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SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells.

Meneses-Morales I, Tecalco-Cruz AC, Barrios-García T, Gómez-Romero V, Trujillo-González I, Reyes-Carmona S, García-Zepeda E, Méndez-Enríquez E, Cervantes-Roldán R, Pérez-Sánchez V, Recillas-Targa F, Mohar-Betancourt A, León-Del-Río A - Nucleic Acids Res. (2014)

Bottom Line: In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor.We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue.These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors.

View Article: PubMed Central - PubMed

Affiliation: Programa de Investigación de Cáncer de Mama, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F. 04510, Mexico Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F. 04510, Mexico.

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NHERF2 is an ERα associated protein. (A) NHERF2 protein sequence obtained from cDNA clones isolated by yeast two-hybrid screening. The PDZ domain1 (PDZ1, amino acids 10–88) and PDZ domain 2 (PDZ2, amino acids 152–238) are highlighted in gray. (B) Subcellular localization of NHERF2 and ERα NHERF2 (left panel, green) and ERα (middle panel, red) were visualized using specific antibodies as described in Materials and Methods. Cellular colocalization is shown by merging NHERF2 and ERα images (right panel, yellow). (C) Estradiol (E2) enhances NHERF2 binding to ERα. Nuclear protein extracts from MCF7 cells were incubated with a GST-NHERF2 fusion protein or a GST control protein. Proteins captured in the presence (E2) or absence (E2−) of estradiol were resolved by PAGE and the presence of ERα was visualized by western blot (WB). Input lane represents 10% of the nuclear extract used in the capture assays. (D) In vitro interaction between NHERF2 and ERα schematic representation of N-terminal (PDZ1), C-terminal (PDZ2) and full-length NHERF2 GST-fusion proteins used in pull-down assays is shown in the top panel. The bottom image shows [35S]methionine-labeled-ERα resolved by PAGE following capture by GST-NHERF2 full length (FL) or protein fragments (PDZ1, PDZ2) or GST control. Input represents 10% of the labeled ERα used in the assay. (E) NHERF2 interacts predominantly with the AF-1 domain of ERα. Schematic representation of N-terminal (AF-1) and C-terminal AF-2 fragments of ERα used in pull-down assays is shown at the top. The bottom image shows [35S]methionine-labeled-NHERF2 resolved by PAGE following capture by GST-AF1 or GST-AF2 ERα fragments or GST control. (F) Coomassie Brilliant Blue staining showed that equimolar amounts of GST proteins were used for the pull-down assay. (G) NHERF2 interacts with ERα in vivo. AD293 cells were transiently transfected with ERα with or without 3× FLAG-NHERF2. AD293 protein extracts were immunoprecipitated with anti-ERα followed by WB to detect FLAG-NHERF2 and ERα. (E) Endogenous NHERF2 interacts with ERα in the MCF7 breast cancer cell line. MCF7 cells were incubated for 45 min with 100 nM E2. Total cell lysates were subjected to immunoprecipitation with anti-ERα or IgG (as negative control) followed by WB with anti-ERα or anti-NHERF2.
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Figure 1: NHERF2 is an ERα associated protein. (A) NHERF2 protein sequence obtained from cDNA clones isolated by yeast two-hybrid screening. The PDZ domain1 (PDZ1, amino acids 10–88) and PDZ domain 2 (PDZ2, amino acids 152–238) are highlighted in gray. (B) Subcellular localization of NHERF2 and ERα NHERF2 (left panel, green) and ERα (middle panel, red) were visualized using specific antibodies as described in Materials and Methods. Cellular colocalization is shown by merging NHERF2 and ERα images (right panel, yellow). (C) Estradiol (E2) enhances NHERF2 binding to ERα. Nuclear protein extracts from MCF7 cells were incubated with a GST-NHERF2 fusion protein or a GST control protein. Proteins captured in the presence (E2) or absence (E2−) of estradiol were resolved by PAGE and the presence of ERα was visualized by western blot (WB). Input lane represents 10% of the nuclear extract used in the capture assays. (D) In vitro interaction between NHERF2 and ERα schematic representation of N-terminal (PDZ1), C-terminal (PDZ2) and full-length NHERF2 GST-fusion proteins used in pull-down assays is shown in the top panel. The bottom image shows [35S]methionine-labeled-ERα resolved by PAGE following capture by GST-NHERF2 full length (FL) or protein fragments (PDZ1, PDZ2) or GST control. Input represents 10% of the labeled ERα used in the assay. (E) NHERF2 interacts predominantly with the AF-1 domain of ERα. Schematic representation of N-terminal (AF-1) and C-terminal AF-2 fragments of ERα used in pull-down assays is shown at the top. The bottom image shows [35S]methionine-labeled-NHERF2 resolved by PAGE following capture by GST-AF1 or GST-AF2 ERα fragments or GST control. (F) Coomassie Brilliant Blue staining showed that equimolar amounts of GST proteins were used for the pull-down assay. (G) NHERF2 interacts with ERα in vivo. AD293 cells were transiently transfected with ERα with or without 3× FLAG-NHERF2. AD293 protein extracts were immunoprecipitated with anti-ERα followed by WB to detect FLAG-NHERF2 and ERα. (E) Endogenous NHERF2 interacts with ERα in the MCF7 breast cancer cell line. MCF7 cells were incubated for 45 min with 100 nM E2. Total cell lysates were subjected to immunoprecipitation with anti-ERα or IgG (as negative control) followed by WB with anti-ERα or anti-NHERF2.

