Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor alpha - PubMed (original) (raw)

Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor alpha

Jun Yan et al. Nucleic Acids Res. 2007.

Abstract

In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ERalpha) in an agonist-dependent manner. The interaction is specific for ERalpha as ERbeta and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ERalpha was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ERalpha is sufficient for interaction with RAP80. RAP80 overexpression reduces ERalpha polyubiquitination, increases the level of ERalpha protein, and enhances ERalpha-mediated transactivation. Knockdown of endogenous RAP80 expression by small-interfering RNA (siRNA) reduced ERalpha protein level and the E2-dependent induction of pS2. In this study, we also demonstrate that RAP80 contains two functional ubiquitin-interaction motifs (UIMs) that are able to bind ubiquitin and to direct monoubiquitination of RAP80. Deletion of these UIMs does not affect the ability of RAP80 to interact with ERalpha, but eliminates the effects of RAP80 on ERalpha polyubiquitination, the level of ERalpha protein, and ERalpha-mediated transcription. These data indicate that the UIMs in RAP80 are critical for the function of RAP80. Our study identifies ERalpha as a new RAP80-interacting protein and suggests that RAP80 may be an important modulator of ERalpha activity.

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Figures

Figure 1.

RAP80 interacts selectively with ERα. The interaction of RAP80 with different nuclear receptors was analyzed by yeast two-hybrid analysis as described in Materials and Methods. RAP80 was used as bait and several full-length (FL) nuclear receptors or their LBD were used as prey. (A) The interaction of RAP80 with different nuclear receptors was analyzed in the presence (+) or absence (−) of corresponding agonist. The following agonists were used: 1,25-dihydroxyvitamin D3 (100 nM) for VDR; dihydrotestosterone (100 nM) for AR; T0901317 (1 µM) for LXRα; GW845 (100 nM) for PPARγ; 17β-estradiol (100 nM) for ERα(FL) and ERα(LBD); the RXR-panagonist SR11217 (1 µM); retinoic acid (1 µM) for RARγ. (B) Interaction of ERα(FL) and ERβ(FL) with RAP80 as a function of the estradiol concentration.

Figure 2.

Analysis of the interaction between RAP80 and ERα by mammalian two-hybrid analysis. (A) CHO cells were co-transfected with (UAS)5-Luc reporter, pM–RAP80, increasing amounts of pVP16–ERα and pcDNA3.1–RAP80 as indicated. Sixteen hours later cells were treated with 100 nM E2 or vehicle. Cells were assayed for reporter activity 24 h after the addition of E2. The relative Luc activity was calculated and plotted. (B) Agonists but not antagonists induce interaction between RAP80 and ERα. CHO cells were co-transfected with pM–RAP80ΔN130, pVP16–ERα and pS5–CAT reporter. Cells were treated with different agonists or antagonists (1 µM) as indicated. Cells were assayed for the reporter activity 24 h after transfection. Ligands used: E2, 17β-estradiol; Tam, tamoxifen; ICI, ICI 182,780; E3, estriol; ZZ-dienestrol; DES, diethylstilbestrol.

Figure 3.

