Genetic Incorporation of a Photo-Crosslinkable Amino Acid Reveals Novel Protein Complexes with GRB2 in Mammalian Cells
Genetic Incorporation of a Photo-Crosslinkable Amino Acid Reveals Novel Protein Complexes with GRB2 in Mammalian Cells
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DOI：
10.1016/j.jmb.2010.12.022
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发表时间：
2011-02-18
影响因子：
5.6
通讯作者：
Sakamoto, Kensaku
中科院分区：
文献类型：
--
作者：
Hino, Nobumasa;Oyama, Masaaki;Sakamoto, Kensaku
细胞信号传导途径基本上是通过信号传导蛋白中各种类型的结合结构域的分布来组织的，其中每个结构域与特定的靶分子结合。虽然这些目标的识别是至关重要的映射的途径，亲和力为基础的或copurification方法是不够的，以区分直接和间接的相互作用，在细胞环境。在本研究中，我们开发了另一种方法，涉及光交联氨基酸的遗传编码。因此，在人胚肾细胞中，将对-三氟甲基-二氮杂环丙烯基-L-苯丙氨酸掺入到衔接蛋白GRB 2的Src同源2（SH2）结构域中的确定位点。这些细胞在表皮生长因子刺激后暴露于365 nm光，可交联的GRB 2-SH 2结构域仅与直接相互作用的蛋白质形成共价键。蛋白质组学质谱分析分别从与GRB 2的Src同源3结构域非共价结合的蛋白质中鉴定出GRB 2-SH 2的这些直接结合剂。除了两个信号相关蛋白（GIT 1和AF6），异质核核糖核蛋白F，H1和H2，因此被确定为新的直接粘合剂。结果揭示了细胞信号蛋白与参与mRNA加工的核机制之间的联系，并证明了遗传编码的光交联剂在绘制细胞中蛋白质相互作用方面的有用性。(C)2010爱思唯尔有限公司版权所有。
Cell signaling pathways are essentially organized through the distribution of various types of binding domains in signaling proteins, with each domain binding to specific target molecules. Although identification of these targets is crucial for mapping the pathways, affinity-based or copurification methods are insufficient to distinguish between direct and indirect interactions in a cellular context. In the present study, we developed another approach involving the genetic encoding of a photo-crosslinkable amino acid. p-Trifluoromethyl-diazirinyl-L-phenylalanine was thus incorporated at a defined site in the Src homology 2 (SH2) domain of the adaptor protein GRB2 in human embryonic kidney cells. These cells were exposed to 365-nm light after an epidermal growth factor stimulus, and the crosslinkable GRB2-SH2 domain exclusively formed covalent bonds with directly interacting proteins. Proteomic mass spectrometry analysis identified these direct binders of GRB2-SH2 separately from the proteins noncovalently bound to the Src homology 3 domains of GRB2. In addition to two signaling-associated proteins (GIT1 and AF6), the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were thus identified as novel direct binders. The results revealed a connection between the cell signaling protein and the nuclear machinery involved in mRNA processing, and demonstrated the usefulness of genetically encoded photo-crosslinkers for mapping protein protein interactions in cells. (C) 2010 Elsevier Ltd. All rights reserved.