Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor
Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor
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DOI：
10.1073/pnas.1503286112
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发表时间：
2015-03
期刊：
影响因子：
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通讯作者：
Ruijun Tian;Haopeng Wang;G. Gish;E. Petsalaki;A. Pasculescu;Yu Shi;M. Mollenauer;R. Bagshaw;
中科院分区：
文献类型：
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作者：
Ruijun Tian;Haopeng Wang;G. Gish;E. Petsalaki;A. Pasculescu;Yu Shi;M. Mollenauer;R. Bagshaw;
意义 在复杂的细胞间相互作用（例如免疫细胞之间）过程中，细胞内信号传导提供了导致细胞反应的重要线索。这些信号事件的整体特征对于系统地探索和理解它们如何最终控制细胞命运至关重要。然而，在细胞间相互作用过程中涉及多个相互作用受体的生理相关条件下细胞间信号传导的蛋白质组表征仍然具有挑战性。我们开发了一种综合蛋白质组策略，用于定量分析蛋白质磷酸化和蛋白质-蛋白质相互作用介导的细胞间信号转导事件。我们应用这种方法通过阻断 CD28 共刺激受体与其配体的相互作用来确定单个受体-配体对在 T 细胞刺激过程中的影响。这种方法通常适用于复杂细胞相互作用过程中参与信号传导的其他跨膜受体。细胞间信号传导的系统表征接近涉及直接细胞间接触的刺激的生理条件是具有挑战性的。我们描述了一种蛋白质组学策略来分析 T 细胞共刺激受体 CD28 介导的生理信号传导。我们通过蛋白质磷酸化的整体分析确定了细胞与细胞直接接触过程中 CD28 激活的信号通路。为了定义直接的 CD28 靶标，我们使用 CD28 细胞质区域的磷酸化形式来获得 CD28 相互作用组。选定的 CD28 相互作用蛋白的相互作用谱在体内进一步表征，以放大 CD28 相互作用组。整体磷酸化和相互作用组分析的结合揭示了磷酸化对 CD28 及其相互作用组的广泛调节。在受 CD28 信号传导影响的细胞磷蛋白中，功能研究涉及 CapZ 相互作用蛋白 (CapZIP)（肌动蛋白细胞骨架的调节因子）。本文应用的组合方法广泛适用于表征与具有短胞质尾部的膜受体相关的信号网络。
Significance Intracellular signaling during complex cell–cell interactions, such as between immune cells, provides essential cues leading to cell responses. Global characterization of these signaling events is critical for systematically exploring and understanding how they eventually control cell fate. However, proteome-wide characterization of intercellular signaling under physiologically relevant conditions involving multiple interacting receptors during cell–cell interactions remains challenging. We developed an integrated proteomic strategy for quantitatively profiling intercellular-signaling events mediated by protein phosphorylation and protein–protein interaction. We applied this approach to determine the influence of a single receptor-ligand pair during T-cell stimulation by blocking the interaction of the CD28 costimulatory receptor with its ligand. This approach is generally applicable to other transmembrane receptors involved in signaling during complex cell interactions. Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell–cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell–cell contact by global analysis of protein phosphorylation. To define immediate CD28 targets, we used phosphorylated forms of the CD28 cytoplasmic region to obtain the CD28 interactome. The interaction profiles of selected CD28-interacting proteins were further characterized in vivo for amplifying the CD28 interactome. The combination of the global phosphorylation and interactome analyses revealed broad regulation of CD28 and its interactome by phosphorylation. Among the cellular phosphoproteins influenced by CD28 signaling, CapZ-interacting protein (CapZIP), a regulator of the actin cytoskeleton, was implicated by functional studies. The combinatorial approach applied herein is widely applicable for characterizing signaling networks associated with membrane receptors with short cytoplasmic tails.