Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry , Am Klopferspitz 18, D-82152 Martinsried, Germany.
J Proteome Res. 2012 Feb 3;11(2):982-94. doi: 10.1021/pr200740a. Epub 2011 Nov 18.
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.
许多重要的调控功能是由动态的多蛋白复合物来执行的，这些复合物能够根据不同的刺激来改变其组成和活性。在这里，我们采用定量亲和纯化结合质谱的方法，有效地将背景与特异性相互作用体分离，但同时又增加了一个定量维度，以明确地描述刺激依赖性相互作用。这是通过 SILAC 三重标记格式来实现的，其中诱饵蛋白、诱饵蛋白加刺激物以及没有诱饵蛋白的 pull-down 相互之间进行定量比较。我们使用全长蛋白融合绿色荧光蛋白，并在内源性控制下表达作为诱饵。我们将这项技术应用于 Wnt 信号通路，该信号通路在发育、组织稳态和癌症中都很重要，并研究了关键成分 APC、Axin-1、DVL2 和 CtBP2 与差异途径激活的相互作用。我们的筛选确定了许多已知的 Wnt 信号复合物成分，并将新的候选物与 Wnt 信号联系起来，包括 FAM83B 和 Girdin，我们发现它们是多个 Wnt 通路成员的相互作用蛋白。Girdin 与 DVL2 的结合不受配体 Wnt3a 的刺激，但与 Axin-1 和 APC 的结合则是刺激依赖性的。作为经典 Wnt 信号通路中调节β-catenin 稳定性的关键步骤，核心破坏复合物本身基本没有变化。
