Rapid identification of 14-3-3-binding proteins by protein microarray analysis

The 14-3-3 protein family consists of acidic 30-kDa proteins composed of seven isoforms in mammalian cells, expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 isoforms form a dimer that acts as a molecular adaptor interacting with key signaling components involved in cell proliferation, transformation, and apoptosis. Until present, more than 300 proteins have been identified as 14-3-3-binding partners, although most of previous studies focused on a limited range of 14-3-3-interacting proteins. Here, we studied a comprehensive profile of 14-3-3-binding proteins by analyzing a high-density protein microarray using recombinant human 14-3-3 epsilon protein as a probe. Among 1752 proteins immobilized on the microarray, 20 were identified as 14-3-3 interactors, most of which were previously unreported 14-3-3-binding partners. However, 11 known 14-3-3-binding proteins, including keratin 18 (KRT18) and mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), were not identified as a 14-3-3-binding protein. The specific binding to 14-3-3 of EAP30 subunit of ELL complex (EAP30), dead box polypeptide 54 (DDX54), and src homology three (SH3) and cysteine rich domain (STAC) was verified by immunoprecipitation analysis of the recombinant proteins expressed in HEK293 cells. These results suggest that protein microarray is a powerful tool for rapid and comprehensive profiling of 14-3-3-binding proteins.