Mentions: To identify novel coregulators that recognize the activation function AF-1 of ERα, we used this region (amino acids 1–180) as bait in a yeast two-hybrid screen of 5 × 106 independent clones of a human mammary gland cDNA library. Eleven cDNA clones were isolated and sequenced. Two showed almost identical sequences encoding a 337 amino acid protein containing two PDZ domains (Figure 1A). Sequence analysis using the BLAST program of the National Center for Biotechnology Information revealed that the candidate protein had been previously described as the human testis determining factor SRY-interacting protein (SIP-1) and as the regulatory factor of the small intestine brush-border membrane Na+/H+ exchanger, NHERF2.


SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells.

Meneses-Morales I, Tecalco-Cruz AC, Barrios-García T, Gómez-Romero V, Trujillo-González I, Reyes-Carmona S, García-Zepeda E, Méndez-Enríquez E, Cervantes-Roldán R, Pérez-Sánchez V, Recillas-Targa F, Mohar-Betancourt A, León-Del-Río A - Nucleic Acids Res. (2014)

NHERF2 is an ERα associated protein. (A) NHERF2 protein sequence obtained from cDNA clones isolated by yeast two-hybrid screening. The PDZ domain1 (PDZ1, amino acids 10–88) and PDZ domain 2 (PDZ2, amino acids 152–238) are highlighted in gray. (B) Subcellular localization of NHERF2 and ERα NHERF2 (left panel, green) and ERα (middle panel, red) were visualized using specific antibodies as described in Materials and Methods. Cellular colocalization is shown by merging NHERF2 and ERα images (right panel, yellow). (C) Estradiol (E2) enhances NHERF2 binding to ERα. Nuclear protein extracts from MCF7 cells were incubated with a GST-NHERF2 fusion protein or a GST control protein. Proteins captured in the presence (E2) or absence (E2−) of estradiol were resolved by PAGE and the presence of ERα was visualized by western blot (WB). Input lane represents 10% of the nuclear extract used in the capture assays. (D) In vitro interaction between NHERF2 and ERα schematic representation of N-terminal (PDZ1), C-terminal (PDZ2) and full-length NHERF2 GST-fusion proteins used in pull-down assays is shown in the top panel. The bottom image shows [35S]methionine-labeled-ERα resolved by PAGE following capture by GST-NHERF2 full length (FL) or protein fragments (PDZ1, PDZ2) or GST control. Input represents 10% of the labeled ERα used in the assay. (E) NHERF2 interacts predominantly with the AF-1 domain of ERα. Schematic representation of N-terminal (AF-1) and C-terminal AF-2 fragments of ERα used in pull-down assays is shown at the top. The bottom image shows [35S]methionine-labeled-NHERF2 resolved by PAGE following capture by GST-AF1 or GST-AF2 ERα fragments or GST control. (F) Coomassie Brilliant Blue staining showed that equimolar amounts of GST proteins were used for the pull-down assay. (G) NHERF2 interacts with ERα in vivo. AD293 cells were transiently transfected with ERα with or without 3× FLAG-NHERF2. AD293 protein extracts were immunoprecipitated with anti-ERα followed by WB to detect FLAG-NHERF2 and ERα. (E) Endogenous NHERF2 interacts with ERα in the MCF7 breast cancer cell line. MCF7 cells were incubated for 45 min with 100 nM E2. Total cell lysates were subjected to immunoprecipitation with anti-ERα or IgG (as negative control) followed by WB with anti-ERα or anti-NHERF2.
