen in all cell lines; however, even in steroid hormone-dependent cell lines like MCF-7 and LNCaP, cell type specificity in p68 gene expression was not detected (Fig. 3B, right panel). The levels of the two transcripts differed among tissues and cell lines, and the smaller transcript (2.4 kb) was expressed at higher levels than the 4.2-kb transcript. However, the biological significance of the difference in p68 transcript size remains to be elucidated.


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  		FIG. 2.   Amino acid sequence of human p68 RNA helicase protein. The five sequences determined by microsequencing are underlined and completely matched to the reported p68 RNA helicase protein (GenBank accession no. X15729 and X52104). The nuclear receptor recognition motif (LXXLL motif [15]) is doubly underlined, and the DEAD box motif is boxed.


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  		FIG. 3.   Expression patterns of p68 RNA helicase transcripts in normal human tissues (A) and cancer cell lines (B). Northern blot analysis was performed as described previously (43). PBL, peripheral blood leukocyte. The cancer cell lines are as follows: HL60, promyelocytic leukemia cell line; HeLa S3, cervical carcinoma cell line; K562, chronic myelogenous leukemia cell line; Raji, Burkitt's lymphoma cell line; SW480, colorectal adenocarcinoma cell line; A549, lung carcinoma cell line; G361, melanoma cell line; MCF-7, breast cancer cell line; T47D, breast cancer cell line; LNCaP, prostate carcinoma cell line; HOS, osteosarcoma cell line; HTOA, ovarian cancer cell line; Ishikawa, uterine body cancer cell line; PC12, rat pheochromocytoma cell line. The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) transcript was used as an internal control. The relative positions of RNA markers (in kilobases) are shown on the right of panel B.

Direct interaction of p68 with the hERalpha A/B domain in vitro and in vivo. A possible p68 interaction with the hERalpha A/B domain was studied in vitro with a recombinant p68 protein produced by in vitro translation in rabbit reticulocyte with the cloned p68 cDNA. The interaction of p68 with the hERalpha A/B domain was tested by the GST pull-down assay (Fig. 1A), used to purify endogenous p68 protein from MCF-7 cells. The recombinant p68 protein interacted with GST-HE15 but not with GST-LBD in both the presence and absence of E2 (Fig. 4A) and the E2 antagonists OHT and ICI (data not shown), consistent with previous data (Fig. 1). Lack of association of p68 with the LBD was further supported by the fact that in vitro-translated SRC-1 binds strongly to this GST-LBD in an E2-dependent manner (Fig. 4A, right panel). The interaction of p68 with the A/B region was further potentiated when the Ser118 residue was replaced with Glu (S118E) (Fig. 4B), suggesting that the MAPK-mediated phosphorylation of Ser118 increases the binding of p68 to the A/B domain. To directly test this idea, GST-HE15 was phosphorylated by MAPK in vitro and subjected to a GST pull-down assay. The binding of p68 to the A/B region appeared to be dependent to some extent on the phosphorylation of the A/B region by MAPK (Fig. 4C). In addition, the binding preference that was increased by the phosphorylation of Ser118 was abrogated by replacement of Ser118 with Ala in the A/B region (S118A) (GST-HE15/457; Fig. 4C).


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  		FIG. 4.   The recombinant p68 RNA helicase specifically binds to the hERalpha A/B domain in vitro. (A) p68 interacts only with the hERalpha A/B domain but not with the LBD, irrespective of the presence of E2. In vitro-translated p68 RNA helicase recombinant protein (left panel, lane 1) was analyzed by SDS-PAGE (5 to 20% polyacrylamide gradient gel). GST-ER fusion proteins, immobilized on beads, were mixed with 15 µl of in vitro translation reaction mixtures of p68; 2 µl of translation reaction mixture was loaded on the input lane. SRC-1 interacts with the hERalpha LBD only in the presence of E2 (right panel; lane 4) as previously reported (39). The open arrowhead indicates the position of a p68 RNA helicase protein, and the solid arrowhead indicates the position of the SRC-1 protein. (B) The p68 interaction is enhanced by replacing the Ser118 residue with Glu (S118E) in the bacterially expressed GST fusion protein [GST-HE15/458]. (C) Phosphorylation of the hERalpha A/B domain by MAPK increases its binding to p68. GST-HE15 or GST-HE15/457 that was incubated with activated MAPK for 0, 15, or 30 min at 30°C was used as the probe for an in vitro pull-down assay with 35S-labeled p68 protein.

