Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1

Abstract

The estrogen receptor (ER) is an important regulator of growth and differentiation of breast epithelium. Transactivation by ER depends on a leucine-rich motif, which constitutes a ligand-regulated binding site for steroid receptor coactivators (SRCs). Cyclin D1 is frequently amplified in breast cancer and can activate ER through direct binding. We show here that cyclin D1 also interacts in a ligand-independent fashion with coactivators of the SRC-1 family through a motif that resembles the leucine-rich coactivator binding motif of nuclear receptors. By acting as a bridging factor between ER and SRCs, cyclin D1 can recruit SRC-family coactivators to ER in the absence of ligand. A cyclin D1 mutant that binds to ER but fails to recruit coactivators preferentially interferes with ER activation in breast cancer cells that have high levels of cyclin D1. These data support that cyclin D1 contributes significantly to ER activation in breast cancers in which the protein is overexpressed. Our present results reveal a novel route of coactivator recruitment to ER and establish a direct role for cyclin D1 in regulation of transcription.

Figure 1

Mapping of the region of cyclin D1 required for ER-mediated transactivation. (A) The effect of cyclin D1 deletion mutants on ER activation in the presence of ligand. The cyclin D1 derivatives used in this study are shown at left; (right) the relative capacity of the mutants to potentiate ERE-dependent transcription. ER-negative Cos-7 cells were transfected with expression vectors for wild-type ER (200 ng), cyclin D1, or cyclin D1 mutants (2.5 μg), an internal control β-galactosidase plasmid (0.5 μg), and an ERE–TATA–luciferase reporter (3 μg). The effect on ER transactivation of wild-type cyclin D1 was set to 100%. These studies were performed in three separate experiments and expressed as mean values with s.d. < 10 % (data not shown). The alignment of leucine-rich motif of ER with D-type cyclins is shown on the bottom; the leucine-rich motif in cyclin D1 is indicated as a solid box. (B) ER activation by D-type cyclins, cyclin D1 L254/255A point mutant (D1–LALA), and SRC-1 in the presence of ligand. Cos-7 cells were transfected with D-type cyclin expression vectors, cyclin D1 leucine-to-alanine point mutant (D1–LALA), or SRC-1 expression vector together with wild-type ER expression vector, an ERE–TATA–luciferase reporter plasmid, and the internal control β-galactosidase construct. Data are expressed as relative luciferase activity compared with basal ERE–luciferase activity of wild-type ER and are normalized for transfection efficiencies.

Figure 1

Mapping of the region of cyclin D1 required for ER-mediated transactivation. (A) The effect of cyclin D1 deletion mutants on ER activation in the presence of ligand. The cyclin D1 derivatives used in this study are shown at left; (right) the relative capacity of the mutants to potentiate ERE-dependent transcription. ER-negative Cos-7 cells were transfected with expression vectors for wild-type ER (200 ng), cyclin D1, or cyclin D1 mutants (2.5 μg), an internal control β-galactosidase plasmid (0.5 μg), and an ERE–TATA–luciferase reporter (3 μg). The effect on ER transactivation of wild-type cyclin D1 was set to 100%. These studies were performed in three separate experiments and expressed as mean values with s.d. < 10 % (data not shown). The alignment of leucine-rich motif of ER with D-type cyclins is shown on the bottom; the leucine-rich motif in cyclin D1 is indicated as a solid box. (B) ER activation by D-type cyclins, cyclin D1 L254/255A point mutant (D1–LALA), and SRC-1 in the presence of ligand. Cos-7 cells were transfected with D-type cyclin expression vectors, cyclin D1 leucine-to-alanine point mutant (D1–LALA), or SRC-1 expression vector together with wild-type ER expression vector, an ERE–TATA–luciferase reporter plasmid, and the internal control β-galactosidase construct. Data are expressed as relative luciferase activity compared with basal ERE–luciferase activity of wild-type ER and are normalized for transfection efficiencies.

