Interaction of 14-3-3 proteins with the Estrogen Receptor Alpha F domain provides a drug target interface

Estrogen receptor alpha (ER alpha) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ER alpha, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ER alpha dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ER alpha dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ER alpha/14-3-3 interaction. Cocrystallization of the trimeric ER alpha/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ER alpha-T-594) for high-affinity interaction. We confirm that T-594 is a distinct ER alpha phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T-594-specific antibody and by mass spectrometry. In line with its ER alpha/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ER alpha dimerization, inhibits ER alpha/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ER alpha are provided, together with a small molecule that selectively targets this ER alpha/14-3-3 interface.