Human 14-3-3 Paralogs Differences Uncovered by Cross-Talk of Phosphorylation and Lysine Acetylation

The 14-3-3 protein family interacts with more than 700 different proteins in mammals, in part as a result of its specific phospho-serine/phospho-threonine binding activity. Upon binding to 14-3-3, the stability, subcellular localization and/or catalytic activity of the ligands are modified. Seven paralogs are strictly conserved in mammalian species. Although initially thought as redundant, the number of studies showing specialization is growing. We created a protein-protein interaction network for 14-3-3, kinases and their substrates signaling in human cells. We included information of phosphorylation, acetylation and other PTM sites, obtaining a complete representation of the 14-3-3 binding partners and their modifications. Using a computational system approach we found that networks of each 14-3-3 isoform are statistically different. It was remarkable to find that Tyr was the most phosphorylatable amino acid in domains of 14-3-3 epsilon partners. This, together with the over-representation of SH3 and Tyr_Kinase domains, suggest that epsilon could be involved in growth factors receptors signaling pathways particularly. We also found that within zeta’s network, the number of acetylated partners (and the number of modify lysines) is significantly higher compared with each of the other isoforms. Our results imply previously unreported hidden differences of the 14-3-3 isoforms interaction networks. The phosphoproteome and lysine acetylome within each network revealed post-transcriptional regulation intertwining phosphorylation and lysine acetylation. A global understanding of these networks will contribute to predict what could occur when regulatory circuits become dysfunctional or are modified in response to external stimuli.哺乳动物体内的14-3-3蛋白家族（14-3-3 protein family）可与700余种不同蛋白质发生相互作用，这一特性部分源于其特异性的磷酸丝氨酸/磷酸苏氨酸结合活性。当结合14-3-3蛋白后，结合配体的稳定性、亚细胞定位以及/或催化活性会发生改变。哺乳动物物种中严格保守存在7种旁系同源基因（paralogs）。尽管最初学界认为这些旁系同源基因功能冗余，但揭示其功能特化的研究数量正不断增加。我们构建了人类细胞中14-3-3蛋白、激酶及其底物信号通路的蛋白质相互作用网络（protein-protein interaction network），纳入了磷酸化、乙酰化及其他翻译后修饰（Post-translational modification, PTM）位点信息，完整呈现了14-3-3结合伴侣及其修饰状态。通过计算系统生物学方法分析，我们发现每种14-3-3亚型的相互作用网络均存在统计学显著性差异。值得注意的是，在14-3-3ε亚型结合伴侣的结构域中，酪氨酸是最易被磷酸化的氨基酸，结合SH3结构域与酪氨酸激酶（Tyr_Kinase）结构域的显著富集现象提示ε亚型可能特别参与生长因子受体信号通路。我们还发现，相较于其他所有亚型，ζ亚型的相互作用网络中乙酰化结合伴侣的数量（以及被修饰的赖氨酸残基数）显著更高。本研究结果揭示了此前未被报道的14-3-3亚型相互作用网络间的隐性差异。各网络中的磷酸化蛋白质组与赖氨酸乙酰化蛋白质组分析结果显示，磷酸化与赖氨酸乙酰化存在转录后调控的相互交织。对这些网络的全局认知将有助于预测当调控环路功能异常或响应外界刺激发生改变时可能出现的生物学变化。