Phosphoproteomic Analysis of Feedback Networks in T cell signaling

信号网络对于响应刺激的细胞功能的协调至关重要。知识 这些网络的结构为理解其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 并且信号在小区中传输的速度使得映射它们成为一个艰巨的挑战。的 用于阐明蜂窝信令网络结构的典型方法包括迭代过程 产生信号蛋白破坏、结构域突变体和定点突变体， 通过一系列的细胞活化试验来检测每一个突变体。作为一种补充方法，现代 使用定量质谱的蛋白质组学方法可以促进假设驱动的表征 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， 通过各种激活状态。 T细胞在针对病毒、各种微生物和癌症的细胞介导的免疫中发挥核心作用。 该建议的重点是阐明的分子细节的T细胞信号通路使用这些 新技术Lck酪氨酸激酶是T细胞活化的中心调节因子，通过其调节T细胞活化。 磷酸化状态Lck在Tyr 394处的自磷酸化激活激酶，而在Tyr 394处的磷酸化激活激酶。 Tyr 505使激酶失活。以前有四种磷酸酶作用于Lck Tyr 394，但每种磷酸酶如何作用于Lck Tyr 394？ 一个被募集到Lck，是否涉及其它负调节分子尚不清楚。的 控制Lck在T细胞受体和下游之间适当分布的分子机制 诸如SLP 76支架信号体的信号传导节点没有被很好地定义。在此前的融资中， 在此期间，我们的研究小组发现，T细胞信号通路的下游成员调节T细胞信号通路。 Lck及其底物的磷酸化。我们发现，支架蛋白SLP-76控制着这两种功能， Lck Tyr 394处T细胞受体信号传导的负反馈和正反馈环。我们还发现PLCγ1 调节TCR和SLP-76复合物内的不同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 一个多学科的团队，应用新的定量蛋白质组学技术，生物化学方法，和小鼠 模型，以提供网络的详细视图。我们在这个项目中要解决的中心问题是 SLP 76和PLCγ1如何设置Lck激活的空间和时间平衡，从而产生适当的T细胞 对抗原的反应这些目标的成功完成将阐明调控蛋白的身份 在每个反馈回路中使用，定义控制Lck细胞定位的分子因子，并定义 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.