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WW Domain Binding Protein-2, an E6-Associated Protein Interacting Protein, Acts as a Coactivator of Estrogen and Progesterone Receptors

Department of Biochemistry and Molecular Biology (S.C.D., S.R., O.Y.K., A.I., J.Su., J.Sl., Z.N.), University of Miami School of Medicine, Miami, Florida 33136; and Department of Molecular and Cellular Biology (B.W.O.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Zafar Nawaz, Ph.D., Department of Biochemistry and Molecular Biology, Braman Breast Cancer Institute (M-877), University of Miami School of Medicine, Batchelor’s Building, Room 416, 1580 Northwest 10th Avenue, Miami, Florida 33136. E-mail: znawaz{at}med.miami.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION Results Discussion Materials and Methods REFERENCES

 
WW domain binding protein-2 (WBP-2) was cloned as an E6-associated protein interacting protein, and its role in steroid hormone receptors functions was investigated. We show that WBP-2 specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER), whereas it did not have any significant effect on the androgen receptor, glucocorticoid receptor, or the activation functions of p53 and VP-16. Depletion of endogenous WBP-2 with small interfering RNAs indicated that WBP-2 was required for the proper functioning of PR and ER. We also demonstrated that WBP-2 contains an intrinsic activation domain. Moreover, chromatin immunoprecipitation assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. Mutational analysis suggests that one of three polyproline (PY) motifs of WBP-2 is essential for its coactivation and intrinsic activation functions. We show that WBP-2 and E6-associated protein each enhance PR function, and their effect on PR action are additive when coexpressed, suggesting a common signaling pathway. In this study, we also demonstrate that the WBP-2 binding protein, Yes kinase-associated protein (YAP) enhances PR transactivation, but YAP’s coactivation function is absolutely dependent on WBP-2. Taken together, our data establish the role of WBP-2 and YAP as coactivators for ER and PR transactivation pathways.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Results Discussion Materials and Methods REFERENCES

 
STEROID HORMONES REGULATE various biological processes via their cognate receptors, which are comprised of a superfamily of structurally related intracellular ligand-activated transcription factors (1, 2). In the absence of hormones, these receptors are transcriptionally inactive and are bound to cellular chaperone proteins. Upon ligand binding, the receptors undergo a conformational change, resulting in their dissociation from cellular chaperones, dimerization, and phosphorylation. Ligand-bound receptor dimers bind to enhancer elements of target genes and subsequently recruit coactivators and general transcription factors to form a preinitiation complex that culminates in target gene transcription (3, 4, 5, 6, 7).

Nuclear hormone receptor coactivators are molecules that interact with activated receptors and stimulate receptor-mediated transcription of target genes (8, 9, 10, 11). The most widely studied coactivators include members of the p160 family of coactivators; steroid receptor coactivator-1 (SRC-1), SRC-2 [transcription intermediary factor-2 (TIF-2)/glucocorticoid receptor interacting protein-1 (GRIP-1)], SRC-3 [p/CIP; p300/CBP interacting protein/activator of thyroid and retinoid acid receptors (ACTR)/amplified in breast cancer-1 (AIB-1)/retinoid acid receptor coactivator-3 (RAC-3)/thyroid receptor activator molecule-1 (TRAM-1)], the cAMP response element-binding protein-binding (CREB) protein (CBP)/p300 family coactivator-associated arginine methyltransferase (CARM-1), and E6-associated protein (E6-AP) (11, 12, 13, 14, 15, 16, 17, 18).

We have previously reported the cloning and characterization of E6-AP as a novel dual-function steroid hormone receptor coactivator. Recently, we demonstrated that the E2 ubiquitin-conjugating enzyme, UbcH7, acts as a coactivator of steroid hormone receptors (19). It has been demonstrated that coactivator proteins form multiprotein complexes to efficiently regulate target gene transcription (15). In this study, our focus was to identify proteins that interact with E6-AP and modulate steroid hormone receptor functions. To identify proteins that interact with E6-AP, we performed a yeast two-hybrid screen using E6-AP mutant (E6-AP-C833S) defective in ubiquitin-ligase function as bait so as to avoid degradation of the prey during the process. WW domain binding protein-2 (WBP-2) was identified as an E6-AP interacting protein. WBP-2 was previously shown to interact with human Yes kinase-associated protein (YAP) via the WW domain of YAP protein (20). The WW domain is characterized by 35–40 semiconserved amino acids, which are involved in protein-protein interaction (21). WBP-2 interacts with the WW domain via a short proline-rich motif (PPXY) with the consensus sequence of four consecutive prolines followed by tyrosine (22). It has been speculated that WBP-2 plays a role in transcription, but its exact function in transcription has not been defined. Additionally, it has been suggested that YAP may regulate transcription (23) by acting as a coactivator for several transcription factors including members of Runx2 family (24) and TEAD/TEF family (25), the proapoptotic protein p73 (26, 27), and is involved in ErbB4 signaling (28).

