Laboratory for Immunochemical Circuits, La Jolla Institute for Immunology, La Jolla, CA, USA.
Center of Autoimmunity and Inflammation, La Jolla Institute for Immunology, La Jolla, CA, USA.
Nat Methods. 2024 Jun;21(6):1033-1043. doi: 10.1038/s41592-024-02256-z. Epub 2024 Apr 29.
Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here we describe the high-throughput, functional assessment of phosphorylation sites through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1, which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
驱动基因表达的信号通路通常被描绘为具有十几个标志性的磷酸化和转录事件。实际上，成千上万的动态翻译后修饰（PTM）协调着几乎每一种细胞功能，而我们缺乏技术来在大规模上找到这些庞大的生化途径和遗传回路之间的因果关系。在这里，我们通过开发以 PTM 为中心的碱基编辑技术并结合表型筛选来描述磷酸化位点的高通量功能评估，该筛选由时间分辨的磷酸蛋白质组学指导。我们以 T 细胞激活作为模型，观察到数百个调节 NFAT 转录活性的未研究磷酸化位点。我们确定了 PHLPP1 的磷酸化介导的核定位，它促进 NFAT 但抑制 NFκB 活性。我们还发现，特定的磷酸位点突变体可以以微妙但不同的模式改变基因表达，这表明了精细调节转录反应的潜力。总的来说，PTM 位点的碱基编辑筛选为在信号通路内剖析 PTM 功能提供了一个强大的平台。