Figure 2

Effect of cyclin D1 on helix 12 mutants of ER. The effect of cyclin D1 on ER mutants was tested in Cos-7 cells (A,B) and in U2-OS cells (C,D) in the presence of ligand. An ERE–TATA–luciferase reporter construct was used in transient transfections together with cyclin D1 and ER 1–535 (A,C) or ER L543/544A mutants (B,D). β-Galactosidase served as an internal control. The reporter activity was determined both in the presence (solid bars) and in the absence (open bars) of 10 nm 17β-estradiol. The relative luciferase activity was calculated by normalizing to the β-galactosidase activity. The relative reporter activity of wild-type ER in the presence of ligand was used as a reference and set at 100%. In the absence of transfected ER plasmid, cyclin D1 did not induce transcriptional activity of the reporter (data not shown). At least five separate transfection experiments were performed (expressed as average ±s.d.).

Figure 2

Effect of cyclin D1 on helix 12 mutants of ER. The effect of cyclin D1 on ER mutants was tested in Cos-7 cells (A,B) and in U2-OS cells (C,D) in the presence of ligand. An ERE–TATA–luciferase reporter construct was used in transient transfections together with cyclin D1 and ER 1–535 (A,C) or ER L543/544A mutants (B,D). β-Galactosidase served as an internal control. The reporter activity was determined both in the presence (solid bars) and in the absence (open bars) of 10 nm 17β-estradiol. The relative luciferase activity was calculated by normalizing to the β-galactosidase activity. The relative reporter activity of wild-type ER in the presence of ligand was used as a reference and set at 100%. In the absence of transfected ER plasmid, cyclin D1 did not induce transcriptional activity of the reporter (data not shown). At least five separate transfection experiments were performed (expressed as average ±s.d.).

Figure 2

Effect of cyclin D1 on helix 12 mutants of ER. The effect of cyclin D1 on ER mutants was tested in Cos-7 cells (A,B) and in U2-OS cells (C,D) in the presence of ligand. An ERE–TATA–luciferase reporter construct was used in transient transfections together with cyclin D1 and ER 1–535 (A,C) or ER L543/544A mutants (B,D). β-Galactosidase served as an internal control. The reporter activity was determined both in the presence (solid bars) and in the absence (open bars) of 10 nm 17β-estradiol. The relative luciferase activity was calculated by normalizing to the β-galactosidase activity. The relative reporter activity of wild-type ER in the presence of ligand was used as a reference and set at 100%. In the absence of transfected ER plasmid, cyclin D1 did not induce transcriptional activity of the reporter (data not shown). At least five separate transfection experiments were performed (expressed as average ±s.d.).

Figure 2

Effect of cyclin D1 on helix 12 mutants of ER. The effect of cyclin D1 on ER mutants was tested in Cos-7 cells (A,B) and in U2-OS cells (C,D) in the presence of ligand. An ERE–TATA–luciferase reporter construct was used in transient transfections together with cyclin D1 and ER 1–535 (A,C) or ER L543/544A mutants (B,D). β-Galactosidase served as an internal control. The reporter activity was determined both in the presence (solid bars) and in the absence (open bars) of 10 nm 17β-estradiol. The relative luciferase activity was calculated by normalizing to the β-galactosidase activity. The relative reporter activity of wild-type ER in the presence of ligand was used as a reference and set at 100%. In the absence of transfected ER plasmid, cyclin D1 did not induce transcriptional activity of the reporter (data not shown). At least five separate transfection experiments were performed (expressed as average ±s.d.).

Figure 3

Role of coactivators in cyclin D1-induced transactivation. (A) Effect of SRC1–DN on ER transactivation. SRC1–DN encoding amino acids 1245–1441 of SRC-1 (1, 2.5, and 5 μg) was introduced by transient transfection, together with wild-type ER (200 ng), and tested for its ability to modulate ERE-dependent transcription. (B) Effect of SRC1–DN on SRC-1- and TIF-2-mediated ER transactivation. SRC-1 (3 μg) or TIF-2 (3 μg) were transfected with SRC1–DN (3 μg) and tested for ER transactivation. (C) Effect of SRC1–DN on cyclin D1-induced transactivation of ER 1–535 mutant. Cyclin D1 was cotransfected with SRC1–DN (1, 2.5, 5 μg) and tested for its effect on an ER harboring a deletion of coactivator-binding site in helix 12 (ER 1–535). (D) Effect of SRC1–DN on cyclin D1-induced transactivation of ER L543/544A mutant. Cyclin D1 and SRC1–DN (1, 2.5, 5 μg) were transfected and tested for the activity of ER helix 12 point mutant (ER L543/544A). The transient transfections (A–C) were performed in Cos-7 cells, which were maintained in DMEM with 10 nm ligand. The relative activity was calculated by normalizing to the internal control and was divided by luciferase activity of ER (mutant) in the presence of ligand. The transfections for each set of conditions were done in at least four independent experiments and expressed as average ±s.d.

