Fluorescent labeling of 14-3-3 proteins by fusicoccins

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14-3-3 proteins play a critical role in serine/ threonine kinase-dependent signaling pathways through protein-protein interactions with multiple phosphorylated ligands. A ligand-dependent 14-3-3 detection technique would facilitate elucidation of 14-3-3-related intracellular signaling networks. Here, we describe phosphopeptide-dependent fluorescent labeling of 14-3-3. using cell penetrating probes derived from a diterpene natural product, fusicoccin. In vitro evaluations demonstrated that these compounds site-specifically attached a fluorescent tag onto the protein surface as a result of ternary complex formation with the 14-3-3 protein and a phosphopeptide ligand. The BODIPY-attached probe labeled human endogenous 14-3-3 in cancer cells under hyper-phosphorylation conditions, proving that 14-3-3 is a primary target of the fusicoccins in mammalian cells. This cell-penetrating labeling agent would be a useful tool to explore the mechanism of antitumor activity associated with fusicoccin-related agents.