Specifically and regulation TCR pathway protein tyrosine kinases and phosphatases

虽然大多数参与TCR信号传导的分子可能已经被确定，但我们对基础信号传导和诱导信号传导状态的理解还相当初级。酪氨酸激酶和磷酸酶维持动态平衡，控制和维持基础状态，其功能活性或空间定位的变化对诱导酪氨酸磷酸化至关重要。本项目的重点是了解这些酪氨酸激酶和磷酸酶在质膜上的特异性和复杂调控。了解TCR细胞质z链和LAT蛋白磷酸化的调控将是我们关注的终点。我们将使用：纯化的重组蛋白；新型基因控制激酶抑制剂；一个模型二维脂质双层系统，包含一定数量的重组蛋白；并且，计算建模来描述和比较简单的双层系统与现有的充分研究的模型细胞系统的完整细胞或膜。我们将以迭代的方式将这种二维模型的生化行为与细胞系统进行比较，增加组件的复杂性并应用计算建模。我们期望这样的分析能够揭示预期的以及意外的行为。我们还希望通过对简单系统与更复杂系统的比较，揭示可能以前未被认识到的调控途径的存在，例如正反馈和负反馈电路。我们清楚地认识到，我们无法模拟TCR信号传导的所有复杂性，但我们的目标是了解控制TCR z链酪氨酸磷酸化的详细机制，以及这些事件如何导致LAT磷酸化。项目#1的总体目标是了解蛋白质酪氨酸激酶的特异性和调控

Although most of the molecules involved in TCR signaling have likely been identified, our understanding of the basal signaling versus the induced signaling states are rather rudimentary. Tyrosine kinases and phosphatases maintain the dynamic equilibrium that controls and maintains the both the basal state and changes in their functional activities or spatial localization are important for Induced tyrosine phosphorylation. This project focuses on understanding the specificity and complex regulation of these tyrosine kinases and phosphatases on at the plasma membrane. Understanding the regulation of phosphorylation of the TCR cytoplasmic Z-chain as well as of LAT proteins will be the endpoints we will focus on. We will use: purified recombinant proteins; novel genetically controlled kinase inhibitors; a model two dimensional lipid bilayer system containing defined quantities of recombinant proteins; and, computational modeling to describe and compare the simple bilayer system to existing well-studied intact cekks or membranes of model cellular systems. We will compare the biochemical behavior of such two dimensional models to cellular systems in an iterative way, increasing component complexity and by applying computational modeling. We expect such analyses to reveal expected as well as unanticipated behaviors. We also expect the comparison of the simple system to more complex systems to reveal the presence of regulatory pathways that may not have previously been appreciated, such as positive and negative feedback circuitry. We clearly recognize that we cannot model all of the complexity of TCR signaling but we aim to understand the detailed mechanisms controlling tyrosine phosphorylation of the TCR Z-chain and how these events lead to LAT phosphorylation. The overall goal of project #1 is to understand the specificity and regulation of protein tyrosine kinases and phosphatases involved In TCR signaling that lead to ITAM and LAT phosphorylation at the membrane surface. We will: 1) define the molecular basis for specificity for Lck and ZAP-70 for ITAMs and LAT, respectively; 2) develop an analog sensitive inhibitor system for Lck; 3) define the mechanisms that control ITAM phosphorylation by Lck; and, 4) define the mechanisms that control LAT phosphorylation by ZAP-70.