Phosphoproteomic Analysis of Feedback Networks in T cell signaling

信号网络对于协调细胞功能以响应刺激是至关重要的。知识 这些网络的结构为理解它们的病理后果提供了基础 并为设计治疗干预措施提供了机会。这些网络的复杂性 而信号在细胞中的传输速度使映射它们成为一项艰巨的挑战。这个 阐明蜂窝信令网络结构的典型方法包括以下迭代过程 产生信号蛋白中断、结构域突变体和定点突变体，然后鉴定 通过一系列的细胞激活分析来确定每个突变体的基因。作为一种补充方法，现代 使用定量质谱学的蛋白质组学方法可以促进假说驱动的表征 通过提供细胞磷酸化和蛋白质-蛋白质相互作用的全局视图来了解信号通路 通过各种激活状态。 T细胞在对抗病毒、各种微生物和癌症的细胞免疫中起着核心作用。 这项建议的重点是阐明分子细节的T细胞信号通路使用这些 新技术。LCK酪氨酸激酶是T细胞活化的中枢调节因子，通过其 磷酸化状态。LCK在Tyr394位的自动磷酸化激活了该激酶，而在Tyr394位的LCK自动磷酸化 Tyr505使该激酶失活。之前有四种磷酸酶被证明作用于Lck Tyr394，但每一种是如何作用的 其中一个被招募到LCK，是否有其他负调控分子参与尚不清楚。这个 控制Lck在T细胞受体和下游之间合理分布的分子机制 像SLP76支架信号体这样的信号节点没有被很好地定义。在之前的资助中 期间，我们的研究小组发现，T细胞信号通路的下游成员调节 Lck及其底物的磷酸化。我们发现支架蛋白SLP-76控制两者 在Lck Tyr394处，T细胞受体信号的负反馈和正反馈循环。我们还发现，PLCγ1 调节TCR和SLP-76复合体内不同的Lck底物磷酸化。 为了获得对调节Lck活性和空间定位的途径的新见解，我们组装了 一个多学科团队应用新的定量蛋白质组学技术、生化方法和小鼠 提供网络详细信息的模型。我们在这个项目中要解决的核心问题是 SLP76和PLCγ1如何设定LCK激活的时空平衡，从而产生适当的T细胞 对抗原的反应。AIMS的成功完成将澄清调控蛋白的身份 在每个反馈环中使用，定义控制LCK细胞定位的分子因素，并定义 它们的生理作用。

Signaling networks are crucial for the orchestration of cellular functions in response to stimuli. Knowledge of the structure of these networks provides a basis for understanding the pathological consequences of their malfunction and offers opportunities for designing therapeutic interventions. The complexity of these networks and the speed with which signals are transmitted in cells makes mapping them a formidable challenge. The typical approach for elucidating the structure of cellular signaling networks involves an iterative process of creating signaling protein disruptions, domain mutants and site-directed mutants followed by characterization of each mutant through a battery of cellular activation assays. As a complementary approach, modern proteomic methods using quantitative mass spectrometry can facilitate the hypothesis-driven characterization of signaling pathways by providing a global view of cellular phosphorylation and protein-protein interactions through a variety of activation states. T cells play a central role in cell-mediated immunity against viruses, a variety of microbes, and cancer. This proposal focuses on the elucidation of the molecular details of the T cell signaling pathway using these new technologies. Lck tyrosine kinase is the central regulator of T cell activation regulated through its phosphorylation state. Lck autophosphorylation at Tyr394 activates the kinase, whereas phosphorylation at Tyr505 inactivates the kinase. Four phosphatases were shown previously to act on Lck Tyr394, but how each one is recruited to Lck and whether other negative regulatory molecules are involved is not understood. The molecular mechanism controlling the proper distribution of Lck between the T cell receptor and downstream signaling nodes such as the SLP76 scaffolded signalosome are not well defined. In the previous funding period, our research team discovered that downstream members of the T cell signaling pathway regulate the phosphorylation of Lck and its substrates. We discovered that the scaffold protein SLP-76 controls both negative and positive feedback loops in T cell receptor signaling at Lck Tyr394. We also discovered that PLCγ1 regulates differential Lck substrate phosphorylation within the TCR and the SLP-76 complex. To gain new insights into the pathways regulating Lck activity and spatial localization, we have assembled a multidisciplinary team to apply novel quantitative proteomic techniques, biochemical methods, and mouse models to provide a detailed view of the network. The central question that we will address in this project is how SLP76 and PLCγ1 set the spatial and temporal equilibrium of Lck activation resulting in appropriate T cell response to antigen. Successful completion of the aims will clarify the identity of the regulatory proteins employed in each feedback loop, define molecular factors controlling the cellular localization of Lck, and define their physiological role.