To further study the p68 interaction in vivo, a mammalian two-hybrid assay in COS-1 cells was performed with a p68 chimeric protein fused to a GAL4 DNA binding domain (DBD) (GAL4-p68) and two hERalpha deletion mutants fused to a VP16 activation domain (VP16-HE15 and VP16-LBD). Interaction of p68 with the A/B domain was detected, whereas the LBD did not interact with p68 even in the presence of E2 (Fig. 5). We also detected an intrinsic transactivation function of p68 by comparing GAL4-p68 with GAL4 DBD only (Fig. 5).


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  		FIG. 5.   p68 interacts with the hERalpha A/B domain but not with the LBD in vivo. p68 interacts with the hERalpha A/B domain in the mammalian two-hybrid system. A mammalian two-hybrid system with GAL4-p68 fusion protein and VP16-ER fusion proteins (VP16-HE15 and VP16-LBD) was used in COS-1 cells. COS-1 cells were cotransfected with 1 µg of either GAL4-DBD, GAL4-p68, VP16-HE15, or VP16-LBD in the presence or absence of E2 (10-7 M), along with 2 µg of 17M2G-CAT reporter plasmid. Significant interaction was detected only between p68 and the hERalpha A/B domain.

p68 acts as a specific coactivator for hERalpha AF-1. From in vivo and in vitro interaction studies, it appeared that p68 RNA helicase acts as a coactivator for the hERalpha AF-1. To address this extra function, the effect of p68 in the transactivation functions of the hERalpha and hERbeta (33) was investigated with ER fusion proteins of the GAL4 DBD. Forced expression of p68 significantly enhanced the AF-1 activity but not the AF-2 activity of the hERalpha in the absence and presence of E2 (Fig. 6A). The enhanced transactivation function of p68 was detected with the full length of hERalpha but was not as marked as that seen with AF-1 alone. However, neither hERbeta AF-1 nor AF-2 was activated by p68 (Fig. 6B). Moreover, the AF-1 activities of other nuclear receptors tested (Fig. 6D), as well as their AF-2 activities (data not shown), were not enhanced by p68. However, overexpression of p68 could not potentiate the activity of the hERalpha AF-1 in HeLa cells, in which the hERalpha AF-1 is known to be very low (Fig. 6C), suggesting that the p68 action is cell type specific. Note that overexpression of p68 does not affect the expression levels of hERalpha (Fig. 6F) or of chimeric receptors (data not shown).


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  		FIG. 6.   p68 potentiates the ligand-induced transactivation function of hERalpha through AF-1. (A) p68 potentiates AF-1 but not AF-2 of hERalpha . COS-1 cells were cotransfected with 0.40 µg of HE15-GAL(AF-1), LBD-GAL(AF-2), or HEGO (AF-1 plus AF-2) (26) and with either 0, 0.3, or 0.6 µg of pSG5-p68 in the presence or absence of E2 (10-7 M), along with 2 µg of 17M2G-CAT or 2 µg of ERE-G-CAT (for HEGO only). p68 potentiated the ligand-induced transactivation of the full-length hERalpha and AF-1 but not AF-2 activated by E2. (B) p68 has no effect in the transactivation function (AF-1 and AF-2) of hERbeta . (C) p68 potentiates the hERalpha AF-1 activity in COS-1 cells but not in HeLa cells. (D) p68 has no effect on the AF-1 activities of the other nuclear receptors. (E) p68 potentiates the hERalpha AF-1 phosphorylated at the Ser118 residue. COS-1 cells were cotransfected with HE15-GAL or HE15/457-GAL along with either 0, 0.3, or 0.6 µg of pSG5-p68 and 2 µg of 17M2G-CAT. (F) p68 does not affect the expression levels of the hERalpha A/B domain. COS-1 cells were transfected with 0.40 µg of HE15-GAL and with either 0, 0.15, 0.3, or 0.6 µg of pSG5-p68. A Western blot analysis shows that the amount of the expressed chimeric protein is not affected by p68 expression. The open arrowhead indicates the position of the protein.

