| Estrogen receptors (ERαand ERβ) are members of the steroid hormone superfamily of nuclear receptors that act as ligand-activated transcription factors. Both of the two receptors regulate gene transcription either by binding directly to estrogen-responsive elements (ERE) located within the promoter regions of target genes or interacting with other transcription factors such as AP1 and SP1. ERαand ERβshare structural similarity characterized by several functional domains. Two distinct activation function (AF) domains contribute to the transcriptional activity of the two receptors. The first activation function AF-1, a ligand-independent transactivation domain, is located at the N terminus, whereas the second, ligand-dependent activation function, AF-2, is located at the C terminus, overlapping the ligand binding domain. The DNA-binding domain (DBD) of the two receptors is centrally located. ERαand ERβpossess similar binding affinities for estrogen and their cognate DNA binding site, which is probably caused by the high degree of sequence homology they share in their ligand and DNA binding domains.ERs mediate the effects of estrogen on the development and progression of breast cancer. ERαhas served as an important diagnostic and therapeutic target for prevention and treatment. Activation of ERs is responsible for many biological processes, including cell proliferation, differentiation, motility, and apoptosis. ERs exert these functions by regulating genes and signaling pathways involved in cell fate. Regulation of gene expression by the ERs requires the coordinate activity of ligand binding, phosphorylation, and cofactor interactions, with particular combinations probably resulting in the tissue-specific responses elicited by the receptors. A growing list of cofactors that regulate ERαincludes coactivators, such as members of the SRC-1 family, p300/CREB-binding protein, p68, PBP, PRMT2, and ARNT, and corepressors, such as N-CoR, SMRT, MTA1, and Smad4. Receptor phosphorylation is another protein modification way to regulate protein stability. Phosphorylation of ERαcan be induced by mitogen-activated protein kinase pathways and contain six phosphorylation sites: Ser104, Ser106, Ser118, Ser167, Ser237 and Tyr537 respectively. So far, very few kinases for ERαphosphorylation have been reported and the intracellular signaling pathways modulating ERαtranscriptional activity by those kinases are not fully elucidated.In this study, we first identified and characterized a novel ERα-interacting protein, c-Abl non-receptor tyrosine kinases. We show that c-Abl interacts with ERαin vitro and in vivo by co-immunoprecipiation, immunoblot, GST pulldown assay,Farwestern blot assay. We also shown that SH3 domain of c-Abl interact with ERα, and pro79 and pro333 of ERαare essential for their interaction, pro79 and pro333 mutation disrupts the interaction between c-Abl and ERα. Secondly, we show that ERαcan be phosphorylated by c-Abl through their interaction and c-Abl increases cellular aboundance of ERαin kinase-dependent manner. The c-Abl mediated effect on the ERαabundance is predominantly dependent on the phosphorylation of ERαat Y52 and Y219. Mutations at these two site of ERαor c-Abl inhibitor STI571 abolishes the c-Abl induced accumulation of ERα, Thirdly, we show that the transcription activity of ER's target gene, such as ERE, was enhanced by c-Abl in ligand dependence and independence manner. We further demonstrate c-Abl kinase mutation(K290R)or STI571 or ERα(Y52FY219F) mutation completely abrogate c-Abl-dependent ERαactivation without changing its basic transcription acitivity.Taken together, these results suggest that the c-Abl increases the cellular ERαabundance through Y52 and Y219 phosphorylation resulting in the enhancement of ERαtranscription activity. Because of the importance of ERαsignaling in the regulation of breast cancer development, c-Abl may play important roles in ERαsignaling and represent a novel target for breast cancer therapy.
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