Figure 4

Direct interaction between cyclin D1 and SRCs. (A) In vivo binding of cyclin D1 to SRCs. HA-tagged SRC-1, AIB-1, or p300 cDNA expression vectors were introduced into Cos-7 cells together with control plasmid (−), plasmids directing the synthesis of wild-type cyclin D1, or D1 L254/255A mutant (D1–LALA), as indicated. Lanes 1 and 2 contain total lysate of cells (5% of amount used in IP) transfected with cyclin D1 and cyclin D1–LALA, respectively. 12CA5 HA antibodies were used for immunoprecipitation of whole-cell extracts prepared from these cells and coimmunoprecipitation of cyclin D1 (mutants) was examined by Western blot analysis using anti-cyclin D1 antibody. Note that wild-type cyclin D1, but not the leucine-to-alanine mutant D1–LALA, coimmunoprecipitates with SRC-1 and AIB-1, whereas binding of both wild-type cyclin D1 and D1–LALA mutant to p300 was hardly detectable. (B) Binding of cyclin D1 and cyclin D1 L254/255A mutant to ER. Cos-7 cells were transfected with ER expression vector, cyclin D1 (mutant), and/or control plasmids. Monoclonal ER antibodies were used for immunoprecipitation of ER of whole-cell extracts prepared from these cells and coimmunoprecipitation of cyclin D1 (mutant) was examined by Western blot analysis using monoclonal cyclin D1 antibodies. (C) Activity of cyclin D1 and D1 L254/255A mutant in phosphorylation of pRb in Rb^−/− 3T3 cells. Cells were transfected with pRb expression vector, cyclin D1 (mutant), and/or control plasmids and were maintained in low serum conditions. After 40 hr, cells were lysed and proteins were separated by low-percentage polyacrylamide gel electrophoresis. Differentially phosphorylated species of pRb were detected by Western blotting using the polyclonal pRb antibody (C15, Santa Cruz).

Figure 4

Direct interaction between cyclin D1 and SRCs. (A) In vivo binding of cyclin D1 to SRCs. HA-tagged SRC-1, AIB-1, or p300 cDNA expression vectors were introduced into Cos-7 cells together with control plasmid (−), plasmids directing the synthesis of wild-type cyclin D1, or D1 L254/255A mutant (D1–LALA), as indicated. Lanes 1 and 2 contain total lysate of cells (5% of amount used in IP) transfected with cyclin D1 and cyclin D1–LALA, respectively. 12CA5 HA antibodies were used for immunoprecipitation of whole-cell extracts prepared from these cells and coimmunoprecipitation of cyclin D1 (mutants) was examined by Western blot analysis using anti-cyclin D1 antibody. Note that wild-type cyclin D1, but not the leucine-to-alanine mutant D1–LALA, coimmunoprecipitates with SRC-1 and AIB-1, whereas binding of both wild-type cyclin D1 and D1–LALA mutant to p300 was hardly detectable. (B) Binding of cyclin D1 and cyclin D1 L254/255A mutant to ER. Cos-7 cells were transfected with ER expression vector, cyclin D1 (mutant), and/or control plasmids. Monoclonal ER antibodies were used for immunoprecipitation of ER of whole-cell extracts prepared from these cells and coimmunoprecipitation of cyclin D1 (mutant) was examined by Western blot analysis using monoclonal cyclin D1 antibodies. (C) Activity of cyclin D1 and D1 L254/255A mutant in phosphorylation of pRb in Rb^−/− 3T3 cells. Cells were transfected with pRb expression vector, cyclin D1 (mutant), and/or control plasmids and were maintained in low serum conditions. After 40 hr, cells were lysed and proteins were separated by low-percentage polyacrylamide gel electrophoresis. Differentially phosphorylated species of pRb were detected by Western blotting using the polyclonal pRb antibody (C15, Santa Cruz).