Phosphorylation of Ser118 by MAPK is essential for enhancement of hERalpha AF-1 activity by p68. Previous studies showed that several serine residues of the ERalpha A/B domain are phosphorylated in cells treated with growth factors, which can potentiate the actions of E2 (1, 3, 19). We further demonstrated that MAPK, which is activated by growth factors, specifically phosphorylates Ser118 of the ERalpha A/B domain and that this phosphorylation enhances the AF-1 activity (26). Taken together with the binding preference of p68 for the A/B domain phosphorylated by MAPK at the Ser118 residue (Fig. 1 and 4), our result indicates that p68 acts as a coactivator for the hERalpha AF-1 in a MAPK-mediated phosphorylation-dependent way. To address this point, the effects of p68 on the AF-1 activity of the phosphorylated A/B domain were examined by comparing the wild-type A/B domain (HE15-GAL) and the A/B domain point mutant (HE15/457-GAL), in which the Ser118 residue is replaced by alanine and is unable to be phosphorylated by MAPK (1, 26). As shown in Fig. 6E, the AF-1 activity enhanced by p68 was abrogated by Ser118 replacement (HE15/457-GAL). Thus, these results demonstrate that the phosphorylation of Ser118 by MAPK is indispensable for the enhanced activity of ERalpha AF-1 by p68. To further clarify the role of p68 in the ERalpha AF-1 activity, the action of p68 in the full-length ERalpha activated by OHT and ICI was examined. Although ICI was shown to suppress ERalpha transactivation function as a pure antagonist, OHT is considered a partial antagonist, blocking only AF-2 function and not AF-1 function. p68 could not induce the transactivation function of hERalpha -bound ICI; however, it potentiated the function of hERalpha -bound OHT (Fig. 7), again supporting the idea that p68 acts as a coactivator specific for hERalpha AF-1.


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  		FIG. 7.   p68 potentiates the transactivation function of hERalpha induced by OHT but not ICI. COS-1 cells were cotransfected with 0.40 µg of HEGO and 2 µg of ERE-G-CAT, along with either 0, 0.3, or 0.6 µg of pSG5-p68, and treated with 100 nM E2, OHT, or ICI.

p68 coactivator activity requires the hERalpha A/B domain but does not require intrinsic RNA helicase activity. To delineate the region of p68 responsible for the interaction with ERalpha A/B domain, a series of p68 deletion mutants were examined by the GST pull-down assay, with the GST-HE15 protein as a probe, and by the transient-expression assay to establish their roles in the ERalpha AF-1 activity (Fig. 8). Truncations of the N-terminal region up to amino acid (aa) 300 (p68-mt4, p68-mt5, and p68-mt6) did not reduce the p68 interaction in vitro, but a further 100-aa deletion caused complete loss of the interaction (p68-mt7) (Fig. 8A, middle panel). Consistent with the N-terminal truncations, the region from aa 300 to 400 was required in the C-terminal truncation mutants (p68-mt1, p68-mt2, and p68-mt3). In the transactivation assay, p68-mt4 and p68-mt5 were as potent as the wild-type p68 but p68-mt1, p68-mt2, and p68-mt6 suppressed the ERalpha AF-1 activity (Fig. 8A, right panel). From these results, it is likely that the regions surrounding the interaction domain are required for the full coactivator activity of p68. Next, to determine the interaction regions for p68 in the A/B domain of ERalpha , a series of truncated mutants of the A/B domain fused to GST were generated and examined for binding to in vitro-translated p68 in GST pull-down assays. As shown in Fig. 8B, p68 directly interacted with the middle region (aa 56 to 127) in the A/B domain, which contains the Ser118 residue and was also essential for the AF-1 activity. Transactivation assays showed that p68 was able to potentiate the transcriptional activities of only the GAL4-fused deletion mutants of the A/B domain which contain the interaction region for p68. From results obtained from in vitro binding assays and in vivo transactivation assays, we concluded that the interaction regions identified in GST pull-down assays were also functional in vivo and that direct binding between p68 and the A/B domain in vivo was absolutely necessary for the potentiation of AF-1 activity by p68.


