Triple SILAC to Determine Stimulus Specific Interactions in the Wnt Pathway

Many important regulatory functions are performed by dynamic multiprotein complexes that adapt their composition and activity in response to different stimuli. Here we employ quantitative affinity purification coupled with mass spectrometry to efficiently separate background from specific interactors but add an additional quantitative dimension to explicitly characterize stimulus-dependent interactions. This is accomplished by SILAC in a triple-labeling format, in which pull-downs with bait, with bait and stimulus, and without bait are quantified against each other. As baits, we use full-length proteins fused to the green fluorescent protein and expressed under endogenous control. We applied this technology to Wnt signaling, which is important in development, tissue homeostasis, and cancer, and investigated interactions of the key components APC, Axin-1, DVL2, and CtBP2 with differential pathway activation. Our screens identify many known Wnt signaling complex components and link novel candidates to Wnt signaling, including FAM83B and Girdin, which we found as interactors to multiple Wnt pathway players. Girdin binds to DVL2 independent of stimulation with the ligand Wnt3a but to Axin-1 and APC in a stimulus-dependent manner. The core destruction complex itself, which regulates beta-catenin stability as the key step in canonical Wnt signaling, remained essentially unchanged.动态多蛋白复合物可响应不同刺激并调整其组成与活性，从而执行诸多重要的调控功能。本研究采用定量亲和纯化联用质谱技术（quantitative affinity purification coupled with mass spectrometry），在有效区分非特异性背景互作与特异性互作蛋白的基础上，新增定量维度以实现刺激依赖性互作关系的明确表征。该实验通过采用三重标记格式的细胞培养氨基酸稳定同位素标记（SILAC）技术得以实现：分别对诱饵蛋白（bait）组、诱饵蛋白+刺激组以及无诱饵对照组的亲和下拉样本进行相互定量比对。本研究以融合绿色荧光蛋白（green fluorescent protein, GFP）且在内源调控下表达的全长蛋白作为诱饵蛋白。我们将该技术应用于在发育、组织稳态及癌症发生中发挥重要作用的Wnt信号通路（Wnt signaling），针对关键组分APC、Axin-1、DVL2及CtBP2在不同通路激活状态下的互作情况展开研究。本筛选实验鉴定出众多已知的Wnt信号通路复合物组分，并将包括FAM83B和Girdin在内的新候选分子关联至Wnt信号通路，我们发现二者可与多个Wnt通路相关分子发生互作。Girdin与DVL2的结合不受配体Wnt3a刺激的影响，但与Axin-1和APC的结合则呈现刺激依赖性。作为经典Wnt信号通路（canonical Wnt signaling）关键步骤的β-连环蛋白（beta-catenin）稳定性调控核心降解复合物，其组成基本未发生改变。