In the present study, we describe a role for WBP-2 and YAP in steroid hormone receptor functions. We show that WBP-2 physically interacts with E6-AP and certain steroid hormone receptors. We demonstrate that WBP-2 specifically modulates the hormone-dependent transcriptional activities of estrogen receptor (ER) and progesterone receptor (PR). Moreover, depletion of endogenous WBP-2 protein with small interfering RNA (siRNA) significantly reduces the transactivation potential of steroid receptors. Our data suggest that the carboxyl-terminal PPXY motif of WBP-2 is required for its coactivation function. Coexpression of WBP-2 and E6-AP enhances steroid receptor transactivation additively. Furthermore, YAP, which has been shown to be physically associated with WBP-2, also is able to enhance receptor function. However, the YAP coactivation function is strictly dependent on the coexpression of functional WBP-2. Together, our results demonstrate the role of WBP-2 as a potent coactivator for a subset of steroid receptors.

	Results

TOP ABSTRACT INTRODUCTION Results Discussion Materials and Methods REFERENCES

 
Isolation and Characterization of WBP-2 as an E6-AP Interacting Protein
Yeast two-hybrid screening system was used to isolate cDNA clones that encode E6-AP interacting proteins. Because E6-AP could activate the degradation of its binding proteins, a catalytically inactive form of E6AP (E6-AP-C833S) was used as bait to avoid potential degradation of interacting proteins. E6-AP (C833S) was fused in-frame with the Gal4 DNA-binding domain and introduced into Saccharomyces cerevisiae. The prey cDNA library, fused to the Gal4 activation domain, was derived from human brain cells. We isolated 12 colonies with cDNAs encoding proteins that strongly interacted with E6-AP. Surprisingly, all 12 colonies contained identical cDNAs. A sequence similarity search in the GenBank database revealed that all colonies encoded the carboxyl terminus of WBP-2.

We subsequently verified that WBP-2 interacts with E6-AP both in vivo and in vitro. As shown in Fig. 1A, in a yeast two-hybrid assay, WBP-2 strongly interacts with E6-AP. To further confirm that the WBP-2 and E6-AP interaction observed in the yeast two-hybrid assay is due to direct physical association of WBP-2 with E6-AP, we performed glutathione-S-transferase (GST) pull-down assays. In this assay, WBP-2 interacted directly with E6-AP (Fig. 1B).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Yeast Two-Hybrid Screen for E6-AP Interacting Proteins A, Yeast cells were transformed with bait plasmid pGAD10-E6-AP (C833S) and prey brain cDNA library, fused to the Gal4 activation domain. The ß-galactosidase activity (Miller units) of clone 69 (WBP-2) was determined. B, In vitro WBP-2 interacts with E6-AP. In vitro translation of E6-AP was performed in the presence of [35S]methionine using the transcription and translation kit (Promega). GST-WBP-2 and GST alone (control) were expressed in E. coli and purified on glutathione-Sepharose beads. The purified and glutathione-bound GST (control) or GST-WBP-2 was incubated with in vitro-translated E6-AP. WBP-2-bound E6-AP was analyzed by autoradiography with 20% of in vitro-translated E6-AP as input.

View larger version (34K): [in this window] [in a new window]   Fig. 2. WBP-2 Interacts with ER A, Interaction of WBP-2 with ER was determined in a GST pull-down assay. ER was radiolabeled with 35S by the in vitro transcription and translation kit. The labeled ER protein was then incubated overnight at 4 C with E. coli expressed GST alone (control) or GST-WBP-2 bound to beads either in the absence of hormone (–H), or presence of E2 or tamoxifen (T). Bound proteins were analyzed by SDS-PAGE and autoradiography with 10% of in vitro-translated ER as input. B, In vivo WBP-2 interacts with E6-AP and ER. MCF-7 cells were treated with either no hormone (–H), E2, or tamoxifen (T) for 24 h, and cells were lysed with RIPA lysis buffer. Lysates were clarified with protein A-Sepharose beads and incubated with either anti-ER antibody or rabbit preimmune serum (Alpha Diagnostic). The lysates were precipitated with protein A-Sepharose beads that bind to rabbit IgGs followed by electrophoresis. Five percent of the lysates were used as input and analyzed by Western blot with either anti-ER, anti-E6-AP, or anti-WBP-2 antibodies.