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  		FIG. 8.   The RNA helicase activity of p68 is not required for the coactivator activity for the hERalpha AF-1. (A) The interaction domain of p68 for the hERalpha A/B domain is essential for the p68 coactivator activity for hERalpha AF-1. Representations of the p68 deletion mutants used for in vitro GST pull-down assay (middle panel) and the transient-expression assay (right panel) are shown. 35S-labeled p68 mutants were assayed with the GST-HE15 protein as a probe, showing that the region (aa 300 to 400) of the ATP binding domain and the DEAD box motif is required for direct interaction with the hERalpha A/B domain. The coactivator activities of p68 deletion mutants were determined in the transient-expression assay. COS-1 cells were cotransfected with 0.40 µg of HE15-GAL, 2 µg of 17M2G-CAT, and 0.6 µg of the expression vector of the p68 mutant. The mean fold induction of the AF-1 activity by the p68 mutant from three independent experiments is shown. (B) The interaction region of the hERalpha A/B domain for p68 is necessary for the potentiation of AF-1 activity by p68. Representations of the hERalpha A/B domain deletion mutants used for in vitro GST pull-down assay (middle panel) and the transient-expression assay (right panel) are shown. 35S-labeled p68 was assayed with the GST-fused A/B domain deletion mutant proteins as a probe, showing that the region (aa 56 to 127) in the A/B domain is required for direct p68 binding. The coactivator activities of p68 for the A/B domain deletion mutants were determined in the transient-expression assay. COS-1 cells were cotransfected with either 0.40 µg of A/B deletion mutants-plus-GAL (pM-A/B-Ms) with 2 µg of 17M2G-CAT and 0.6 µg of the expression vector of p68. The mean fold induction of the AF-1 activity by p68 from three independent experiments is shown. (C) A p68 mutant with a mutation in the ATP binding domain essential for the RNA helicase activity still potentiates the hERalpha AF-1. The ATP binding domain and DEAD box motif are indicated by solid and shaded boxes, respectively. The amino acid residues (AXXGXGKT), which are highly conserved among helicases, are boxed. The asterisk shows the replaced amino acid. COS-1 cells were cotransfected with 0.40 µg of pM-HE15 and 2 µg of 17M2G-CAT and with 0, 0.3, or 0.6 µg of the expression vector for the p68 mutant. The activity of hERalpha AF-1 was enhanced by both the wild-type p68 and the K144R mutant of p68. (D) p68 binds to CBP. To test the binding between p68 and CBP, GST control protein, GST-p68N (aa 1 to 387) fusion protein, or GST-p68C (aa 388 to 614) fusion protein was immobilized on beads and mixed with 15 µl of in vitro CBP translation reaction mixtures. Binding proteins were analyzed by SDS-PAGE. In vitro-translated CBP binds to both GST-fused p68N and p68C but not to the GST control protein.

Previous reports demonstrating that the p68 protein is an ATP-dependent RNA helicase raised the question of whether RNA helicase activity (16) was required for the p68 coactivator activity for the hERalpha AF-1. To address this issue, a point mutation (Lys144 to Arg) in the p68 ATP binding site was introduced to abolish RNA helicase activity (12, 13) (Fig. 8C, upper panel). This mutation had no effect on the p68 coactivator activity, leading us to conclude that p68 helicase activity is not required for its hERalpha coactivator function.

Since p68 itself exhibited only weak intrinsic activity in transactivation (Fig. 5), we explored a possible interaction of p68 with known coactivators. For this study, we used two major classes of coactivators, the SRC-1/TIF2 family proteins and the CBP/p300 class (44). All SRC-1/TIF2 family proteins showed no interaction with p68 in a GST pull-down assay (data not shown). However, both the N-terminal (aa 1 to 387) and C-terminal (aa 388 to 614) domains of p68 fused to GST bound to in vitro-translated CBP (Fig. 8D). These results demonstrate that the coactivator activity of p68 for the hERalpha AF-1 is mediated at least by CBP.

