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Department of Women’s Health and Bone Research (F.B., C.M., B.S.K., B.J.C.), Wyeth Research, Collegeville, Pennsylvania 19426; Department of Molecular and Integrative Physiology (B.K.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801-3704
Address all correspondence and requests for reprints to: Dr. Boris J. Cheskis, Department of Women’s Health and Bone Research, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426. E-mail: cheskib{at}wyeth.com.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ER) and a newly identified scaffold protein, called MNAR (modulator of nongenomic activity of ER). Under basal condition, Src enzymatic activity is inhibited by intramolecular interactions. The enzyme can be activated by interaction between the SH2 domain of Src and phosphotyrosine-containing sequences and/or by interaction between the SH3 domain of Src and proteins containing PXXP motifs. Mutational analysis and functional evaluation of MNAR and the use of ER and cSrc mutants revealed that MNAR interacts with Src’s SH3 domain via its N-terminal PXXP motif. Mutation of this motif abolished both the MNAR-induced activation of Src and the stimulation of ER transcriptional activity. ER interacts with Src’s SH2 domain using phosphotyrosine 537, and this complex was further stabilized by MNAR-ER interaction. Mapping studies reveal that both the A/B domain and Y537 of ER are required for MNAR-induced activation of ER transcriptional activity. The region responsible for MNAR interaction with ER maps to two N-terminal LXXLL motifs of MNAR. Mutation of these motifs prevented ER-MNAR complex formation and eliminated activation of the Src/MAPK pathway. These data explicate how the coordinate interactions between MNAR, ER, and Src lead to Src activation. Our findings also demonstrate that MNAR is a scaffold protein that mediates ER-Src interaction and plays an important role in the integration of ER action in Src-mediated signaling. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ß (ER and -ß) are usually described as ligand-inducible transcription factors that control expression of target genes involved in regulation of metabolism, development, and reproduction (1, 2). However, there is increasing evidence that not all the biological effects of estrogens are mediated by direct receptor control of target gene expression. Some actions of estrogens appear to be attributed to estrogenic regulation of cell-signaling cascades. Estrogens affect intracellular calcium mobilization (3) and stimulate adenylate cyclase activity and cAMP production (4, 5). In the ovary they activate G protein-coupled receptors and stimulate production of inositol phosphate (4). In endothelial and breast cancer cells they activate the phosphatidylinositol 3-kinase pathway (6, 7, 8, 9). In vascular endothelial (10), neuroblastoma (11), mammary carcinoma (12), and bone cell lines (13, 14) they stimulate the MAPK signaling pathway. Other steroid hormones can also affect cell signaling (for review see Refs.15 and 16).
These and other rapid effects suggest that estrogens and other steroids can interact with receptors that are localized in close proximity to the plasma membrane. The nature of these receptors remains to be elucidated, although some studies have suggested the existence of ERs unrelated to conventional ER and -ß (17, 18). However, cloning or isolation and confirmation of bona fide novel membrane ER have not been accomplished. Others have demonstrated that a subpopulation of the classical ER is associated with the cell membrane and is responsible for some manifestations of estrogenic signaling action (12, 19, 20).
The molecular mechanism of ER integration into cellular signaling is not well understood. However, a physical association of ER with IGF receptor, cSrc, phosphatidylinositol 3-kinase, and caveolin-1 has been reported (19, 21, 22, 23). Multiple evidence suggests that activation of the tyrosine kinase, cSrc, represents one of the initial steps in ER-mediated cell signaling (24). The essential role of Src kinase in the nongenomic action of steroid receptors was demonstrated in experiments with embryonic fibroblasts derived from Src–/– mice. These cells did not show rapid activation of the MAPK pathway in response to androgen receptor and ER activation, whereas wild-type Src+/+ cells did (13).
The Src kinases share common structural organization differing in the amino-terminal 60–80 amino acids (aa) (25). There are several functional motifs common to all Src family members. The amino-terminal region, Src homology 4 domain (SH4), contains consensus sequences for myristoylation and palmethylation (26). The SH3 domain binds polyproline motifs (27), and the SH2 domain binds to phosphotyrosine-containing sequences (26). The carboxyl-terminal SH1 domain contains the catalytic region and a short regulatory domain with major regulatory tyrosine Y527 (25). Under basal conditions, the catalytic domain of Src is constrained in an inactive state through intramolecular interactions. Binding of the SH2 domain to the C-terminal phosphorylated tyrosine and the SH3 domain to the proline-rich region in the Src linker domain locks the molecule in an inhibited conformation (28). Full catalytic activation requires release of these constraints. The kinase activity of Src can be enhanced by binding of phosphotyrosine-containing sequences to the SH2 domain and binding of proline-rich sequences to the SH3 domain (29). Activation of Src kinase is known to influence many pathways, including the MAPK pathway.
