Proteomic characterization of proteins associated with the 14-3-3 family members

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The 14-3-3 protein is a family of acidic, dimeric proteins distributed widely among eukaryotic cells. This protein family binds to a variety of proteins in a phosphorylation-dependent manner and participants in the regulation of cell proliferation, differentiation and function. In this study, we have attempted to screen 14-3-3-binding proteins in PC12 cells by means of proteomic techniques. We developed a novel multiple affinity tag, called myc-TEV-FLAG (MEF), and transected the MEF-tagged 14-3-3η cDNA into PC12 cells. The proteins associated with the expressed MEF-14-3-3η protein were pulled from the PC12 extracts and were analyzed by SDS-PAGE and mass spectrometry. This procedure allowed us to identify 121 proteins that include 14 proteins already reported as known targets of 14-3-3 and 107 novel interacting partners. These proteins include a series of components in the growth factor signaling pathways such as the growth factor receptor in the plasma membrane and the effectors of small GTP-binding proteins, suggesting that the 14-3-3 protein may act as a coordinator that creates functional signaling complexes in the coupled with the action of protein kinases. It was also observed that one of the identified proteins, kinesin light chain 2 (KLC2), was directly associated with 14-3-3, and their association was dependent on the phosphorylation of KLC2 at Ser575.