WBP-2 Selectively Modulates the Transcriptional Activities of Progesterone and Estrogen Receptors
Because E6-AP acts as a coactivator for nuclear hormone receptors and WBP-2 physically associates with E6-AP and steroid hormone receptors, we next tested whether WBP-2 regulates receptor-dependent activation of target gene expression using transient transfection assays in HeLa cells. HeLa cells were cotransfected with mammalian expression plasmids containing one of the following receptors, PR, ER, glucocorticoid (GR) and androgen (AR) receptors along with the reporter plasmids driven by their cognate hormone response element. These assays were carried out with or without coexpression of an expression vector for WBP-2. WBP-2 had little effect on the transactivation functions of PR and ER in the absence of hormone. However, WBP-2 significantly enhanced (4- to 5-fold) the hormone-dependent transcriptional activities of PR and ER (Fig. 3A). WBP-2 had only minimal effect on the transcriptional activities of GR and AR both with and without their cognate ligands (Fig. 3B). Similarly WBP-2 had only negligible effect on other nuclear receptors like retinoic acid receptor- and thyroid receptor (data not shown). These data suggest that WBP-2 selectively modulates the ligand-dependent transcriptional activities of ER and PR. Because HeLa cells express the viral E6 protein that could functionally interact with WBP-2 via E6-AP, we wanted to assay the coactivation function of WBP-2 in cells that lack E6 expression. As shown in Fig. 3C, WBP-2 stimulated the hormone-dependent transcriptional activity of PR in the E6-negative T47D cells. Similarly, WBP-2 could also stimulate the hormone-dependent transcriptional activity of ER in the E6-negative MCF-7 cells. Furthermore, WBP-2 has no significant effect on the transcriptional activity of ER in the presence of antiestrogen (tamoxifen), suggesting that WBP-2 only enhance the hormone-dependent transcriptional activity of receptor (Fig. 3D). These data also indicate that the coactivation function of WBP-2 is not dependent on the E6 protein. This is consistent with our previously published data, indicating that the coactivation function of E6-AP is not dependent on the viral E6 protein (16).

View larger version (18K): [in this window] [in a new window]   Fig. 3. WBP-2 Specifically Modulates the Hormone-Dependent Transcriptional Activity of ER and PR A, HeLa cells were transiently transfected with receptor expression plasmid for PR, ER, and their cognate hormone response elements in the presence or absence of WBP-2. Later, cells were treated with respective hormones as follows: PR, progesterone (10–7 M); ER, estradiol (10–8 M). Cells were harvested after 24 h and assayed for luciferase activity, and bars represent mean from three different determinations. The data are presented as fold activation. The activity of each receptor in the presence of hormone and in the absence of WBP-2 was defined as 1-fold, and the data for other bars were scaled accordingly. B, WBP-2 has no significant effect on the hormone-dependent transcriptional activity of AR and GR. LNCaP cells were transfected with androgen-responsive reporter in the presence or absence of WBP-2. HeLa cells were transiently transfected with GR expression plasmid and its cognate hormone response elements in the presence or absence of WBP-2. Later, LNCaP and HeLa cells were treated with R1881 (2.5 x 10–10 M) for AR and dexamethasone (10–7 M) for GR, respectively. Cells were harvested after 24 h and assayed for luciferase activity, and bars represent mean from three different determinations. The data are presented as fold activation. The activity of each receptor in the presence of hormone and in the absence of WBP-2 was defined as 1-fold, and the data for other bars were scaled accordingly. C, WBP-2 enhances PR transactivation in T47D cells. T47D cells were transiently transfected with progesterone response element containing reporter plasmid in the presence or absence of WBP-2. Later, cells were treated with progesterone (10–7 M). Cells were harvested after 24 h and assayed for luciferase activity, and bars are mean and SD from three different determinations. The data are presented as fold activation. The activity of receptor in the presence of hormone and in the absence of WBP-2 was defined as 1-fold, and the data for other bars were scaled accordingly. D, WBP-2 enhances ER transactivation in MCF-7 cells. MCF-7 cells were transiently transfected with estrogen response element containing reporter plasmid in the presence or absence of WBP-2. Later, cells were treated with either vehicle (–H), estradiol (10–8 M), or antiestrogen, tamoxifen (10–6 M). Cells were harvested after 24 h and assayed for luciferase activity, and bars are mean and SD from three different determinations. The data are presented as fold activation. The activity of receptor in the presence of hormone and in the absence of WBP-2 was defined as 1-fold, and the data for other bars were scaled accordingly. E, WBP-2 had no significant effect on the transcriptional activity of nonnuclear hormone receptor transcription factors p53 and VP-16 activation domain. HeLa cells were transiently transfected with expression plasmid for either p53 or VP-16 activation domain along with their respective reporter plasmids, p21 promoter-LUC or 17-mer-LUC in the presence or absence of WBP-2. Data are expressed as mean and SD of three different transfection assays. The data are presented as fold activation. The activity of each transcription factor in the absence of WBP-2 was taken as 1-fold, and the other bar was scaled accordingly.