Activation of the Src/Ras/Erk kinase pathway has been shown to promote ER-mediated transcription (30, 31, 32, 33). ER contains two transcriptional activation domains: activation function (AF)-1 and AF-2. Ligand binding controls the AF-2 activity, whereas phosphorylation provides the important mechanism that regulates the AF-1 functions. Phosphorylation of serines 104, 106, 118, and 167 regulates AF-1 activity (34, 35, 36, 37, 38, 39), and their mutation results in reduced transactivation by ER (35, 36). Activation of Erk 1/2 kinases in vitro and in vivo leads to ER phosphorylation on serine 118 (S118). Moreover, MAPK activation enables ligand-independent activation of ER (30, 32, 40). An interesting explanation for the ability of the AF-1 domain to synergize with AF-2 action was proposed by the demonstration that AF-1 and AF-2 domains can interact with separate surfaces of the same coactivator molecule (41). Therefore, activation of Src can stimulate ER activity through activation of the Raf/MAPK kinase/ERK signaling cascade resulting in phosphorylation of S118.
Using affinity purification, we have recently isolated a new scaffold protein, termed MNAR (modulator of nongenomic activity of ER) that promotes ligand-dependent interaction between the ERs and members of the Src family of tyrosine kinases. We have shown that this interaction leads to stimulation of cSrc enzymatic activity and activation of the MAPK pathway activation of (Erk1 and Erk 2 kinases) (42).
In this study, we examined the molecular mechanism of ER-MNAR-Src interaction leading to Src activation. Our data indicate that coordinate binding of MNAR and ER to Src’s SH3 and SH2 domains, respectively, stabilized by ER-MNAR interaction through MNAR’s LXXLL motifs, leads to activation of cSrc and Src-mediated signaling.
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RESULTS |
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). We have designated these motifs nos. 1–10 starting from the most N-terminal motif. Similar motifs in other transcription factors have been shown to interact with a hydrophobic groove on the surface of the ligand-binding domain of nuclear hormone receptors (43) and could potentially be used for MNAR interaction with ER. Due to the presence of these motifs and their putative function, we termed the N-terminal region of MNAR the nuclear receptor interaction domain (NRID). The NRID also contains three PXXP motifs. These motifs may interact with Src homology domain 3 (SH3) present in multiple signal-transducing molecules (44). An interesting feature of the MNAR molecule is an extended proline and glutamic acid-rich domain localized in the C-terminal part of the MNAR molecule, which imparts a strong negative charge to the MNAR molecule, bringing the predicted pI to 4.30 (Fig. 1). We found no homology in gene databases for this domain.
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, wild-type MNAR and MNAR truncation mutants (encoded by aa 1–120, 1–189, 1–278, 1–469, 1–1130, 80–594, and 595-1130) were expressed, 35S radiolabeled by a coupled transcription /translation reaction, and incubated with purified glutathione-S-transferase (GST)-ER ligand-binding domain. MNAR-ER complexes were isolated using glutathione-sepharose and separated by 10% SDS-PAGE (Fig. 2A). Full-length MNAR and MNAR truncation mutants encoded by aa 1–189, 1–278, 1–469, and 80–594 all interacted with ER ligand dependently. However, the MNAR mutants encoded by aa 1–120 and 595-1130 were both unable to interact with ER. Sequence analysis revealed that the common element between all MNAR mutants interacting with ER includes LXXLL 4 and 5, suggesting that the MNAR-ER interaction is mediated by one or the combination of these two motifs.
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), biotinylated peptides corresponding to each of the MNAR LXXLL motifs were synthesized and immobilized on a neutravidin-coated microplate via biotin-neutravidin interaction. Flag-tagged ER was incubated with 17ß-estradiol (E2, 1 µM) or vehicle and allowed to interact with the immobilized peptides. ER binding was detected using anti-Flag antibodies fused to horseradish peroxidase (HRP). ER interacted in a ligand-dependent manner with peptides corresponding to LXXLL motifs 4 and 5 (L4 and L5), respectively (Fig. 2C). These results are in agreement with data obtained using the GST pull-down assay (Fig. 2A) and suggest that MNAR binding to ER is potentially mediated by LXXLL 4 and 5 or a combination of these motifs.
