Triple SILAC to determine stimulus specific interactions in the Wnt pathway.
Triple SILAC to determine stimulus specific interactions in the Wnt pathway.
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三席拉克（Triple Silac）确定Wnt途径中的刺激特定相互作用。
DOI：
10.1021/pr200740a
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发表时间：
2012-02-03
影响因子：
4.4
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
--
作者：
Hilger, Maximiliane;Mann, Matthias
关键词：
许多重要的调节功能是由动态的多蛋白复合物来完成的，这些复合物根据不同的刺激来调整它们的组成和活性。在这里，我们采用定量亲和纯化结合质谱法，有效地分离特定的相互作用背景，但增加了一个额外的定量维度，明确表征刺激依赖的相互作用。这是通过SILAC以三重标记形式实现的，其中对具有诱饵、具有诱饵和刺激以及不具有诱饵的下拉进行相互量化。作为诱饵，我们使用全长蛋白融合的绿色荧光蛋白和内源性控制下表达。我们将这项技术应用于Wnt信号传导，这在发育，组织稳态和癌症中很重要，并研究了关键组分APC，Axin-1，DVL 2和CtBP 2与差异途径激活的相互作用。我们的筛选鉴定了许多已知的Wnt信号传导复合物组分，并将新的候选物与Wnt信号传导联系起来，包括FAM 83 B和Girdin，我们发现它们是多个Wnt通路参与者的相互作用物。Girdin与DVL 2的结合不依赖于配体Wnt 3a的刺激，但以刺激依赖性方式与Axin-1和APC结合。核心破坏复合物本身，其调节β-连环蛋白稳定性作为经典Wnt信号传导的关键步骤，基本上保持不变。蛋白质相互作用通常根据细胞环境进行调节，但通常以静态形式进行研究。在这里，我们开发了一个基于三重SILAC的动态交互屏幕，并将其应用于Wnt信号通路。我们的筛选鉴定了许多已知的Wnt信号传导复合物组分，并将新的候选物与Wnt信号传导联系起来，包括FAM 83 B和Girdin，我们发现它们是多个Wnt通路参与者的相互作用物。
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. Protein interactions are often regulated depending on the cellular context but are generally studied in a static format. Here we develop a triple-SILAC based dynamic interaction screen and apply it to the Wnt signaling pathway. 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.
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影响因子：
46.9
作者：
Blagoev, B;Ong, SE;Mann, M
通讯作者：
Mann, M
影响因子：
3.3
作者：
Guarguaglini, G;Duncan, PI;Nigg, EA
通讯作者：
Nigg, EA
影响因子：
21.3
作者：
Angers, S;Thorpe, CJ;Moon, RT
通讯作者：
Moon, RT
影响因子：
64.8
作者：
Gavin, AC;Aloy, P;Superti-Furga, G
通讯作者：
Superti-Furga, G
DOI：
10.1093/bioinformatics/btn615
发表时间：
2009-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Carbon S;Ireland A;Mungall CJ;Shu S;Marshall B;Lewis S;AmiGO Hub;Web Presence Working Group
通讯作者：
Web Presence Working Group