Liu Huadong, Li Lei, Voss Courtney, Wang Feng, Liu Juewen, Li Shawn Shun-Cheng
From the ‡Department of Biochemistry and the Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1;
§Department of Chemistry, University of Waterloo, Waterloo, ON, N2L 3G1, Canada.
Mol Cell Proteomics. 2015 Jul;14(7):1846-58. doi: 10.1074/mcp.M115.047951. Epub 2015 Apr 23.
Cells of the immune system communicate with their environment through immunoreceptors. These receptors often harbor intracellular tyrosine residues, which, when phosphorylated upon receptor activation, serve as docking sites to recruit downstream signaling proteins containing the Src Homology 2 (SH2) domain. A systematic investigation of interactions between the SH2 domain and the immunoreceptor tyrosine-based regulatory motifs (ITRM), including inhibitory (ITIM), activating (ITAM), or switching (ITSM) motifs, is critical for understanding cellular signal transduction and immune function. Using the B cell inhibitory receptor CD22 as an example, we developed an approach that combines reciprocal or bidirectional phosphopeptide and SH2 domain array screens with in-solution binding assays to identify a comprehensive SH2-CD22 interaction network. Extending this approach to 194 human ITRM sequences and 78 SH2 domains led to the identification of a high-confidence immunoreceptor interactome containing 1137 binary interactions. Besides recapitulating many previously reported interactions, our study uncovered numerous novel interactions. The resulting ITRM-SH2 interactome not only helped to fill many gaps in the immune signaling network, it also allowed us to associate different SH2 domains to distinct immune functions. Detailed analysis of the NK cell ITRM-mediated interactions led to the identification of a network nucleated by the Vav3 and Fyn SH2 domains. We showed further that these SH2 domains have distinct functions in cytotoxicity. The bidirectional protein-peptide array approach described herein may be applied to the numerous other peptide-binding modules to identify potential protein-protein interactions in a systematic and reliable manner.
免疫系统的细胞通过免疫受体与周围环境进行通信。这些受体通常含有细胞内酪氨酸残基，当受体激活时这些残基发生磷酸化，作为招募含有Src同源2（SH2）结构域的下游信号蛋白的对接位点。系统研究SH2结构域与基于免疫受体酪氨酸的调节基序（ITRM）之间的相互作用，包括抑制性（ITIM）基序、激活性（ITAM）基序或转换性（ITSM）基序，对于理解细胞信号转导和免疫功能至关重要。以B细胞抑制性受体CD22为例，我们开发了一种方法，将相互或双向磷酸肽和SH2结构域阵列筛选与溶液内结合测定相结合，以鉴定一个全面的SH2 - CD22相互作用网络。将该方法扩展到194个人类ITRM序列和78个SH2结构域，从而鉴定出一个包含1137个二元相互作用的高可信度免疫受体相互作用组。除了重现许多先前报道的相互作用外，我们的研究还发现了许多新的相互作用。由此产生的ITRM - SH2相互作用组不仅有助于填补免疫信号网络中的许多空白，还使我们能够将不同的SH2结构域与不同的免疫功能联系起来。对自然杀伤细胞ITRM介导的相互作用的详细分析导致鉴定出一个以Vav3和Fyn SH2结构域为核心的网络。我们进一步表明，这些SH2结构域在细胞毒性中具有不同的功能。本文所述的双向蛋白质 - 肽阵列方法可应用于许多其他肽结合模块，以系统且可靠的方式鉴定潜在的蛋白质 - 蛋白质相互作用。