Analysis of the interaction between RAP80 and ERα by co-immunoprecipitation and GST pull-down assays. (A) Co-immunoprecipitation analysis. HeLa cells were transfected with pcDNA3.1–ERα–Myc-His and pLXIN–3×FLAG–RAP80 expression plasmids (1 μg each) and treated with 100 nM E2 or ethanol as indicated. After 24 h, cell lysates were prepared and FLAG–RAP80 protein complexes were isolated using anti-FLAG M2 agarose affinity resin. Proteins in the total cellular lysates and immunoprecipitated (IP) proteins were examined by western blot analysis with anti-FLAG M2 and anti-ERα antibodies. Western blot analysis of IP proteins is shown in the left panel and that of 10% of cell lysates in the right panel. (B) Interaction between endogenous RAP80 and ERα. MCF-7 cells were grown in phenol-red-free medium with 10% charcoal-stripped serum for 2 days and subsequently treated with or without 100 nM E2 or 1 μM ICI 182,780. After 3 h incubation, cells were collected and nuclear lysates prepared. RAP80 protein complexes were immunoprecipitated with an anti-RAP80 antibody and examined by western blot analysis with anti-RAP80 and anti-ERα antibodies. Lower panel shows input ERα. (C) MCF-7 cells were grown and treated with 100 nM E2 as described under (B) before cell lysates were prepared. One part of the lysates was analyzed by western blot analysis using an anti-RAP80 antibody (input RAP80). The remaining lysates were subjected to immunoprecipitation with an anti-ERα antibody or control rabbit IgG (Santa Cruz). The immunoprecipitated ERα protein complexes were then examined by western analysis with anti-RAP80 and anti-ERα antibodies. (D) GST pull-down assay. GST and GST–RAP80Δ110 fusion protein were bound to glutathione-Sepharose 4B beads and then incubated with [35S]-methionine radiolabeled ERα in the presence or absence of 1 μM E2. After 1 h incubation, beads were washed extensively and bound proteins solubilized. Radiolabeled proteins were analyzed by PAGE and visualized by autoradiography. Lane 1: 20% input of radiolabeled ERα. (E) GST pull-down assays were carried out as under (C) using three ERα deletion mutants, ERαΔN248, ERαΔN180 and ERαΔC248.

Figure 4.

Effect of C-terminal deletions on the interaction of RAP80 with ERα. (A) Schematic of RAP80 deletion mutants. UIM and ZF indicate the two ubiquitin-interacting and zinc fingerlike motifs, respectively. (B) HeLa cells were co-transfected with pcDNA3.1–ERα–Myc-His and various pLXIN–3×FLAG–RAP80 plasmids as shown in (A). Cells were treated with E2 (0.1 μM) for 24 h before cell lysates were prepared and FLAG–RAP80 protein complexes isolated using anti-FLAG M2 agarose affinity resin. Proteins in the cellular lysates and immunoprecipitated (IP) proteins were examined by western blot analysis with anti-FLAG M2 and anti-ERα antibodies.

Figure 5.

Effect of RAP80 on ERα-mediated transcriptional activation. RAP80 increased transcriptional activation by endogeneous ERα. MCF-7 cells were transfected with different amounts of (ERE)3-Luc, pLXIN–3×FLAG-RAP80 or pLXIN–3×FLAG–RAP80ΔC204 as indicated. Cells were treated with 100 nM E2 or ethanol for 24 h. Cells were then collected and assayed for luciferase activity. RAP80 protein levels were examined by western blot analysis with anti-FLAG M2 antibody (lower panel).

Figure 6.

RAP80 expression increases the level of ERα protein. (A) HeLa cells were transfected with 2 μg pERα and 1 μg pLXIN–3×FLAG–RAP80 plasmid DNA. The cells were treated with 100 nM E2 or ethanol for 24 h before cell lysates were prepared and FLAG–RAP80 protein complexes isolated with anti-FLAG M2 affinity resin. The isolated complexes were examined by western blot analysis using anti-FLAG M2 and anti-ERα antibodies. About 5% of the cell lysates were used for direct Western blot analysis. (B) Effect of RAP80 knockdown by RAP80 siRNA on the expression of ERα protein and pS2 induction in MCF-7 cells. MCF-7 cells transfected with RAP80 or scrambled siRNAs, were grown in phenol-red-free medium with 10% charcoal-stripped serum for 2 days, and subsequently treated for 24 h with or without 100 nM E2. Cell lysates were prepared and examined by western blot analysis with antibodies against ERα, pS2, RAP80, and actin.

Figure 7.

