Transgenic mouse proteomics identifies new 14-3-3-associated proteins involved in cytoskeletal rearrangements and cell signaling
Transgenic mouse proteomics identifies new 14-3-3-associated proteins involved in cytoskeletal rearrangements and cell signaling
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DOI：
10.1074/mcp.m600147-mcp200
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发表时间：
2006-12-01
影响因子：
7
通讯作者：
Acker-Palmer, Amparo
中科院分区：
文献类型：
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作者：
Angrand, Pierre-Olivier;Segura, Inmaculada;Acker-Palmer, Amparo
蛋白质-蛋白质相互作用的鉴定对于解开细胞过程和信号转导的生化机制至关重要。在这里，我们描述了，第一次，应用串联亲和纯化（TAP）和LC-MS方法来表征转基因小鼠的蛋白质复合物。在转基因小鼠中开发的TAP策略允许复合物在其生理环境中与可能仅在某些组织中特异性表达的蛋白质接触，同时确保TAP蛋白质与其结合伴侣的正确化学计量，并且代表了迄今为止使用的蛋白质组学方法的新奇。产生表达TAP标记的14-3-3 ζ蛋白的小鼠系，并测定蛋白质相互作用。14-3-3蛋白质是细胞信号传导的一般调节因子，占大脑总蛋白质的1%。该研究允许鉴定近40种新的14-3-3 ζ结合蛋白。这些相互作用的生化和功能表征揭示了14-3-3 zeta在几种信号通路中的新作用机制，例如谷氨酸受体信号传导通过与Homer同源物3（Homer 3）结合，以及在细胞骨架重排和棘形态发生中通过结合和调节由G蛋白偶联受体激酶-相互作用物1（GIT 1）和p21-活化的激酶相互作用交换因子β（beta PIX）。
Identification of protein-protein interactions is crucial for unraveling cellular processes and biochemical mechanisms of signal transduction. Here we describe, for the first time, the application of the tandem affinity purification ( TAP) and LC-MS method to the characterization of protein complexes from transgenic mice. The TAP strategy developed in transgenic mice allows the emplacement of complexes in their physiological environment in contact with proteins that might only be specifically expressed in certain tissues while simultaneously ensuring the right stoichiometry of the TAP protein versus their binding partners and represents a novelty in proteomics approaches used so far. Mouse lines expressing TAP-tagged 14-3-3 zeta protein were generated, and protein interactions were determined. 14-3-3 proteins are general regulators of cell signaling and represent up to 1% of the total brain protein. This study allowed the identification of almost 40 novel 14-3-3 zeta-binding proteins. Biochemical and functional characterization of some of these interactions revealed new mechanisms of action of 14-3-3 zeta in several signaling pathways, such as glutamate receptor signaling via binding to homer homolog 3 (Homer 3) and in cytoskeletal rearrangements and spine morphogenesis by binding and regulating the activity of the signaling complex formed by G protein-coupled receptor kinase-interactor 1 (GIT1) and p21-activated kinase-interacting exchange factor beta(beta PIX).