Figure 4

Direct interaction between cyclin D1 and SRCs. (A) In vivo binding of cyclin D1 to SRCs. HA-tagged SRC-1, AIB-1, or p300 cDNA expression vectors were introduced into Cos-7 cells together with control plasmid (−), plasmids directing the synthesis of wild-type cyclin D1, or D1 L254/255A mutant (D1–LALA), as indicated. Lanes 1 and 2 contain total lysate of cells (5% of amount used in IP) transfected with cyclin D1 and cyclin D1–LALA, respectively. 12CA5 HA antibodies were used for immunoprecipitation of whole-cell extracts prepared from these cells and coimmunoprecipitation of cyclin D1 (mutants) was examined by Western blot analysis using anti-cyclin D1 antibody. Note that wild-type cyclin D1, but not the leucine-to-alanine mutant D1–LALA, coimmunoprecipitates with SRC-1 and AIB-1, whereas binding of both wild-type cyclin D1 and D1–LALA mutant to p300 was hardly detectable. (B) Binding of cyclin D1 and cyclin D1 L254/255A mutant to ER. Cos-7 cells were transfected with ER expression vector, cyclin D1 (mutant), and/or control plasmids. Monoclonal ER antibodies were used for immunoprecipitation of ER of whole-cell extracts prepared from these cells and coimmunoprecipitation of cyclin D1 (mutant) was examined by Western blot analysis using monoclonal cyclin D1 antibodies. (C) Activity of cyclin D1 and D1 L254/255A mutant in phosphorylation of pRb in Rb^−/− 3T3 cells. Cells were transfected with pRb expression vector, cyclin D1 (mutant), and/or control plasmids and were maintained in low serum conditions. After 40 hr, cells were lysed and proteins were separated by low-percentage polyacrylamide gel electrophoresis. Differentially phosphorylated species of pRb were detected by Western blotting using the polyclonal pRb antibody (C15, Santa Cruz).

Figure 5

Cyclin D1 interacts directly with SRC-1. (A) In vitro interaction between SRC-1 and cyclin D1. A series of GST fusion proteins containing SRC-1 (GST–RC1 361–441; GST–RC1 361-782; GST-SRC1 361-568) or GST–p300 (1–95) were tested for direct binding to His-tagged cyclin D1 (His–D1); the GST–SRC1 derivatives are shown at bottom. The conserved LxxLL motifs are boxed and the amino acid boundaries are demonstrated. In the in vitro binding assay, a series of GST-containing proteins were incubated with bacterially expressed His-tagged cyclin D1 and immobilized on glutathione–agarose. Cyclin D1 binding was detected by Western blot analysis using anti-cyclin D1 antibody. (B) Competition of cyclin D1–SRC1 binding by SRC1 peptides. Peptides (0.3 and 3 μg) derived from the four LxxLL motifs of SRC1 were used in a GST pull-down assay using GST–SRC1(361–1441) and His-tagged cyclin D1 as described in A. The sequence and the position of the peptides in SRC-1 are shown at bottom. (C). Competition of P2 and P3 in cyclin D1–SRC-1 and ER–SRC-1 interaction. Peptides P2 and P3 (0.1, 0.2, 0.4, or 0.8 μg) and control peptide P4M (0.4 and 0.8 μg) were tested for their ability to compete the binding between GST–SRC1 and cyclin D1 (top) or GST–SRC1 and ER (bottom). Input of cyclin D1 and ER proteins shown represents 20% of the amount of protein used in the binding assay.