Depletion of Endogenous WBP-2 Levels Reduces the Transcriptional Activity of PR and ER
To confirm that WBP-2 is indeed required for PR activation, cellular WBP-2 expression was down-regulated in HeLa cells using WBP-2-directed siRNA. HeLa cells were transiently transfected with siRNAs directed against either WBP-2 or against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control, together with PR and PR-responsive reporter plasmids. Expression of siRNA directed against WBP-2 reduced the level of WBP-2 expression, whereas the control siRNA had no effect on WBP-2 expression (Fig. 4A). Depletion of endogenous WBP-2 reduced PR transcriptional activity by 75.3% (Fig. 4B), indicating that WBP-2 is required for fully functional PR activity.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Endogenous WBP-2 Expression Is Essential for Steroid Hormone Receptor Function A, Expression analysis of WBP-2 protein after siRNA treatment. HeLa cells were transiently transfected with PR expression plasmid and its response element either in presence of WBP-2 siRNA (in vitro synthesized) or control GAPDH siRNA. Four hours post transfection, cells were treated either with progesterone (10–7 M) or vehicle (ethanol). Twenty-four hours after transfection, cells were harvested and lysed. Cell lysates were run on a 4–20% gradient gel and transferred onto nitrocellulose paper. Protein levels were assessed by Western blot using WBP-2-specific antibodies. Equal loading of samples was confirmed using ß-actin-specific antibodies. B, Depletion of endogenous WBP-2 levels reduces transcriptional activity of PR. Part of the cell lysates was used to measure luciferase activity. The activity of PR in the presence of progesterone and control siRNA (GAPDH) was taken as 100-fold activation, and the data for other bars were scaled accordingly. C, Depletion of endogenous WBP-2 levels reduces the expression levels of ER target gene pS2. MCF-7 cells were transfected with either control siRNA (siScrambled), siGAPDH, or siWBP-2. Then, cells were treated with vehicle (–H) or E2 (+H) for 12 h before being collected for RNA preparation. RT-PCR was performed with specific primers for pS2. The results were normalized against GAPDH transcripts, and plotted as relative fold mRNA levels of pS2. The relative mRNA level of pS2 gene when treated with siRNA against GAPDH is taken as 1-fold, and other bars are scaled accordingly.

Recruitment of WBP-2 onto an Estrogen-Responsive Promoter in MCF-7 Cells
To further substantiate the coactivation function of WBP-2, we employed chromatin immunoprecipitation (ChIP) to assay the recruitment of WBP-2 onto an ER-responsive promoter in MCF-7 cells. Formaldehyde cross-linked protein-chromatin complexes were immunoprecipitated from MCF-7 that were treated with or without estrogen using the appropriate specific antibodies. The precipitated genomic DNA associated with ER or WBP-2 was amplified by PCR using primers specific for estrogen receptor binding site within the pS2 promoter. ChIP analyses demonstrated the recruitment of WBP-2 onto ER-responsive promoter in the presence of estrogen (Fig. 5, A and B); thus, as is the case for known ER coactivators, WBP-2 is recruited to the pS2 promoter in a hormone-dependent manner (29). This assay also demonstrates that WBP-2 is physically present on an ER target gene promoter at a time when the gene is activated.

View larger version (11K): [in this window] [in a new window]   Fig. 5. WBP-2 Is Recruited onto ER-Responsive Promoter ChIP was performed using MCF-7 cells in the presence of E2 (+) or absence of estrogen hormone (–). A, Primers specific for pS2 promoter were used to amplify the genomic DNA associated with ER in MCF-7 cells. B, Primers specific for pS2 promoter were used to amplify the genomic DNA associated with WBP-2 in MCF-7 cells. The PCR products were separated on agarose gel and the band intensities were quantified using the Image J (NIH) software. Input band intensity was considered as 100%, and the other bands were scaled accordingly.

View larger version (12K): [in this window] [in a new window]   Fig. 6. WBP-2 Reverses the Transcriptional Interference between PR and ER HeLa cells were transfected with 0.2 µg of PR expression plasmid, 0.3 µg of ER expression plasmid, 1.0 µg of PRE.TATA.LUC, and increasing concentrations (0, 25, 50, 100, 150, 200 ng) of WBP-2. Cells were then treated with progesterone (Prog.) alone or progesterone and estradiol together (each at 10–8 M). The last bar corresponds to control cells transfected with WBP-2, ER and PR expression plasmids but treated with progesterone alone. Data are expressed as mean and SD of three independent transfections. The data are presented as fold activation. The activity in the presence of hormone and in the absence of WBP-2 was defined as 1-fold, and the data for other bars were scaled accordingly.