We have previously shown that MNAR overexpression in the presence of E2, through ER-MNAR-Src interaction and activation of Src and MAPK pathway (Erk 1 and 2), augments ER transcriptional activity and ER-mediated gene expression (42). We used a transient cotransfection assay to determine whether leucine to alanine mutations of MNAR LXXLL motifs 4, 5, or 4 and 5 would affect MNAR-induced activation of ER transcriptional activity (Fig. 2D). To address this question, HEPG2 cells were cotransfected with a 2XERE-tk-luc-reporter gene, ER expression vector, and a plasmid for expression of wild-type or mutated MNAR. Cells were untreated, or treated with E2 at 10 nM or a combination of E2 and PP2 (Src kinase inhibitor at 10 µM). Consistent with our previous data (42), MNAR overexpression in cells treated with E2 stimulated ER transcriptional activity, which was abolished by cotreatment of cells with PP2. Mutation of the leucine residues to alanine in either LXXLL motif 4 or 5 did not affect MNAR-mediated stimulation of ER activity. However, when leucine residues in both LXXLL motifs 4 and 5 were mutated to alanines, MNAR was unable to stimulate ER transcriptional activity (Fig. 2D). These results, together with the binding studies (Fig. 2, A–C), indicate LXXLL motifs 4 and 5 are interchangeable and can both mediate ER-MNAR interaction.
N-Terminal Portion of MNAR Is Necessary and Sufficient for Stimulation of ER Transcriptional Activity
To determine the regions of the MNAR molecule necessary and sufficient for activation of ER, we have generated a library of MNAR truncation mutants (Fig. 3A) that were evaluated for their ability to stimulate ER-mediated transcription. HepG2 cells were transfected with expression plasmids for ER and various MNAR mutants together with a 2xERE-tk-Luc reporter plasmid.
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transcriptional activity in HEPG2 cells treated with 10 nM E2 (Fig. 3B). MNAR1–189 was the smallest fragment that retained an ability to stimulate ER activity. This portion of the MNAR molecule contains LXXLL motifs 1–6. In contrast, MNAR1–120 that does not contain LXXLL motifs 4, 5, or 6 was silent in the cotransfection assay. These data are in agreement with our previous results indicating that MNAR’s LXXLL motifs 4 and 5 are required for MNAR-induced stimulation of ER activity (Fig. 2, A–D). However, MNAR80–594 and MNAR80–574 that contain LXXLL motifs 3–10 or 3–9, respectively, were both inactive in this assay. These data indicate that whereas the region of MNAR that comprises LXXLL motifs 4 and 5 is required, it is not sufficient for MNAR-induced activation of ER, and that other structural elements localized in the very N-terminal portion of the MNAR molecule encoded by aa 1–80 are also essential for ER activation. MNAR1–80 contains two PXXP motifs (PXXP nos. 1 and 2) that could potentially mediate MNAR interaction with the Src SH3 domain.
MNAR Src Interaction Analysis. PXXP motif number 1 is required for MNAR Stimulation of ER Activity
To evaluate this hypothesis, we used a GST pull-down approach. Src SH3 domain was expressed in a bacterial system as a GST-fusion protein and purified using glutathione agarose. Beads (10 µl) with immobilized Src-SH3 domains were incubated with transcribed/translated, 35S-radiolabeled full-length MNAR. The formed complex was isolated using glutathione agarose. Our data indicate that MNAR interacts with the SH3 domain (Fig. 4A). To evaluate which of MNAR’s N-terminal PXXP motifs is required for MNAR-Src interaction and activation of ER transcriptional activity, we used a reporter gene assay. HepG2 cells were transfected with expression plasmids for ER alone or in combination with full-length MNAR, MNAR1–469, or MNAR1–469, in which both prolines in the first, second, or both first and second PXXP motifs were mutated to alanines. Luciferase activity was evaluated in cells treated with E2 at 10 nM for 24 h. Mutation of the prolines to alanines in the first, or both the first and second PXXP motifs, abolished the MNAR stimulation of ER transcriptional activity, whereas the mutation of the second PXXP motif alone had no effect (Fig. 4B). These data indicate that PXXP motif number 1 is required for ER activation and suggest that this motif is used for the interaction of MNAR with the SH3 domain of Src.