RAP80 contains two functional ubiquitin-interacting motifs (UIMs). (A) Sequence comparison of the UIM1 and UIM2 of RAP80 with those of epsin, hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), the proteasome subunit PSMD4, the ubiquitin-specific peptidase 25 (USP25) and the consensus UIM sequence (Φ is a hydrophobic residue, e is a negatively charged residue and x is any amino acid). (B) UIMRAP80 is able to bind ubiquitin. GST (lane 1) or a GST–UIMRAP80 fusion protein (lane 2) was bound to glutathione-Sepharose 4B beads and then incubated with 500 ng of purified Ub2-7. After 1 h incubation, beads were washed extensively and bound proteins solubilized. Bound proteins were examined by western blot analysis with anti-Ub antibody (upper panel). The input for GST and GST–UIMRAP80 was also shown (lower panel). (C) UIMRAP80 promotes monoubiquitination of EGFP–UIMRAP80. pEGFP or pEGFP–UIMRAP80 was transfected in HeLa cells with or without pCMV–HA–Ub. Forty-eight hours later, the EGFP proteins were isolated with anti-GFP antibody. The proteins were separated with SDS-PAGE and blotted with anti-HA (upper panel) and anti-GFP (lower panel) antibodies respectively. The IgGH and a non-specific band (NS) are indicated.

Figure 8.

Role of UIMRAP80 in RAP80 ubiquitination. (A) HEK293 cells were transfected with pLXIN–3×FLAG–RAP80, pCMV–HA–Ub as indicated. After 48 h incubation, cells were treated with or without 25 µM of MG132 for 4 h before cell lysates were prepared. Ubiquitinated proteins were isolated with anti-HA antibody and examined by western blot analysis with anti-FLAG antibody. The input RAP80 is shown in the lower panel. ‘ns’ indicates nonspecific pull-down. (B) UIMRAP80 promotes the ubiquitination of the amino terminus of RAP80. HeLa cells were transfected with pLXIN–3×FLAG–RAP80ΔC78 or pLXIN–3×FLAG–RAP80Δ122 with or without pCMV–HA–Ub. Forty-eight hours later, FLAG–RAP80 was isolated with FLAG M2 resin and examined by western blot analysis with anti-HA and anti-FLAG antibodies. Non-specific staining of IgG is indicated on the right. (C) HEK293 cells were transfected with wild type or mutant pLXIN–3×FLAG–RAP80ΔC122 and pCMV–HA–Ub as indicated. Cells were treated and processed as described under A.

Figure 9.

Role of UIMRAP80 on the interaction of RAP80 with ERα. (A) UIM is required for the RAP80-induced increase in the level of ERα protein. HeLa cells were transfected with pLXIN–3×FLAG–RAP80, pERα and pCMV–HA–Ub. Forty-eight hours after transfection, cells were collected and protein cell lysates examined by western blot analysis using anti-ERα, anti-FLAG M2 and anti-actin antibodies. (B) Effect of RAP80 on ERα polyubiquitination. HeLa cells were transfected with pcDNA3–ERα–Myc-His, wild type pLXIN–3×FLAG–RAP80 or FLAG–RAP80ΔUIM1,2 and pCMV–HA–Ub for 48 h and treated with or without E2 for 24 h. The cells were treated with MG132 for 4 h before collection and ERα proteins immunoprecipitated with an anti-ERα antibody. Western blot was performed with an anti-HA or anti-ERα antibody to detect ERα ubiquitination and the level of immunoprecipitated ERα, respectively. The level of FLAG–RAP80 expression was determined with anti-FLAG M2 antibody (lower panel). (C) UIM is required for the RAP80-induced increase in ERα-mediated transactivation. MCF-7 cells were transfected with different amounts of pLXIN–3×FLAG–RAP80 or pLXIN–3×FLAG–RAP80ΔUIM1,2 and then treated with 100 nM E2 or ethanol for 24 h. Cells were collected and assayed for luciferase activity. The relative Luc activity was calculated and plotted. RAP80 expression was also detected with anti-FLAG M2 antibody (lower panels).

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Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor alpha - PubMed (original) (raw)