View larger version (27K): [in this window] [in a new window]   Fig. 7. PY Motif 3 of WBP-2 Is Indispensable for Its Coactivation Function A, Schematic representation of mutated WBP-2 PY motifs. The PY motif 1, 2, and 3 of WBP-2 were mutated by in vitro site-directed mutagenesis. B, The PY motif 3 of WBP-2 is required for its coactivation function. HeLa cells were transfected with PR expression and reporter plasmids in the presence or absence of either wild-type or PY3 mutant WBP-2. Cells were treated with progesterone (10–7 M), and luciferase activity was measured. The data are presented as fold activation. The activity of PR in the presence of hormone and in the absence of WBP-2 was defined as 1-fold, and the data for other bars were scaled accordingly. C, Wild-type and PY3 mutant WBP-2 expressed at an equal level. Protein levels were analyzed by Western blot using anti-WBP-2 antibody. Control lane represents cells that were transfected with empty vector. ß-Actin expression was used as loading control. D, Wild-type and PY3 mutant WBP-2 interact with ER. Interaction of wild-type and PY3 mutant WBP-2 with ER was determined in a GST pull-down assay. ER was labeled with 35S by in vitro transcription and translation kit. The labeled ER protein was then incubated overnight at 4 C with E. coli expressed GST alone (control), GST-WBP-2 (wild type), or GST-PY3 mutant WBP-2 bound to beads either in the absence of hormone (–H) or presence of E2. Bound proteins were analyzed by SDS-PAGE and autoradiography with 10% of in vitro-translated ER as input.

View larger version (8K): [in this window] [in a new window]   Fig. 8. PY Motif 3 of WBP-2 Is Essential for Its Intrinsic Activation Function HeLa cells were transiently transfected with either pBIND (empty), pBIND-WBP-2 (wild type), or pBIND-WBP-2 (PY 3 mutant) and its specific reporter pGS5. pBIND.SRC-1 was used as a positive control. Cells were harvested after 24 h and assayed for luciferase activity, and bars are mean and SD from three different determinations. The data are presented as fold activation. The activity of Gal4 DNA binding domain (control) in the absence of WBP-2 (wild type and mutant) or SRC-1 was defined as 1-fold, and the data for other bars were scaled accordingly.

View larger version (9K): [in this window] [in a new window]   Fig. 9. WBP-2 and E6-AP Additively Enhance PR Transactivation Cells were transiently transfected with PR expression plasmid and progesterone-responsive reporter plasmid (PRE.TATA.LUC) in the absence or presence of WBP-2 or E6-AP or both. Cells were treated with or without progesterone (10–7 M). Data are expressed as mean and SD of three different transfections and plotted as fold activation.

View larger version (8K): [in this window] [in a new window]   Fig. 10. YAP Shows an Absolute Dependence on Wild-Type WBP-2 to Enhance PR Transactivation HeLa cells were transiently transfected with receptor expression plasmid for PR and its specific reporter (PRE.TATA.LUC). Wild-type WBP-2, mutant WBP-2 (PY3), and YAP expression vectors were also coexpressed alone or in combination. Empty vector of the above proteins was transfected as control. Later, cells were treated with progesterone (10–7 M). Cells were harvested after 24 h and assayed for luciferase activity, and bars are mean and SD from three different determinations. The data are presented as fold activation. The activity of receptor in the presence of hormone and in the absence of WBP-2/ mutant WBP-2/ YAP was defined as 1-fold, and the data for other bars were scaled accordingly.

	Discussion

TOP ABSTRACT INTRODUCTION Results Discussion Materials and Methods REFERENCES

 
In this study, we describe the isolation of WBP-2 as an E6-AP interacting protein and its role in transactivation by steroid hormone receptors. Our data demonstrate that WBP-2 interacts with E6-AP as well as with the liganded form of ER and PR both in vitro and in vivo. This result is consistent with previously published reports indicating that coactivators form multiprotein complexes (15, 29) and interact with receptors primarily in the presence of hormone. It has been suggested that most coactivators interact with receptors via the LXXLL motifs contained within the coactivators (31). WBP-2 is distinct from these coactivators, because it does not contain LXXLL motifs. It also has been reported that most of the cloned coactivators exhibit little receptor specificity and are able to coactivate a wide variety of nuclear hormone receptors. Unlike these coactivators, WBP-2 exhibits receptor selectivity and preferentially coactivates the hormone-dependent transcriptional activities of PR and ER, having little effect on the transactivation functions of GR and AR. In addition, as described for several recently characterized coactivators (16), WBP-2 contains an intrinsic transactivation function.

