Specificity of cellular signaling scaffolds

14-3-3蛋白是一个高度保守和丰富的调节分子家族。它们以磷酸化依赖的方式与大量靶蛋白（客户）相互作用。通过这样做，它们是大多数真核生物信号通路的关键组成部分。14-3-3客户在结构和功能上高度多样化。它们参与例如细胞周期、凋亡、DNA损伤、转录和细胞运动。有趣的是，14-3-3蛋白的确切作用模式取决于客户蛋白。它们可以调节其客户的酶活性、构象、组装成复合物、亚细胞定位或介导聚集。因此，14-3-3s代表了真核生物中异常通用的信号转导调控平台。 蛋白质组学研究已经开始解开14-3-3蛋白质的客户库。然而，由于不同的实验方法，细胞系和检测灵敏度，这些研究之间的重叠是相当适度的。然而，也许更重要的是，标准的非定量方法无法解决该领域的主要问题之一：亚型特异性。几乎所有的生物体表达多种14-3-3亚型，但它们的特异性在体外几乎是相同的。然而，小鼠研究揭示了引人注目的亚型特异性表型，这表明当前的蛋白质组学方法不够灵敏和定量。 我建议结合联合收割机定量国家的最先进的质谱与定量成对相互作用分析，以全面表征客户端的所有七个人14-3-3异构体在同基因细胞系的蛋白库。我们将发现新的客户端蛋白，确定异构体特异性相互作用，并在分子水平上剖析特异性的基础。这些实验将辅以详细的功能随访研究。我将利用我在分子伴侣方面的丰富经验，分子伴侣就像14-3- 3一样，是一种丰富的分子，有数千种瞬时关联的客户蛋白质。特别是，我作为博士后研究员开发的高通量相互作用测定LUMIER与BACON，为瞬时14-3-3/客户端相互作用提供了灵敏和定量的读数。 总之，我们的研究将导致更全面地了解磷酸化依赖的信号通路，这些通路是发育中的关键介质，并在各种病理中受到错误调节。

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.