title: Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface.
abstract: Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, 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 ERα 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 ERα 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 ERα/14-3-3 interaction. Cocrystallization of the trimeric ERα/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERα-T(594)) for high-affinity interaction. We confirm that T(594) is a distinct ERα 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 ERα/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERα dimerization, inhibits ERα/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERα are provided, together with a small molecule that selectively targets this ERα/14-3-3 interface.
authors.affiliation: Department of Structural Biology, Faculty Earth and Life Sciences, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands.
authors.forename: Ingrid J
authors.initials: IJ
authors.lastname: De Vries-van Leeuwen
authors.forename: Daniel
authors.initials: D
authors.lastname: da Costa Pereira
authors.forename: Koen D
authors.initials: KD
authors.lastname: Flach
authors.forename: Sander R
authors.initials: SR
authors.lastname: Piersma
authors.forename: Christian
authors.initials: C
authors.lastname: Haase
authors.forename: David
authors.initials: D
authors.lastname: Bier
authors.forename: Zeliha
authors.initials: Z
authors.lastname: Yalcin
authors.forename: Rob
authors.initials: R
authors.lastname: Michalides
authors.forename: K Anton
authors.initials: KA
authors.lastname: Feenstra
authors.forename: Connie R
authors.initials: CR
authors.lastname: Jiménez
authors.forename: Tom F A
authors.initials: TF
authors.lastname: de Greef
authors.forename: Luc
authors.initials: L
authors.lastname: Brunsveld
authors.forename: Christian
authors.initials: C
authors.lastname: Ottmann
authors.forename: Wilbert
authors.initials: W
authors.lastname: Zwart
authors.forename: Albertus H
authors.initials: AH
authors.lastname: de Boer
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000188:drug therapy*; D003460:Crystallization; D019281:Dimerization; D016503:Drug Delivery Systems / Q000379:methods*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005454:Fluorescence Polarization; D040461:Gene Components; D005786:Gene Expression Regulation / Q000187:drug effects; D006027:Glycosides / Q000494:pharmacology*; D006801:Humans; D061986:MCF-7 Cells; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D020033:Protein Isoforms / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D048948:14-3-3 Proteins; D047628:Estrogen Receptor alpha; D006027:Glycosides; D020033:Protein Isoforms; C007808:fusicoccin