| Estrogen plays essential roles in the growth, development, and homeostasis of a number of tissues, and can also be linked to the growth of breast cancer. The biological activities of estrogen are mediated by estrogen receptors (ERs) ERα and ERβ and also orphan G-protein-coupled receptor 30 (GPR30). In order to identify novel proteins that are involved in ER-mediated actions of estrogen, we used mass spectrometry-based quantitative proteomic methods to systematically profile global protein expression in responses to E2 (17β-estradiol) stimulation in human breast cancer cell, and identify and characterize cellular novel proteins that are associated with ERs in breast cancer cells. Specifically, through a SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomic method, we found that multiple components of the PKA-LKB1/STRAD/MO25-AMPK pathway were significantly upregulated in addition to the commonly observed phosphorylation of the AMPK (Thr172). In addition to the PKA-LKB1/STRAD/MO25-AMPK pathway, several other pathways were also significantly affected by E2.;With regard to identifying the cellular proteins that are associated with ERs, first, we used a two-dimensional gel electrophoresis (2-DE) based approach to identify proteins that are associated with ERα. One of the identified proteins was mitochondrial protein, trifunctional protein β-subunit (HADHB). We verified the interaction between ERα and HADHB by coimmunoprecipitation and also in-vitro binding assay. The ligands of ERα such as 17β-estradiol and TAM were demonstrated to affect the association of ERα with HADHB in human cell extract. We also demonstrated that ERβ associates and colocalizes with HADHB, and affects HABHB enzyme activity. We then used a SILAC-based quantitative proteomic method to identify cellular proteins that are associated with ERα. One identified protein is a histone acetyltransferase isoform, designed as x (HATx). Functional studies demonstrated that HATx inhibits the ERα-mediated transcription in human breast cancer cells potentially through directly binding to ERα at DNA binding domain (DBD) and blocking binding of ERα to the ERE.;In summary, through quantitative proteomic, biochemical, molecular, and cell biology methods, we have identified novel pathways that are affected by estrogen in breast cancer cells, and several novel cellular proteins that bind to ERs and affect the biological functions of the receptors. |
