Splicing potentiation by growth factor signals via estrogen receptor phosphorylation

Abstract

Mitogen-activated protein kinase-mediated growth factor signals are known to augment the ligand-induced transactivation function of nuclear estrogen receptor alpha (ERalpha) through phosphorylation of Ser-118 within the ERalpha N-terminal transactivation (activation function-1) domain. We identified the spliceosome component splicing factor (SF)3a p120 as a coactivator specific for human ERalpha (hERalpha) activation function-1 that physically associated with ERalpha dependent on the phosphorylation state of Ser-118. SF3a p120 potentiated hERalpha-mediated RNA splicing, and notably, the potentiation of RNA splicing by SF3a p120 depended on hER Ser-118 phosphorylation. Thus, our findings suggest a mechanism by which growth factor signaling can regulate gene expression through the modulation of RNA splicing efficiency via phosphorylation of sequence-specific activators, after association between such activators and the spliceosome.

Fig. 1.

SF3a p120 directly binds phosphorylated Ser-118 hERα (A/B) domains. (A) Endogenous interactants of the hERα (A/B) domain. Three endogenous interactants (p120, p72, and p68, as indicated) were detected in 10 μg of nuclear extracts from HeLa, COS-1, and MCF7 cells lines with Ser-118 phosphorylated or nonphosphorylated hERα (A/B) domain probes by using the Far-Western technique (20). (Left) Coomassie brilliant blue R-250 (CBB)-stained gel. (B) Identification of phosphorylated hERα (A/B) domain-interacting proteins. Nuclear extracts prepared from HeLa S3 cells were incubated with immobilized GST-hERα (A/B) or GST-p-Ser-118 hERα (A/B) domains. (Left) Proteins eluted from the columns by 1 M KCl were subjected to SDS/PAGE followed by staining with CBB. (Right) Products of FLAG-M2 resin affinity purification from nuclear extracts of HeLa cells stably expressing FLAG-hERα or FLAG-Ser-118–Ala hERα were examined by MS. Identified proteins are indicated at the right. (C) Selective binding of SF3a p120 to hERα in a pull-down assay. In vitro-translated SF3a p120 protein was tested for direct interaction with biotin-tagged hERα (A/B), chimeric GST-fused A/B domain of hERβ, or D/E/F domains of hERα or hERβ. DRIP205/TRAP220 was used as a positive control and exhibited ligand-induced association with the hERα D/E/F domain.

Fig. 2.

Association of hERα AF-1 domain with spliceosome complex through SF3a p120. (A) In vivo association of hERα and SF3a p120. The 293T cells were transfected with hERα expression vectors (0.1 μg) and then incubated with or without E2 (10^-8 M). Cells were then lysed in TNE buffer (10 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) and immunoprecipitated with anti-hERα or anti-Flag SF3a p120 Ab. Immunoprecipitates were subjected to SDS/PAGE followed by Western blotting with the indicated Abs. (B) Ligand-induced association of full-length hERα with U1/U2 components was further characterized by using endogenous proteins in MCF7 cells. Cells were treated with E2 (10–8 M), EGF (100 ng/ml), and MAPK inhibitor U0126 (20 μM), as indicated, and then immunoprecipitated with anti-hERα or anti-SF3a p120 Ab. Immunoprecipitates were subjected to SDS/PAGE followed by Western blotting with the indicated Abs. (C) hERα associates with a complex containing SF3a spliceosome components. Nuclear extracts from a stable transformant of FLAG-tagged full-length hERα with or without E2 (10^-7 M) were applied to FLAG-M2 resin, and eluted proteins were separated by using Superose 6 gel filtration column (10, 14) and detected by the indicated Abs.

Fig. 3.

SF3a p120 coactivates the transactivation function of hERα AF-1. (A and B) SF3a p120 potentiates the transactivation function of hERα AF-1 in a Ser-118 phosphorylation-dependent manner. The 293T cells were transfected with expression vectors of full-length hERα (50 ng), hERα (A/B/C) (50 ng), Ki-Ras^val12 (R¹²) (100 ng), MAPKK (100 ng), SF3a p120 (300 ng), or combinations as indicated, in either the absence or presence of E2 (10^-8 M), TAM (10^-7 M), U0126 (20 μM), or combinations as indicated, along with pGL-estrogen response element-tk (1.0 μg) and pRL-CMV (10 ng). Cultured cells were also transfected with 100 pmol dsRNA for SF3a p120 siRNA (5′-AGACGGAAUGGAAACUGAAUGGGCAAG-3′ and 5′-UGCCCAUUCAGUUUCCAUUCCGUCUAU-3′) by using Lipofectamine 2000 (Invitrogen). Assays were performed 24 h after transfection. (Upper) Cell extracts were used in luciferase assays (20) and Western blotting. (C) SF3a p120 enhances the association of hERα AF-1 with p68, p72, and p300. The 293T cells were transfected with indicated plasmids. The complex associated with A/B/C domains of hERα were analyzed to detect SF3a p120, p68, p72, and p300 by purification from the cell extracts with anti-Flag M2 resin and Ni resin, followed by Western blotting by using the anti-hERα, anti-Flag, anti-His, anti-Myc, or anti-p300 Abs (lanes 1–10). (D) Occupancy of human oxytocin promoter by hERα and spliceosome components in MCF7 cells was determined by chromatin immunoprecipitation analysis (14, 23).

Fig. 4.

hERα AF-1-specific potentiation of mRNA processing by SF3a p120. (A) Schematic representation of the estrogen response element-CD44 construct used in the in vivo splicing assay (22). (B and C) SF3a p120 regulation of CD44 mRNA processing depends on hERα Ser-118 phosphorylation state. The 293T cells were transfected with the indicated plasmids and siRNA (SF3a p120; 100 pmol). After transfection, cells were treated with E2 (10–8 M), TAM (10–7 M), EGF (100 ng/ml), and U0126 (20 μM) as indicated. Total RNA was extracted with Isogen 24 h after transfection and subjected to RT-PCR analysis (22). (D) Schematic representation of the human oxytocin gene exon 1 and 2 used in the in vivo splicing assay. (E) MCF7 cells were transfected with the indicated plasmids and treated as in B and C. After the treatment, total RNA was extracted. Splicing patterns were evaluated by RT-PCR by using oxytocin-specific primers.