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). The region of MNAR, common between these mutants, is encoded by aa 888–962 (Fig. 4C) and contains tyrosine 920 (Y920), which when phosphorylated could potentially serve as an interaction site for the SH2 domain. However, a 14-mer peptide corresponding to this region of MNAR with phosphorylated Y920 was unable to block the SH2-MNAR binding, suggesting that Y920 is not the site of MNAR interaction (data not shown). The portion of the MNAR molecule encoded by aa 888–962 contains multiple glutamic acid residues; hence this interaction could potentially be mediated by a strong negative charge confined to this portion of MNAR. The functional consequence of this interaction is presently unclear. MNAR truncation mutants lacking the SH2 interaction site (MNAR1–469, 1–278, 1–189, 80–594, 80–574), in the presence of ER and E2, were able to activate the Src/MAPK cascade and stimulate ER transcriptional activity. However, MNAR interaction with both Src SH2 and SH3 domains could potentially explain why in vitro, in the absence of ER-E2, MNAR was able to stimulate cSrc activity. Notably, Src activation by MNAR in vitro was strongly augmented by addition of ER-E2 (42).
Y537 Mediates ER Interaction with SH2 Domain of cSrc
It has been previously shown that ER can interact with Src’s SH2 domain in a ligand-dependent manner (20). We next asked which ER tyrosines are required for this interaction. To address this question, full-length ER was expressed and 35S radiolabeled in a coupled transcription-translation reaction and incubated with GST-SH3, GST-SH2, or GST-SH32 of cSrc. In accordance with previously published data (20), ER interacted with SH2 in a ligand-dependent manner but did not interact with the SH3 domain of Src. 14 mer peptides were synthesized that correspond to the regions of the ER molecule that contain tyrosine residues. These peptides were used in a pull-down/competition assay (all peptides at 30 µM) with 35S-radiolabeled full-length transcribed/translated ER and the GST-SH2 (Fig. 5B). ER binding to the SH2 domain was abolished by the phosphorylated peptide corresponding to the portion of the ER molecule encoded by aa 530–544 that contained a Y537 residue. Interestingly, control peptide that was not phosphorylated on Y537 had no effect on the ER-SH2 interaction. These results demonstrate that the ER interaction with Src’s SH2 domain is mediated by phosphorylated Y537 and suggests that ER-Y537 phosphorylation status is important for the action of Src.
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Activity Requires the Intact AB Domain and Tyrosine 537 of ER transcriptional activity (Ref.42 ; see also Figs. 2D, 3, and 4C). To define which portions of the ER molecule are required for MNAR-induced ER activation, HepG2 cells were transfected with MNAR and wild-type ER, or one of the ER mutants (Fig. 6).
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, MNAR was unable to activate the ER mutant lacking the AB domain (encoded by aa 263–595), or ER in which serines 104, 106, and 118 were mutated to alanines. These data suggest that MNAR-induced ER activation is mediated by ER phosphorylation. It has been previously established that activation of the Src/MAPK pathway by epidermal growth factor leads to ER phosphorylation on S118, thereby enhancing activity of the receptor (30, 32). Treatment of cells with E2 also leads to ER phosphorylation at S118 (35, 36). These data, therefore, are in agreement with our previous results that MNAR mediates E2-induced activation of Src/MAPK pathway and activation of ER transcriptional activity. Mutation of the 537 tyrosine to alanine also abrogated ER activation by MNAR. These results substantiate our findings that phosphorylated tyrosine 537 is required for ER interaction with Src in the MNAR-ER-Src complex (Fig. 6). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and controls ER-induced Src activation. We have shown that in cells treated with E2, MNAR overexpression activates the Src/MAPK cascade and augments ER transcriptional activity. Inhibitors of Src and MAPK kinase kinases blocked MNAR-induced activation of ER (42). In this study, we have used this assay as readout for MNAR-ER-induced Src activation. cSrc can be activated either by dephosphorylation of the C-terminal inhibitory phosphotyrosine site (or in oncogenic variants by loss of the C-terminal tail) or by binding of high-affinity ligands to the SH2 or SH3 domains. The consequent unraveling of the autoinhibited structure results in activation of the kinase domain, potentially aided by decoupling of the SH3 and SH2 domains, and binding of the these domains to cellular proteins, thereby targeting the kinase domain to its substrates (45). SH2 and SH3 domains are modular polypeptide units that mediate protein-protein interactions and are found together on many proteins, suggesting that their activities can be coordinated and that they can cooperate in Src regulation (27).