Our ChIP analyses demonstrate the hormone-mediated recruitment of WBP-2 onto an endogenous ER-responsive pS2 promoter. Thus, as is the case for coactivator proteins such as E6-AP, UbcH7, and SRC family members, WBP-2 is also recruited to target promoters by receptors in a hormone-dependent manner. Because WBP-2 binds to both receptor and E6-AP, it is likely that WBP-2 could be recruited to the target promoters in a hormone-dependent manner by its association with receptors and/or E6-AP.

Targeted depletion of coactivators in mice and cell lines has demonstrated that coactivators are required for proper functioning of steroid hormone receptors (32, 33). Our siRNA-mediated depletion experiments suggest that, like other coactivators, WBP-2 is important for the proper functioning of steroid hormone receptors. The existence of modulatory proteins in the nuclear hormone receptor transactivation pathway is supported by the findings that the transcriptional activity of one receptor can be squelched by the overexpression of another receptor, indicating that both receptors compete for a limited pool of common factor. Our results indicate that overexpression of WBP-2 reverses the squelching effect of ER on PR transactivation in a dose-dependent manner and are consistent with previously published studies indicating that authentic coactivators usually can reverse squelching between two receptors (19). Taken together, our data support the observation that WBP-2 is a bona fide coactivator of PR and ER.

The WBP-2 protein contains three PPXY sequences known as the PY motifs. The PY motifs are present in the transcriptional activation domains of several transcription factors, including c-Jun, AP-2, C/EBP, NF-E2, KROX-20, MEF2B, and PEBP2, suggesting that the PY motifs may play vital role in gene transcription. PY motifs in these proteins have been previously shown to mediate protein-protein interactions and they represent potential transactivation domains that could function by recruiting additional strong transactivators to the promoters of target genes. Our data support the hypothesis that the PY motif is involved in gene transcription because mutations in one of the three PY motifs abolish both the intrinsic activation function of the molecule as well as the coactivation function of WBP-2 in ER- and PR-mediated gene transcription.

Initially, WBP-2 was proposed as a ligand for the WW domain of YAP. In this report, we have shown that YAP also acts as a coactivator of steroid hormone receptors. However, the coactivation function of YAP is dependent on the presence of WBP-2 (carboxyl-terminal PY motif of WBP-2). Our data are consistent generally with previously published reports that YAP stimulates gene transcription by binding to the PY motif of ErbB4 protein. In conclusion, the results presented in this study substantiate the role of WBP-2 (contains PY motif) and YAP (contains WW domain) in female steroid hormone receptor function. Based on our data, we postulate that the PY motif of WBP-2 binds to the WW domain of YAP and recruits YAP to the target gene promoter by interacting with receptor and E6-AP. When the receptor-E6-AP-WBP-2-YAP complex is recruited to hormone-responsive promoters, it acts at one of the many substeps required to modulate the transactivation functions of a steroid hormone-responsive target gene.

	Materials and Methods

TOP ABSTRACT INTRODUCTION Results Discussion Materials and Methods REFERENCES

 
Plasmid Construction
The mammalian expression plasmids for progesterone receptor-B (pCR3.1.PR-B), glucocorticoid receptor (pCR3.1.GR), estrogen receptor (pCR3.1.ER), p53, SRC-1 (pBIND.SRC-1), E6-AP (pCR3.1.E6-AP), YAP65 (pCDNA3.1.YAP65), pGEM.E6-AP (3003), and GAL-VP16 have been described previously (1, 16, 22, 34, 35). The progesterone/glucocorticoid/androgen-responsive reporter (PRE-TATA.LUC), estrogen-responsive reporter (ERE-TATA.LUC), p53-responsive reporter (p21 promoter-LUC), and VP-16-responsive reporter (17mer-LUC) plasmids also have been described previously (17, 36, 37, 38).

To reconstitute the ubiquitin-protein ligase defective E6-AP in a yeast two-hybrid plasmid, HindIII-digested (and filled) pGEM E6-AP (C833S) was redigested with BamHI. The resulting BamHI-HindIII (filled) fragment was inserted into the BamHI-EcoRI (filled) sites of pGAD10 (Clontech, Cambridge, UK). To fuse WBP-2 with GST, the BamHI-EcoR1 fragment of full-length WBP-2 was subcloned in-frame with GST into plasmid pGEX4T (Amersham Biosciences, Piscataway, NJ). To fuse WBP-2 in-frame with Gal4 DNA binding domain, the BamHI-EcoRI fragment of WBP-2 was subcloned into the corresponding sites of pBIND (Invitrogen, San Diego, CA) vector and pBK-RSV (Stratagene, La Jolla, CA) vector. The reporter plasmid pGS5 with multiple copies of Gal4 response element was purchased from Invitrogen.

