Isoform-Selective Fluorescent Labeling of 14-3-3σ by Acrylamide-Containing Fusicoccins.

ABSTRACT

The 14-3-3 family of proteins is central to the regulation of signaling pathways driven by serine/threonine kinases. In humans, 14-3-3 consists of seven highly conserved isoforms, yet the function of each isoform remains to be fully elucidated. Synthetic agents capable of isoform-specific fluorescent labeling of 14-3-3 would provide a useful tool for studying in depth the biological roles of isoforms. In this study, the 14-3-3σ isoform was evaluated, which possesses a unique Cys38, and a natural product-based fluorescent labeling agent was designed by introducing an acrylamide group and a fluorescent dye to fusicoccin (FC). In vitro evaluation demonstrated that 12-hydroxy 1 and 2 exhibit 14-3-3σ selective labeling activity over 14-3-3ζ in the presence of a mode-3 phospholigand. Furthermore, 2 was shown to label 14-3-3σ in cell lysate in the presence of a C-terminal mode-3 phosphopeptide derived from ERα, with no apparent nonspecific labeling. These results indicate that 2 is capable of selective fluorescent detection of 14-3-3σ upon binding to mode-3 phospholigand under biologically relevant conditions.