Mammary gland tumorigenesis as well as normal breast development are strongly influenced by estrogens, whose genomic responses are mediated by intracellular receptors. The transcriptional activity of the estrogen receptor (ER) is stimulated by compounds which increase intracellular cAMP, dopamine and estradiol, and this receptor activation is accompanied by increased phosphorylation. Antiestrogens, such as tamoxifen or 4-hydroxytamoxifen (4HT), are used clinically and experimentally to inhibit estrogen-stimulated ER function. However, the signal transduction pathways which ligand-independently activate the ER are not blocked by 4HT and the resulting gene expression is greater than that observed for either 4HT, cAMP or dopamine alone. Protein-protein interactions also contribute to ER activity and the coactivator proteins, steroid receptor coactivator-1 (SRC1) and CREB binding protein (CBP), synergistically increase ER transcriptional activity. Interestingly, both of these coactivators are phosphoproteins, and phosphorylation events are known to regulate the ability of CBP to bind to specific transcription factors and stimulate gene expression. I therefore propose to test the following hypothesis: ER-regulated gene expression is dependent upon phosphorylation of the ER and/or SRC-1 and CBP, and that phosphorylation events contribute to the protein-protein interactions between receptor and coactivators.
The specific aims of this proposal are to: 1) establish whether mutation of known or potential ER, SRC-1 or CBP phosphorylation sites alters the ability of SRC-1 or CBP to activate ER-dependent gene expression stimulated by estradiol, dopamine or elevated intracellular cAMP using trans- activation assays in cultured cells; 2) determine if mutation of ER, SRC- l or CBP phosphorylation sites alters the ability of these factors to interact with one another by mammalian two-hybrid or co- immunoprecipitation assays, and 3) assess the contribution of phosphorylation to the ability of 4HT to stimulate transcription activated by dopamine and cAMP. It is anticipated that these studies will further our understanding of the regulation of ER action and provide insight that may allow ER-coactivator interactions to be exploited in the development of new strategies to block ER action.
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