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Scaffold proteins regulate cell signalling by promoting the proximity of putative interaction partners. Although they are frequently applied in cellular settings, fundamental understanding of them in terms of, amongst other factors, quantitative parameters has been lagging behind. Here we present a scaffold protein platform that is based on the native 14-3-3 dimeric protein and is controllable through the action of a small-molecule compound, thus permitting study in an in vitro setting and mathematical description. Robust small-molecule regulation of caspase-9 activity through induced dimerisation on the 14-3-3 scaffold was demonstrated. The individual parameters of this system were precisely determined and used to develop a mathematical model of the scaffolding concept. This model was used to elucidate the strong cooperativity of the enzyme activation mediated by the 14-3-3 scaffold. This work provides an entry point for the long-needed quantitative insights into scaffold protein functioning and paves the way for the optimal use of reengineered 14-3-3 proteins as chemically inducible scaffolds in synthetic systems., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

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.