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Figure 1: NHERF2 is an ERα associated protein. (A) NHERF2 protein sequence obtained from cDNA clones isolated by yeast two-hybrid screening. The PDZ domain1 (PDZ1, amino acids 10–88) and PDZ domain 2 (PDZ2, amino acids 152–238) are highlighted in gray. (B) Subcellular localization of NHERF2 and ERα NHERF2 (left panel, green) and ERα (middle panel, red) were visualized using specific antibodies as described in Materials and Methods. Cellular colocalization is shown by merging NHERF2 and ERα images (right panel, yellow). (C) Estradiol (E2) enhances NHERF2 binding to ERα. Nuclear protein extracts from MCF7 cells were incubated with a GST-NHERF2 fusion protein or a GST control protein. Proteins captured in the presence (E2) or absence (E2−) of estradiol were resolved by PAGE and the presence of ERα was visualized by western blot (WB). Input lane represents 10% of the nuclear extract used in the capture assays. (D) In vitro interaction between NHERF2 and ERα schematic representation of N-terminal (PDZ1), C-terminal (PDZ2) and full-length NHERF2 GST-fusion proteins used in pull-down assays is shown in the top panel. The bottom image shows [35S]methionine-labeled-ERα resolved by PAGE following capture by GST-NHERF2 full length (FL) or protein fragments (PDZ1, PDZ2) or GST control. Input represents 10% of the labeled ERα used in the assay. (E) NHERF2 interacts predominantly with the AF-1 domain of ERα. Schematic representation of N-terminal (AF-1) and C-terminal AF-2 fragments of ERα used in pull-down assays is shown at the top. The bottom image shows [35S]methionine-labeled-NHERF2 resolved by PAGE following capture by GST-AF1 or GST-AF2 ERα fragments or GST control. (F) Coomassie Brilliant Blue staining showed that equimolar amounts of GST proteins were used for the pull-down assay. (G) NHERF2 interacts with ERα in vivo. AD293 cells were transiently transfected with ERα with or without 3× FLAG-NHERF2. AD293 protein extracts were immunoprecipitated with anti-ERα followed by WB to detect FLAG-NHERF2 and ERα. (E) Endogenous NHERF2 interacts with ERα in the MCF7 breast cancer cell line. MCF7 cells were incubated for 45 min with 100 nM E2. Total cell lysates were subjected to immunoprecipitation with anti-ERα or IgG (as negative control) followed by WB with anti-ERα or anti-NHERF2.
Mentions: To identify novel coregulators that recognize the activation function AF-1 of ERα, we used this region (amino acids 1–180) as bait in a yeast two-hybrid screen of 5 × 106 independent clones of a human mammary gland cDNA library. Eleven cDNA clones were isolated and sequenced. Two showed almost identical sequences encoding a 337 amino acid protein containing two PDZ domains (Figure 1A). Sequence analysis using the BLAST program of the National Center for Biotechnology Information revealed that the candidate protein had been previously described as the human testis determining factor SRY-interacting protein (SIP-1) and as the regulatory factor of the small intestine brush-border membrane Na+/H+ exchanger, NHERF2.

Bottom Line: In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor.We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue.These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors.

View Article: PubMed Central - PubMed

Affiliation: Programa de Investigación de Cáncer de Mama, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F. 04510, Mexico Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F. 04510, Mexico.

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Related in: MedlinePlus