MNAR contains three perfect PXXP motifs localized in the N-terminal portion of the molecule and an extended proline-rich region at the C-terminal end that could potentially mediate MNAR interaction with the Src SH3 domain. A GST-SH3 pull-down assay demonstrated that in vitro transcribed and translated full-length MNAR specifically interacted with Src’s SH3 domain (Figs. 4A and 7). The N-terminal portion of the MNAR molecule, encoded by aa 1–189, which contains two PXXP motifs, was essential for MNAR-induced Src activation. We therefore generated MNAR mutants in which the prolines in the first, second, or both PXXP motifs were substituted to alanines and evaluated them in a cotransfection assay with ER. Mutation of the first PXXP motif abrogated ER activation, whereas the second PXXP motif was dispensable. These data suggested that PXXP motif 1 is used for MNAR interaction with Src’s SH3 domain (Figs. 4B and 7). MNAR also interacted with Src’s SH2 domain. Using a pull-down experiment with GST-SH2 domain, we mapped this interaction to the C-terminal region of MNAR encoded by aa 887–962 (Fig. 4C). This region of MNAR contains only one tyrosine (Y920), which when phosphorylated, could potentially serve as an interaction site for the Src SH2 domain. However, the functionality of this interaction is unknown, because deletion of the C-terminal portion of MNAR did not affect its ability to activate the Src/MAPK cascade and stimulate ER transcriptional activity (Fig. 3B). In vitro analysis with purified Src and MNAR demonstrated that MNAR itself was able to potentiate Src enzymatic activity; this stimulation was strongly augmented in the presence of ER-E2 (42). Potentially, MNAR binding to Src’s SH2 domains could contribute to Src activation. Evaluation of its role in regulation of Src activity is an important goal for future studies.
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using LXXLL motifs 4 and/or 5 (Figs. 2 and 7). LXXLL motifs are present in multiple transcriptional factors, including members of the SRC/p160 family of coactivators (46, 47, 48, 49, 50, 51, 52), p300/cAMP response element binding protein (CREB)-binding protein (53, 54), and the thyroid hormone receptor-associated protein/vitamin D receptor-interacting protein/mediator complex (55, 56, 57, 58). These domains are sufficient for ligand-dependent interaction with nuclear hormone receptors (59, 60). The number of LXXLL motifs in nuclear receptor-interacting proteins varies. SRC/p160 family members have three LXXLL motifs (61) and receptor-interacting protein 140 contains nine of these motifs (62). MNAR contains 10 LXXLL motifs, including two doublets. The presence of all of these motifs may indicate that MNAR can interact with multiple nuclear hormone receptors. Indeed, our data indicate that, in addition to ER and -ß, androgen receptor, glucocorticoid receptor, and progesterone receptor also interact with MNAR in a ligand-dependent manner (42). Peptides corresponding to LXXLL motifs 4 and 5 were both able to interact with ER. Substitution of leucines to alanines in only one of the LXXLL motifs did not affect Src activation by MNAR and ER (Fig. 2D). However, mutation of both LXXLL motifs abrogated Src activation. These data indicate that motifs 4 and 5 are interchangeable for MNAR-ER interaction.
Binding to MNAR could potentially bring ER in proximity with Src. It has been previously shown that ER interacts with Src in a ligand-dependent manner (20). Our data indicate that phosphotyrosine 537Y of ER is required for this interaction, because phosphorylated peptide corresponding to this region of ER blocked ER binding to Src’s SH2 domain (Figs. 5 and 7). Substitution of this residue by several different amino acids has been shown to result in ER activation and coactivator recruitment in the absence of ligand, but not in the presence of tamoxifen or ICI 182,780. Therefore, it has been postulated that this tyrosine at the N terminus of a conserved helix, which forms a major part of the ligand-dependent activation function in the ER, is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone (63, 64). MNAR overexpression did not affect transcriptional activity of the ER mutant in which 537Y was substituted to phenylalanine, an amino acid with a hydrophobic side chain similar in size and structure to tyrosine but which lacks a hydroxyl group and cannot be modified by phosphorylation (Fig. 6). This mutant of ER is responsive to E2, and its transcriptional activity is similar to that of the wild-type receptor (63, 64).
Similarly, MNAR overexpression had no effect on transcriptional activity of ER in which Ser 104, 106, and 118 were substituted with alanines (Fig. 6), or a ER mutant in which the A/B domain was deleted. Serine 118 can be phosphorylated by the MAPKs Erk 1 and Erk 2 (30) in response to growth factor treatment or overexpression/activation of Src (33), resulting in ligand-independent activation of ER and stimulation of ligand-induced ER activity (32). These data substantiate our previous findings that MNAR-induced ER activation is mediated by stimulation of the Src/MAPK pathway and presumably by ER phosphorylation.
In summary, this study has demonstrated that coordinate interaction between Src’s SH3 and SH2 domains and MNAR and ER correspondingly, stabilized by ER-MNAR interaction, leads to Src activation. These data provide additional information and support to our previous hypothesis that MNAR is a new scaffolding protein that incorporates ER action into the Src-mediated cell signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cloning, Protein Expression, and Purification
A series of constructs encoding MNAR C-terminal truncation mutants fused to N-terminal flag peptide were constructed by designing oligonucleotides to amplify appropriate fragments of MNAR coding region from cDNA template and subcloning into pcDNA3.1 expression vectors through restriction enzyme sites. Appropriate clones were confirmed by sequencing and tested in reporter assays as described. Src SH2 (aa 151–253) and SH3 (aa 81–150) domains were cloned into pGEX-5X-3 vector and expressed in bacteria and purified using glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ). The ER mutants have been previously described (36, 64, 65, 66, 67, 68).