The p68 interaction region in the hERalpha A/B domain is essential for full activity of the hERalpha AF-1. To assess if the p68 interaction with the hERalpha A/B domain in vivo reflects the p68 coactivator activity for the hERalpha AF-1, we studied the relationship between p68 interaction and the enhanced AF-1 activity mediated by p68 with a series of the hERalpha A/B domain deletion mutants. In vitro GST pull-down assays demonstrated that the central region (from aa 56 to 127) in the hERalpha A/B domain is required for the in vitro p68 interaction (Fig. 8B). In agreement with this interaction, p68 potentiated the transactivation functions of the hERalpha A/B domain deletion mutants which retain the central region. Interestingly, this interaction region was also essential for the full activity of the hERalpha AF-1 (Fig. 8B). Thus, from these findings, it is likely that the full activity of hERalpha AF-1 requires p68 interaction through a central region in the hERalpha A/B domain.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Identification of ERalpha AF-1 specific coactivator, p68. Several classes of putative nuclear receptor coactivators such as the SRC-1/TIF2 (2, 9, 11, 39, 47) and CBP/p300 (24) family proteins, TIF1 (29), ARA70 (52), and many others (22, 35, 36, 40) have been investigated in terms of transcriptional mediation between nuclear receptors and the basal transcriptional machinery (5, 17). Significant enhancement of ligand-induced transactivation of many nuclear receptors is observed in the SRC-1/TIF2 family proteins when they are expressed in mammalian cells. Moreover, overexpression of the SRC-1/TIF2 family proteins can reduce transcriptional interference among nuclear receptors and nuclear receptor autosquelching (11, 39, 47). By biochemical approaches, direct interactions of the SRC-1/TIF2 family proteins with the LBD have been further demonstrated to occur in a ligand-dependent manner (2, 9, 11, 47), and ligand-induced interactions of ERalpha with the SRC-1/TIF2 family proteins are induced by agonists but not by antagonists (9, 14). Analysis of the interacting domains of coactivators led to the identification of consensus motifs (LXXLL) for the direct interaction with nuclear receptors (15). From these observations, an SRC-1/TIF2 family of proteins was recognized as containing the best-characterized coactivators for AF-2 of various nuclear receptors, including ERalpha and ERbeta . Like the SRC-1/TIF2 family proteins, the reported coactivators were found while searching for a coactivator for AF-2 but not for AF-1. To our knowledge, there is no report of a coactivator specific for AF-1 of nuclear receptors. However, since the ratios between the AF-1 and AF-2 activities, at least in ERalpha , are cell type specific (6, 46), the existence of a coactivator that specifically interacts with and activates AF-1 is a distinct possibility (37, 45). More recently, the SRC-1/TIF2 family proteins have been reported to stimulate the ERalpha AF-1 activity of nuclear receptors synergistically with the AF-2 (34, 49), but these coactivators seem only to partially support the AF-1 activity of ERalpha (49). Thus, even if this family of proteins are coactivators for AF-1, an additional coactivator directly interacting with the ERalpha A/B domain is believed to exist. When recombinant A/B and LBD domains of the hERalpha were used as probes to purify interactants from the nuclear extracts of various cell lines, no interactant for the 160-kDa protein of the ERalpha A/B domain was found in the present study, while a ligand-dependent interaction with the LBD was seen in the interactant for the 160-kDa protein, presumably SRC-1/TIF2 family proteins (9, 14). In contrast to the 160-kDa interactant, we found that p68 interacts with the A/B domain but not the LBD of ERalpha even in the presence of E2 and E2 antagonists. Purification and cloning identified this p68 protein as a previously reported p68 RNA helicase protein. The recombinant p68 protein interacted in vivo and in vitro specifically with the ERalpha A/B domain. The overexpression of p68 potentiated the hERalpha AF-1 activity but not other AF-1 and AF-2 activities of tested nuclear receptors, including ERbeta . Interestingly, deletion of the hERalpha A/B domain showed that p68 interacts with the central region, which is essential for the hERalpha AF-1 activity (Fig. 8B). Although the p68 coactivator activity was less potent in the full length of hERalpha bound to E2 than in AF-1 alone, p68 could potentiate the ligand-induced transactivation function of the full-length hERalpha by OHT, which is considered to be an ERalpha AF-1 agonist and an AF-2 antagonist (1, 6). Taken together, these results indicate that p68 is a coactivator that specifically interacts with and activates the AF-1 of ERalpha .