Figure 5

Cyclin D1 interacts directly with SRC-1. (A) In vitro interaction between SRC-1 and cyclin D1. A series of GST fusion proteins containing SRC-1 (GST–RC1 361–441; GST–RC1 361-782; GST-SRC1 361-568) or GST–p300 (1–95) were tested for direct binding to His-tagged cyclin D1 (His–D1); the GST–SRC1 derivatives are shown at bottom. The conserved LxxLL motifs are boxed and the amino acid boundaries are demonstrated. In the in vitro binding assay, a series of GST-containing proteins were incubated with bacterially expressed His-tagged cyclin D1 and immobilized on glutathione–agarose. Cyclin D1 binding was detected by Western blot analysis using anti-cyclin D1 antibody. (B) Competition of cyclin D1–SRC1 binding by SRC1 peptides. Peptides (0.3 and 3 μg) derived from the four LxxLL motifs of SRC1 were used in a GST pull-down assay using GST–SRC1(361–1441) and His-tagged cyclin D1 as described in A. The sequence and the position of the peptides in SRC-1 are shown at bottom. (C). Competition of P2 and P3 in cyclin D1–SRC-1 and ER–SRC-1 interaction. Peptides P2 and P3 (0.1, 0.2, 0.4, or 0.8 μg) and control peptide P4M (0.4 and 0.8 μg) were tested for their ability to compete the binding between GST–SRC1 and cyclin D1 (top) or GST–SRC1 and ER (bottom). Input of cyclin D1 and ER proteins shown represents 20% of the amount of protein used in the binding assay.

Figure 5

Cyclin D1 interacts directly with SRC-1. (A) In vitro interaction between SRC-1 and cyclin D1. A series of GST fusion proteins containing SRC-1 (GST–RC1 361–441; GST–RC1 361-782; GST-SRC1 361-568) or GST–p300 (1–95) were tested for direct binding to His-tagged cyclin D1 (His–D1); the GST–SRC1 derivatives are shown at bottom. The conserved LxxLL motifs are boxed and the amino acid boundaries are demonstrated. In the in vitro binding assay, a series of GST-containing proteins were incubated with bacterially expressed His-tagged cyclin D1 and immobilized on glutathione–agarose. Cyclin D1 binding was detected by Western blot analysis using anti-cyclin D1 antibody. (B) Competition of cyclin D1–SRC1 binding by SRC1 peptides. Peptides (0.3 and 3 μg) derived from the four LxxLL motifs of SRC1 were used in a GST pull-down assay using GST–SRC1(361–1441) and His-tagged cyclin D1 as described in A. The sequence and the position of the peptides in SRC-1 are shown at bottom. (C). Competition of P2 and P3 in cyclin D1–SRC-1 and ER–SRC-1 interaction. Peptides P2 and P3 (0.1, 0.2, 0.4, or 0.8 μg) and control peptide P4M (0.4 and 0.8 μg) were tested for their ability to compete the binding between GST–SRC1 and cyclin D1 (top) or GST–SRC1 and ER (bottom). Input of cyclin D1 and ER proteins shown represents 20% of the amount of protein used in the binding assay.

Figure 6

Cyclin D1 mediates ligand-independent recruitment of SRC-1 to ER. (A) Ligand-independent in vitro binding of SRC-1, ER, and cyclin D1. The purified proteins GST–SRC1, His-tagged cyclin D1, and baculovirus-produced ER were tested for in vitro binding in a GST pull-down assay. GST protein served as negative control. Cyclin D1 and ER were incubated with GST–SRC1 in the presence or absence of 1 μm 17β-estradiol and binding was detected by Western blot analysis using anti-cyclin D1 and anti-ER monoclonal antibodies. Lane 1 represents 10% of the input for cyclin D1 and 20% of input of ER proteins. (B) Cyclin D1 and SRC-1 can interact with DNA-bound ER. Oligonucleotide-containing ER binding sequence was biotin 5′-end labeled and bound to paramagnetic particles coated with streptavidin. Purified GST–SRC1, baculovirus-produced ER, and His-tagged cyclin D1 proteins were tested for DNA binding using these ERE-containing beads and analyzed by Western blotting using antibodies directed against GST, ER, and cyclin D1, respectively.

