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鉴定，但我们对基础信号传导与诱导信号传导状态的理解是相当初步的。酪氨酸激酶和磷酸酶维持动态平衡，控制和维持基础状态和它们的功能活性或空间定位的变化对于诱导的酪氨酸磷酸化是重要的。该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的调节。了解TCR胞浆Z链和LAT蛋白磷酸化的调节将是我们重点关注的终点，我们将用途：纯化的重组蛋白，新型的基因控制的激酶抑制剂，含有定量重组蛋白的模型二维脂质双层系统，和计算建模，以描述和比较简单的双层系统与现有的充分研究的完整的细胞膜或模型细胞系统的膜。我们将以迭代的方式比较这种二维模型与细胞系统的生化行为，增加组件的复杂性并应用计算建模。我们希望这样的分析能够揭示预期和未预期的行为。我们还希望将简单系统与更复杂系统进行比较，以揭示以前可能未被认识到的调节途径的存在，例如正反馈和负反馈回路。我们清楚地认识到，我们不能模拟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酪氨酸激酶的特异性和调节， TCR信号传导中涉及的磷酸酶，导致膜表面的ITAM和LAT磷酸化。我们将：1）定义Lck和ZAP-70分别对ITAM和LAT的特异性的分子基础; 2）开发Lck的类似物敏感性抑制剂系统; 3）定义通过Lck控制ITAM磷酸化的机制;和4）定义通过ZAP-70控制LAT磷酸化的机制。

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.