Interaction Analysis Using GST Pull Down
Wild-type MNAR and MNAR truncation mutants (encoded by aa 1–189, 1–278, 1–469, 80–594, 595-1130, and 1–1130), and full-length ER were transcribed/translated and 35S radiolabeled using TNT Quick Coupled Transcription /Translation System (Promega, Madison, WI). For interaction analysis the indicated GST fusion protein bound to glutathione-Sepharose 4B and indicated transcribed and translated protein were incubated for 3 h at 4 C in binding buffer (50 mM Tris-HCl, pH. 8; 150 mM NaCl; 10% glycerol, 0.05% Nonidet P-40; 1 mM phenylmethylsulfonylfluoride; 1 mM dithiothreitol) in the absence or presence of ligand at the indicated concentration. For competition experiments, the peptides were used at a final concentration of 50 µM. Beads were washed four times with binding buffer, and bound proteins were eluted by addition of SDS buffer and analyzed by SDS-PAGE and autoradiography.
Cell Culture and Transfection
HEPG-2 human liver hepatocellular carcinoma cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum from Atlanta Biologicals, Inc. (Norcross, GA). Cells were grown in a humidified atmosphere of 95% O2-5% CO2 at 37 C; 75,000 cells per well were plated in 96-well plates and transfected in DMEM without L-glutamine or phenol red supplemented with 1% charcoal-stripped serum. A 2xERE-tk-luciferase reporter (100 ng), 1 ng pcDNA3.1 ER, 0.2 ng, 1 ng, or 5 ng of pcDNA 3.1 MNAR expression vectors, and 10 ng pCMV b-galactosidase (Stratagene, La Jolla, CA) as an internal control were introduced into cells using Lipofectamine 2000 following manufacturer’s instructions (Life Technologies, Inc., Gaithersburg, MD). Sixteen hours after transfection, cells were incubated for 24 h with the indicated treatments. Cells were harvested and ß-Gal and luciferase activities were evaluated according to manufacturer’s instructions.
ELISA-Based Interaction Analysis
We used a rapid, nonisotopic ELISA-type method for characterization of receptor-coactivator interactions. Biotinylated peptides corresponding to different MNAR’s LXXLL motifs (designated 1–9, sequentially, from the N-terminal most motif) were synthesized and immobilized on a Reacti-Bind NeutrAvidin-coated microplate (Pierce Biotechnology, Rockford, IL). The micoplate was washed twice with binding buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1 mM dithiothreitol; 1 mM EDTA; 0.01% Nonidet P-40; and 0.01% BSA). Peptides were diluted in 100 µl binding buffer to a final concentration of 50 µM, incubated with the Reacti-Bind NeutrAvidin-coated microplate for 1 h at room temperature, and washed four times with the binding buffer. Flag-tagged ER, preincubated with vehicle or 1 µM E2 for 1 h at room temperature, was allowed to interact with the immobilized peptide corresponding to one of the MNAR’s LXXLL motif for 2 h at room temperature. The plate was washed four times with binding buffer and incubated with anti-Flag antibodies fused to HRP (Sigma) for 1 h and washed four more times. SuperSignal ELISA Pico Chemiluminescent Substrate (Pierce Biotechnology) was used to detect antigen-antibody complex, and signal was read using a Wallac Victor2 1420 Multilabel Counter (Perkin-Elmer Lifesciences, Boston, MA).
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FOOTNOTES |
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Present address for C.-W.W.: Metabolex, Inc., 3876 Bay Center Place, Hayward, California 94545.
Abbreviations: aa, Amino acids; AF, activation function; E2, 17ß-estradiol; ER, estrogen receptor; GST, glutathione-S-transferase; HRP, horseradish peroxidase; MNAR, modulator of nongenomic activity of ER; NRID, nuclear receptor interaction domain; S118, serine 118; SH2, Src homology 2.
Received for publication September 2, 2003. Accepted for publication February 2, 2004.