Figure 6

Cyclin D1 mediates ligand-independent recruitment of SRC-1 to ER. (A) Ligand-independent in vitro binding of SRC-1, ER, and cyclin D1. The purified proteins GST–SRC1, His-tagged cyclin D1, and baculovirus-produced ER were tested for in vitro binding in a GST pull-down assay. GST protein served as negative control. Cyclin D1 and ER were incubated with GST–SRC1 in the presence or absence of 1 μm 17β-estradiol and binding was detected by Western blot analysis using anti-cyclin D1 and anti-ER monoclonal antibodies. Lane 1 represents 10% of the input for cyclin D1 and 20% of input of ER proteins. (B) Cyclin D1 and SRC-1 can interact with DNA-bound ER. Oligonucleotide-containing ER binding sequence was biotin 5′-end labeled and bound to paramagnetic particles coated with streptavidin. Purified GST–SRC1, baculovirus-produced ER, and His-tagged cyclin D1 proteins were tested for DNA binding using these ERE-containing beads and analyzed by Western blotting using antibodies directed against GST, ER, and cyclin D1, respectively.

Figure 7

Role of cyclin D1 in ER transactivation in breast cancer cells. (A) Dominant-negative activity of cyclin D1‘LALA’. The ER-containing T47D and MCF-7 breast cancer cells were maintained in 17β-estradiol-enriched medium with 10% fetal calf serum after cotransfection with cyclin D1 (1.5 μg) in the presence and absence of the cyclin D1 L254/255A mutant (cyclin D1–LALA, 1.5 μg), together with an ERE-reporter gene for testing its effect on ER activation. ER transactivation in the absence of coexpression of cyclin D1 was set at 100%. (B). Cyclin D1–LALA inhibits ER activation preferentially in cyclin D1-overexpressing breast cancer cells. T47D and MCF-7 breast cancer cells were maintained in 17β-estradiol-enriched medium with 10% fetal calf serum and transfected with increasing amounts of cyclin D1–LALA expression vector. ER activity was measured by cotransfection with the ERE-reporter plasmid. Transactivation in the absence of co-expression of the cyclin D1–LALA was set at 100%. (Right) The expression levels of endogenous cyclin D1 in both breast tumor cell lines compared with α-tubulin, which served as an internal control.

Figure 7

Role of cyclin D1 in ER transactivation in breast cancer cells. (A) Dominant-negative activity of cyclin D1‘LALA’. The ER-containing T47D and MCF-7 breast cancer cells were maintained in 17β-estradiol-enriched medium with 10% fetal calf serum after cotransfection with cyclin D1 (1.5 μg) in the presence and absence of the cyclin D1 L254/255A mutant (cyclin D1–LALA, 1.5 μg), together with an ERE-reporter gene for testing its effect on ER activation. ER transactivation in the absence of coexpression of cyclin D1 was set at 100%. (B). Cyclin D1–LALA inhibits ER activation preferentially in cyclin D1-overexpressing breast cancer cells. T47D and MCF-7 breast cancer cells were maintained in 17β-estradiol-enriched medium with 10% fetal calf serum and transfected with increasing amounts of cyclin D1–LALA expression vector. ER activity was measured by cotransfection with the ERE-reporter plasmid. Transactivation in the absence of co-expression of the cyclin D1–LALA was set at 100%. (Right) The expression levels of endogenous cyclin D1 in both breast tumor cell lines compared with α-tubulin, which served as an internal control.

Figure 8

Model for cyclin D1-mediated ER transactivation. In the absence of ligand, ER is unable to interact with SRCs directly as its leucine-rich coactivator interaction motif is sterically unavailable for interaction. Ligand-independent binding of cyclin D1 to ER provides a single leucine-rich interaction motif for SRCs which is present in the carboxyl terminus of cyclin D1. This results in partial activation of ER (left). Subsequent ligand binding of ER induces a conformational change in ER that also exposes the leucine-rich motif in AF-2 of ER for SRC interaction, allowing higher affinity binding of SRCs to the liganded D1/ER complex (right). The observed synergism between estradiol and cyclin D1 in ER activation results from their cooperative recruitment of SRCs to the D1/ER complex. The protein interaction motifs are shown in italics.