The 14-3-3 proteins are a family of phospho-binding proteins ubiquitously expressed in all eukaryotic cells and are essential for the regulation of enormous number of cellular events, such as proliferation, autophagy, and stress responses. Diterpene glucoside fusicoccin A (FC-A) is a phytotoxic fungal metabolite that stabilizes 14-3-3 interactions by binding to a hydrophobic cavity of the protein complex of 14-3-3 bound to the phosphorylated C-terminus of plasma membrane H+-ATPase, leading to stomatal dysfunction and eventually plant death. While FC-A is inactive in cancer cells, our previous study revealed that removal of the 12-hydroxy group of FC-A converted it into an antitumor agent [1], and that this compound is capable of stabilizing 14-3-3 interactions with mode-1 phospholigands due to less steric repulsion with the conserved i+2 Pro residue in the mode-1 [2,3]. In order to elucidate the mode of action, we conducted target-ID study based on coimmunoprecipitation of 14-3-3-binding proteins followed by quantitative mass spectrometry analysis. As a result, a scaffold protein involved in the regulatory protein complex formation responsible for translational repression was identified. Biochemical evaluation showed that FC upregulates binding of 14-3-3 to the protein and recruitment of TTP, thereby suppressing protein synthesis. Further experiments using deletion mutants identified a potential binding site of 14-3-3, which possesses a typical mode-1 consensus motif with i+1 residue suitable for interacting with FC. These results indicate that formation of the translational repression machinery requires 14-3-3-binding and suggest that the process of translational regulation may be a part of environmental cellular responses controlled by 14-3-3-mediated kinases / phosphatases signaling pathways, which may provide a new set of drug targets for the future therapeutics. Details of the biological evaluations will be discussed.
[1] Anticancer Agents Med. Chem. 2012, 12, 791.
[2] Angew. Chem. Int. Ed. 2012, 51, 509.
[3] Chem. Eur. J. 2018, 24, 16066.