3-3 proteins play a critical role in serine/threonine kinase dependent signaling pathways through protein-protein interactions (PPIs) with multiple phosphorylated ligands. [1] A ligand-dependent 14-3-3 detection technique would facilitate elucidation of 14-3-3-related intracellular signaling networks. Herein, we describe phosphopeptide-dependent fluorescent labeling of 14-3-3 z by using cell-penetrating probes derived from the diterpene natural product fusicoccin.
The 14-3-3 proteins are a family of dimeric conserved regulatory proteins that are expressed in all eukaryotic cells. Each of the highly helical monomers possesses a shallow groove (approximately 25 in length) that recognizes consensus phosphopeptide motifs containing either phosphoserine (pS) or phosphothreonine (pT) residues. [1b,c] Hundreds of intracellular ligand proteins that possess 14-3-3 consensus motifs have been identified, including proteins involved in signaling and cell-cycle control, such as the Raf family, p53, and Cdc25 phosphatases. [1b] A number of studies have revealed that the disruption of 14-3-3 PPIs results in suppression of tumor growth, [2] thus suggesting that 14-3-3 proteins could potentially serve as new therapeutic targets. However, many details regarding intracellular regulatory processes that involve 14-3-3 proteins remain unknown. Low-molecular-weight agents that detect 14-3-3 proteins are desirable for elucidating intracellular 14-3-3 functions, however, such chemical probes are not currently available. Recent studies have successfully identified small molecule stabilizers [3a] and inhibitors [3b-c] of 14-3-3 PPIs, thus providing useful reagents for cell-based analysis. We anticipated that development of a cell-penetrating chemical probe that is capable of detecting 14-3-3 proteins in response to their binding to