Site-Directed Mutagenesis of the PY Motifs of WBP-2
The GeneEditor in vitro Site-Directed Mutagenesis System from Promega Corporation (Madison, WI) was used to generate WBP-2 PY motif mutants. Mutations within the three poly-proline motifs (PY motifs) were generated in pBlueScript vector. The oligonucleotide primers used in the process are as follows: 5'-GGA ATG TAC CCC TGC GCT GCT GGC GCC CCC TAT CCA CCG CCC-3' coding for AAGA (first PY motif); 5'-TAC GTG CAG CCC CCA GCA GCG CCC GCC CCT GGG CCC ATG GAA-3' coding for AAPA (second PY motif); 5'-AGC CAG CCG CCG CCA GCT GCC TAC GCC CCA CCG GAA GAT AAG coding for AAYA (third PY motif). Mutants were screened and confirmed by sequencing. The cDNAs of PY mutants of WBP-2 were then digested with BamHI-EcoRI and inserted into the corresponding sites of pBK-RSV, pBIND (in-frame with Gal4 DNA binding domain), and pGEX-4T (in-frame with GST).

Yeast Two-Hybrid Screening
The yeast two-hybrid screening assay was performed as described previously (16). A catalytically inactive form of E6-AP (C833S), in which the active site cysteine residue is substituted with serine, was used as bait. The prey cDNA library fused to the Gal4 activation domain was derived from human brain cells (Clontech).

WBP-2 Antibody Generation and Western Blot Analysis
Alpha Diagnostic International (San Antonio, TX) generated the antibody against WBP-2 protein. A unique 17 amino acid (N'-NDMKNVPEAFKGTKKGT-C') peptide sequence was selected within WBP-2 protein based on hydrophilicity, antigenicity, and accessibility scale using various bioinformatics protein-profiling programs. Cross-reactivity of the peptide sequence was checked by basic local alignment search tool (BLAST) analysis and was conserved in WBP-2 proteins across species. This peptide was synthesized in vitro and covalently attached to a carrier protein (KLH) via a cysteine residue added to the amino terminus and was injected into rabbits for polyclonal antibody generation. Antibodies generated were concentrated by affinity purification. Anti-WBP-2 was used at 1:500 dilutions in immunoblotting, and at 1:50 for ChIP assay.

In Vitro Interaction Assay
In vitro expression of radiolabeled E6-AP and ER were performed by in vitro transcription and translation from rabbit reticulocyte extract in the presence of [³⁵S]methionine according to the manufacturer’s recommended conditions (Promega). GST-WBP-2 was expressed in Escherichia coli DH-5 cells and purified on glutathione-Sepharose beads. The purified and glutathione-bound WBP-2 was incubated with in vitro-translated E6-AP and ER in NETN buffer [50 mM NaCl, 1 mM EDTA, 20 mM Tris (pH 8.0), 0.1% Nonidet P-40] overnight at 4 C. After washing four times with NETN buffer, WBP-2-bound E6-AP and ER were eluted and separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels.

Immunoprecipitation Assay
Twenty-four hours after growth, cells were washed in TEN buffer [40 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl] and lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing salt [400 mM NaCl, 1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonylfluoride (10 µl/ml), aprotinin (30 µl/ml), and 100 nM sodium orthovanadate (10 µl/ml)] by pipetting up and down. Thereafter, cell lysates were placed on ice for 30 min. To bring the salt concentration of cell lysates to 150 mM NaCl, 150 µl of NaCl-free RIPA buffer [1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonylfluoride (10 µl/ml), aprotinin (30 µl/ml), and 100 nM sodium orthovanadate (10 µl/ml)] was added to the lysates. After centrifugation at 4 C (21,000 x g), lysates were incubated with 20 µl of protein A-Sepharose and rocked at 4 C for 30 min. After centrifugation, supernatants were transferred to fresh tubes and lysates were mixed either with serum or anti-ER antibody (HC-20; Santa Cruz Biotechnology, Santa Cruz, CA) at 4 C for 2 h on a rocker. Afterward, 20 µl of protein A-Sepharose beads were added, and lysates were incubated for an additional hour at 4 C on a rocker. Finally, after extensive washing with NaCl-free RIPA buffer, immunoprecipitates were subjected to SDS-PAGE and analyzed by Western blotting using either an anti-ER, anti-E6-AP, or anti-WBP-2 antibody.

