Specificity of cellular signaling scaffolds

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14-3-3 proteins comprise a family of highly conserved and abundant regulatory molecules. They interact with a large number of target (client) proteins in a phosphorylation-dependent manner. By so doing, they are critical components of most eukaryotic signaling pathways. 14-3-3 clients are structurally and functionally highly diverse. They are involved in e.g. cell cycle, apoptosis, DNA damage, transcription, and cell motility. Intriguingly, the exact mode of action of 14-3-3 proteins varies depending on the client protein. They can regulate their clients' enzymatic activity, conformation, assembly into complexes, subcellular localization, or mediate aggregation. Consequently, 14-3-3s represent an unusually versatile regulatory platform for signaling in eukaryotes. Proteomic studies have begun to unravel the client repertoire of 14-3-3 proteins. However, due to different experimental methods, cell lines, and assay sensitivity, the overlap between these studies is rather modest. Perhaps more importantly, however, standard non-quantitative approaches cannot address one of the major questions in the field: isoform specificity. Almost all organisms express multiple 14-3-3 isoforms, but their specificity is virtually identical in vitro. However, mouse studies have revealed striking isoform-specific phenotypes, suggesting that current proteomic methods are insufficiently sensitive and quantitative. I propose to combine quantitative state-of-the-art mass spectrometry with quantitative pairwise interaction assays to comprehensively characterize the client protein repertoire of all seven human 14-3-3 isoforms in isogenic cell lines. We will uncover novel client proteins, identify isoform-specific interactions, and dissect the basis of specificity at the molecular level. These experiments will be complemented with detailed functional follow-up studies. I will take advantage of my extensive experience with chaperones, which, like 14-3-3s, are abundant molecules with thousands of transiently associating client proteins. In particular, the high-throughput interaction assay LUMIER with BACON, which I developed as a postdoctoral fellow, provides a sensitive and quantitative readout for transient 14-3-3/client interactions. Taken together, our studies will lead to a much more comprehensive understanding of phosphorylation-dependent signaling pathways that are key mediators in development and misregulated in diverse pathologies.