The p68 RNA helicase acts as a coactivator. The p68 RNA helicase protein was first reported to have immunological cross-reactivity with an antibody against the simian virus 40 large T antigen (12). p68 is a nuclear protein, and its localization in the nucleus varies during the cell cycle. At telophase, it translocates from the nucleoplasm to the nucleoli (18). p68 is a member of the DEAD-box protein family of putative RNA helicases (16) and contains an ATPase A motif that is responsible for RNA helicase activity (12). p68 is proposed to be important in diverse cellular processes, including RNA processing, transcription, translation, cell growth, and division. Moreover, p68 binds calmodulin in a Ca2+-dependent manner and is phosphorylated by protein kinase C through the IQ domain in the C-terminal region (7). Since the p68 ATPase activity is inhibited by both calmodulin binding and protein kinase C phosphorylation, the RNA-unwinding activity of p68 is supposed to be modulated by dual Ca2+-signaling pathways (7). In the present study, we first found that this p68 also functions as a transcriptional coactivator but that the coactivator activity of p68 does not require the RNA helicase activity. Since p68 itself possesses weak intrinsic transactivation activity, a region other than that containing the RNA-unwinding activity appears to regulate the p68 coactivator function of the ERalpha AF-1. Since CBP exhibited in vitro affinity to the N-terminal and C-terminal domains of p68 (Fig. 8D), p68 may serve as an adapter protein to associate with AF-2 coactivators. Since the ERalpha AF-1 activity is cell type specific and p68 is ubiquitiously expressed (Fig. 3), it is possible to speculate that upon binding ERalpha AF-1, p68 recruits an unknown coactivator(s) which acts in a cell-type-specific manner. This idea was further supported by the present observation that p68 overexpression cannot potentiate the ERalpha AF-1 activity in HeLa cells (Fig. 6C).

p68 potentiates ERalpha AF-1 phosphorylated by MAPK. It was first reported in 1993 that ligand-dependent phosphorylation occurs on the Ser118 residue of ERalpha (1) and this phosphorylation supports the full activity of ERalpha AF-1. We further demonstrated that MAPK, which is activated by growth factors, undergoes this phosphorylation at Ser118 and potentiates the ERalpha AF-1 activity (26). In the present study, we found that the p68 binding affinity for the the hERalpha A/B region is increased by MAPK-mediated phosphorylation. Moreover, p68 does not potentiate the ERalpha AF-1 mutant, in which Ser118 is replaced by alanine and cannot be phosphorylated. Thus, it is most likely that when the Ser118 residue is phosphorylated, p68 associates tightly with the ERalpha A/B region to potentiate the ERalpha AF-1 function.

Like the ERalpha AF-2, the ERalpha AF-1 function is also induced by ligand binding to the ERalpha LBD (6, 46). During this process, physical and functional interactions between the AF-1 and AF-2 of ERalpha induced by ligand binding are believed to occur. More recently, it was reported that SRC-1 mediates the ligand-dependent interaction of AF-1 and AF-2 (34, 49). Since p68 potentiated the ligand-bound ERalpha but not unbound ERalpha , the possibility exists that p68 binds to the A/B domain only when the AF-2 is activated by ligand binding. Alternatively, it is speculated that like the CBP/p300 proteins, a coactivator(s) bound to the LBD in a ligand-dependent manner recruits p68 to associate with the A/B region. To test these hypothesis, further studies of p68 relating to the ligand-induced interaction of the A/B domain and LBD of hERalpha are required.


    ACKNOWLEDGMENTS

We thank Pierre Chambon for helpful discussions throughout the study, Hiroaki Fuse for discussions and technical assistance, and Eisuke Nishida for the kind gift of purified MAPK.

This work was supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Medicale, and the Human Frontier Science Program and by a grant-in-aid for priority areas from the Ministry of Education, Science, Sports and Culture of Japan (S.K.)


    FOOTNOTES

* Corresponding author. Mailing address: Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan. Phone: 81-3-5841-8478. Fax: 81-3-5841-8477. E-mail: uskato{at}hongo.ecc.u-tokyo.ac.jp.


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