Transient Transfection
HeLa and MCF-7 cells were maintained in DMEM containing 10% fetal bovine serum. T47D and LNCaP cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. A total of 3 x 10⁵ cells was plated 24 h before transfection in six-well plates containing 5% dextran-coated charcoal-stripped serum. Cells were transfected with the indicated amount of DNA using FuGene 6 transfection reagent (Roche Diagnostics, Indianapolis, IN). After 4 h, cells were treated with the indicated hormones and harvested 24 h later. Luciferase assays were performed using Promega’s luciferase assay system.

Design, Construction, and Transfection of siRNA against WBP-2
The siRNA target finder program from Ambion (Austin, TX) was used to design siRNA against WBP-2. The sequences used are as follows from the amino terminus: Seq#1(AS) 5'-AACGTGCCAGAAGCCTTCAAACCTGTCTC-3', Seq#1(S) 5'-AATTTGAAGGCTTCTTCTGGCACGCCTGTCTC-3'. Ambion siRNA construction kit was used to construct and purify scrambled and WBP-2-specific siRNA molecules for transfection assays. JetSI transfection reagent (Qbiogene/MP Biochemicals, Carlsbad, CA) was used for transfection in HeLa and MCF-7 cells followed by luciferase reporter assay (described previously) and real-time PCR analysis (described below), respectively. The GAPDH (control) siRNA was purchased from Ambion.

Real-Time PCR Analysis
The real-time PCR analysis was carried out according to previous studies in MCF-7 cells. RNA was isolated using the TRIzol (Invitrogen) reagent as per the supplier’s protocol. cDNA synthesis (iScript cDNA Synthesis kit) and real-time PCR analysis (iQ SYBR Green Supermix) were performed using the protocol provided with the products (Bio-Rad, Hercules, CA). The DNA was quantified by real-time quantitative PCR using pS2 promoter-specific primers (forward, 5'-GCGCCCTGGTCCTGGTGTCCAT-3'; reverse, 5'-GAAACCACAATTCTGTCTTT CAC-3'). The results were normalized to PCR product amplified with a pair of GAPDH-specific primers (forward, 5'-GAAGGTGAAGGTCGGAGTC-3'; reverse, 5'-GAAGATGGTGATGGGATTTC-3'). Real-time PCR were performed using the iCycler iQ multicolor real-time PCR detection system (Bio-Rad). To avoid variations from different samples, the relative pS2 mRNA levels were normalized against GAPDH mRNA content of the same sample.

ChIP
The ChIP analysis was performed as described previously using MCF cells. The DNA was purified using QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted in 50 µl of H₂O. Total input samples were eluted in 100 µl of H₂O and diluted 1:10 before PCR analysis. Each PCR contains 6 µl of immunoprecipitate or input, 0.5 µM of each primer, 0.4 mM dNTP mixture, 1x Titanium Taq PCR buffer (Clontech), and 1x Titanium TaqDNA polymerase (Clontech) in a total volume of 25 µl. The primers for the pS2 promoter were as follows: forward, 5'-GGCCATCTCTCACTATGAATCACTTCTGC-3', and reverse, 5'-GGCAGGCTCTGTTTGCTTAAAGAGCG-3'. PCR was performed for 29 cycles with 1 min of denaturing at 94 C, annealing at 62 C, and extension at 68 C.

	ACKNOWLEDGMENTS

 
We thank Drs. Fred Pereira and David Lonard for critical reading of the manuscript. We also extend our gratitude to Dr. Marius Sudol (Department of Medicine, Mount Sinai Medical Center, New York, NY) for providing us with the cDNA for full-length WBP-2 and full-length YAP protein.

	FOOTNOTES

 
This work was supported by National Institutes of Health Grants DK56833, DK060907, and funds from the Braman Family Breast Cancer Institute (to Z.N.) and NIH-HD (to B.W.O.).

The authors have nothing to disclose.

First Published Online June 13, 2006

Abbreviations: AR, Androgen receptor; ChIP, chromatin immunoprecipitation; E2, estradiol; E6-AP, E6-associated protein; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, glucocorticoid receptor; GST, glutathione-S-transferase; PR, progesterone receptor; RIPA, radioimmunoprecipitation assay; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; SRC, steroid receptor coactivator; WBP-2, WW domain binding protein-2; YAP, Yes kinase-associated protein.

Received for publication December 27, 2005. Accepted for publication June 1, 2006.

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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  GR  |  PR  |  AR

Coregulators:   E6AP

Ligands:   Dexamethasone  |  17β-Estradiol  |  Progesterone  |  R1881

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