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REFERENCES |
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 L. Li, K. Hisamoto, K. H. Kim, M. P. Haynes, P. M. Bauer, A. Sanjay, M. Collinge, R. Baron, W. C. Sessa, and J. R. Bender Variant estrogen receptor c-Src molecular interdependence and c-Src structural requirements for endothelial NO synthase activation PNAS, October 16, 2007; 104(42): 16468 - 16473. [Abstract] [Full Text] [PDF]
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 W. K. Sumanasekera, G. U. Sumanasekera, K. A. Mattingly, S. M. Dougherty, R. S. Keynton, and C. M. Klinge Estradiol and dihydrotestosterone regulate endothelial cell barrier function after hypergravity-induced alterations in MAPK activity Am J Physiol Cell Physiol, August 1, 2007; 293(2): C566 - C573. [Abstract] [Full Text] [PDF]
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C. M. Silva and M. A. Shupnik Integration of Steroid and Growth Factor Pathways in Breast Cancer: Focus on Signal Transducers and Activators of Transcription and Their Potential Role in Resistance Mol. Endocrinol., July 1, 2007; 21(7): 1499 - 1512. [Abstract] [Full Text] [PDF]
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R. Rajhans, S. Nair, A. H. Holden, R. Kumar, R. R. Tekmal, and R. K. Vadlamudi Oncogenic Potential of the Nuclear Receptor Coregulator Proline-, Glutamic Acid-, Leucine-Rich Protein 1/Modulator of the Nongenomic Actions of the Estrogen Receptor Cancer Res., June 1, 2007; 67(11): 5505 - 5512. [Abstract] [Full Text] [PDF]
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 A. A. Kazi and R. D. Koos Estrogen-Induced Activation of Hypoxia-Inducible Factor-1{alpha}, Vascular Endothelial Growth Factor Expression, and Edema in the Uterus Are Mediated by the Phosphatidylinositol 3-Kinase/Akt Pathway Endocrinology, May 1, 2007; 148(5): 2363 - 2374. [Abstract] [Full Text] [PDF]
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 P. Galluzzo, F. Caiazza, S. Moreno, and M. Marino Role of ER{beta} palmitoylation in the inhibition of human colon cancer cell proliferation Endocr. Relat. Cancer, March 1, 2007; 14(1): 153 - 167. [Abstract] [Full Text] [PDF]
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 J. G. Greger, N. Fursov, N. Cooch, S. McLarney, L. P. Freedman, D. P. Edwards, and B. J. Cheskis Phosphorylation of MNAR Promotes Estrogen Activation of Phosphatidylinositol 3-Kinase Mol. Cell. Biol., March 1, 2007; 27(5): 1904 - 1913. [Abstract] [Full Text] [PDF]
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N. Vasudevan and D. W. Pfaff Membrane-Initiated Actions of Estrogens in Neuroendocrinology: Emerging Principles Endocr. Rev., February 1, 2007; 28(1): 1 - 19. [Abstract] [Full Text] [PDF]
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K. Moriarty, K. H. Kim, and J. R. Bender Estrogen Receptor-Mediated Rapid Signaling Endocrinology, December 1, 2006; 147(12): 5557 - 5563. [Abstract] [Full Text] [PDF]
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 M. H. Herynk, A. R. Beyer, Y. Cui, H. Weiss, E. Anderson, T. P. Green, and S. A.W. Fuqua Cooperative action of tamoxifen and c-Src inhibition in preventing the growth of estrogen receptor-positive human breast cancer cells Mol. Cancer Ther., December 1, 2006; 5(12): 3023 - 3031. [Abstract] [Full Text] [PDF]
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R. X-D Song, P. Fan, W. Yue, Y. Chen, and R. J Santen Role of receptor complexes in the extranuclear actions of estrogen receptor {alpha} in breast cancer Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S3 - S13. [Abstract] [Full Text] [PDF]
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S. Kraus, D. Gioeli, T. Vomastek, V. Gordon, and M. J. Weber Receptor for Activated C Kinase 1 (RACK1) and Src Regulate the Tyrosine Phosphorylation and Function of the Androgen Receptor. Cancer Res., November 15, 2006; 66(22): 11047 - 11054. [Abstract] [Full Text] [PDF]
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 K. Ohshiro, S. K. Rayala, M. D. Williams, R. Kumar, and A. K. El-Naggar Biological Role of Estrogen Receptor {beta} in Salivary Gland Adenocarcinoma Cells. Clin. Cancer Res., October 15, 2006; 12(20): 5994 - 5999. [Abstract] [Full Text] [PDF]
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 R. X.-D. Song and R. J. Santen Membrane Initiated Estrogen Signaling in Breast Cancer Biol Reprod, July 1, 2006; 75(1): 9 - 16. [Abstract] [Full Text] [PDF]
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A. E. Gururaj, S. K. Rayala, R. K. Vadlamudi, and R. Kumar Novel Mechanisms of Resistance to Endocrine Therapy: Genomic and Nongenomic Considerations Clin. Cancer Res., February 1, 2006; 12(3): 1001s - 1007s. [Abstract] [Full Text] [PDF]
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M. M. Khan, M. Hadman, C. Wakade, L. M. De Sevilla, K. M. Dhandapani, V. B. Mahesh, R. K. Vadlamudi, and D. W. Brann Cloning, Expression, and Localization of MNAR/PELP1 in Rodent Brain: Colocalization in Estrogen Receptor-{alpha}- But Not in Gonadotropin-Releasing Hormone-Positive Neurons Endocrinology, December 1, 2005; 146(12): 5215 - 5227. [Abstract] [Full Text] [PDF]
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R. K. Vadlamudi, B. Manavathi, S. Balasenthil, S. S. Nair, Z. Yang, A. A. Sahin, and R. Kumar Functional Implications of Altered Subcellular Localization of PELP1 in Breast Cancer Cells Cancer Res., September 1, 2005; 65(17): 7724 - 7732. [Abstract] [Full Text] [PDF]
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D. Haas, S. N. White, L. B. Lutz, M. Rasar, and S. R. Hammes The Modulator of Nongenomic Actions of the Estrogen Receptor (MNAR) Regulates Transcription-Independent Androgen Receptor-Mediated Signaling: Evidence that MNAR Participates in G Protein-Regulated Meiosis in Xenopus laevis Oocytes Mol. Endocrinol., August 1, 2005; 19(8): 2035 - 2046. [Abstract] [Full Text] [PDF]
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B. Manavathi, S. S. Nair, R.-A. Wang, R. Kumar, and R. K. Vadlamudi Proline-, Glutamic Acid-, and Leucine-Rich Protein-1 Is Essential in Growth Factor Regulation of Signal Transducers and Activators of Transcription 3 Activation Cancer Res., July 1, 2005; 65(13): 5571 - 5577. [Abstract] [Full Text] [PDF]
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 K. H. Kim and J. R. Bender Rapid, Estrogen Receptor-Mediated Signaling: Why Is the Endothelium So Special? Sci. Signal., June 14, 2005; 2005(288): pe28 - pe28. [Abstract] [Full Text] [PDF]
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D. Rai, A. Frolova, J. Frasor, A. E. Carpenter, and B. S. Katzenellenbogen Distinctive Actions of Membrane-Targeted Versus Nuclear Localized Estrogen Receptors in Breast Cancer Cells Mol. Endocrinol., June 1, 2005; 19(6): 1606 - 1617. [Abstract] [Full Text] [PDF]
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C. M. Klinge, K. A. Blankenship, K. E. Risinger, S. Bhatnagar, E. L. Noisin, W. K. Sumanasekera, L. Zhao, D. M. Brey, and R. S. Keynton Resveratrol and Estradiol Rapidly Activate MAPK Signaling through Estrogen Receptors {alpha} and {beta} in Endothelial Cells J. Biol. Chem., March 4, 2005; 280(9): 7460 - 7468. [Abstract] [Full Text] [PDF]
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K. L. Chambliss, L. Simon, I. S. Yuhanna, C. Mineo, and P. W. Shaul Dissecting the Basis of Nongenomic Activation of Endothelial Nitric Oxide Synthase by Estradiol: Role of ER{alpha} Domains with Known Nuclear Functions Mol. Endocrinol., February 1, 2005; 19(2): 277 - 289. [Abstract] [Full Text] [PDF]
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 R. K. Vadlamudi, S. Balasenthil, R. R. Broaddus, J.-A. Gustafsson, and R. Kumar Deregulation of Estrogen Receptor Coactivator Proline-, Glutamic Acid-, and Leucine-Rich Protein-1/Modulator of Nongenomic Activity of Estrogen Receptor in Human Endometrial Tumors J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6130 - 6138. [Abstract] [Full Text] [PDF]
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 J. C. Fleet Rapid, Membrane-Initiated Actions of 1,25 Dihydroxyvitamin D: What Are They and What Do They Mean? J. Nutr., December 1, 2004; 134(12): 3215 - 3218. [Abstract] [Full Text] [PDF]
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S. S. Nair, S. K. Mishra, Z. Yang, S. Balasenthil, R. Kumar, and R. K. Vadlamudi Potential Role of a Novel Transcriptional Coactivator PELP1 in Histone H1 Displacement in Cancer Cells Cancer Res., September 15, 2004; 64(18): 6416 - 6423. [Abstract] [Full Text] [PDF]
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