Deconstructing and Reconstructing the T Cell Signaling Network

描述（由申请人提供）：T细胞-抗原呈递细胞相互作用的复杂性和目前用于研究它的简单生物化学方法需要不同的方法来理解TCR信号传导及其调控。共同的兴趣，现有的合作和独特的专业知识，为我们提供了一个独特的机会（博士Chakraborty，格罗夫斯，Kuriyan，鲁塞和韦斯）研究这个复杂的生物系统与更复杂和新颖的方法。我们在这里提出了一个全面的计划，以了解相互作用的分子的特异性和调节，并开发一个系统来研究TCR调节的信号转导事件的脂质双层系统，以模拟基本上发生在质膜的二维空间的事件。这种方法可能会产生新的见解分子相互作用和动力学不能在复杂的细胞系统中获得，而不是模仿的反应，发生在溶液中，扩散不限于2维。事实上，格罗夫斯博士和Kuriyan博士应用双层系统研究了将Ras和SOS蛋白定位在双层表面的影响，获得了与溶液动力学相比催化活性显著增加的意外结果。我们的总体目标是开发一个简单但强大的TCR信号传导的生物化学系统和计算模型，帮助我们理解调节的关键机制：（项目#1）TCR相关的免疫受体酪氨酸激活基序（ITAM）和LAT的酪氨酸磷酸化;以及（项目#2）通过鸟嘌呤核苷酸交换因子（GEF）RasGRP和SOS激活LAT下游的Ras。通过研究二维系统中分子的特异性、调节和活性，我们试图模拟质膜内小叶的表面。我们将从一个最小的简单系统开始，迭代地增加复杂性。我们希望能够增加空间复杂性和分子复杂性。我们将研究定义明确和重要的输出，并将承担不仅是生物化学和生物物理措施，但也计算工具来描述这个系统。我们将使用建模来比较简单和更复杂的系统，但也研究这些简单的系统如何偏离那些可在解决方案或更复杂的细胞系统。

DESCRIPTION (provided by applicant): The complexity of the T cell - antigen presenting cell interaction and the current simple biochemical approaches being used to study it requires different approach to understand TCR signaling and its regulation. Common interests, existing collaborations and unique expertise, provide us (Drs. Chakraborty, Groves, Kuriyan, Roose and Weiss) with a unique opportunity to study this complex biological system with more sophisticated and novel approaches. We present here a comprehensive program to understand the specificity and regulation of the interacting molecules and the development of a system to study TCR regulated signaling events on a lipid bilayer system to simulate the events occurring essentially on the two dimensional space of the plasma membrane. Such an approach is likely to yield novel insights into molecular interactions and kinetics not obtainable in complex cellular systems and not mimicked by reactions that occur in solution, where diffusion is not limited to 2 dimensions. Indeed, unanticipated results, with marked increase in catalytic activity compared to solution kinetics, were obtained by Dr. Groves and Kuriyan who applied a bilayer system to study the influence of localizing Ras and SOS proteins together at the surface of a bilayer. Our overall objective is to develop a simple but robust biochemical system and computational model of TCR signaling that helps us understand the critical mechanisms that regulate: (Project #1) tyrosine phosphorylation of the TCR-associated immunoreceptor tyrosine-based activation motifs (ITAMs) and of LAT; and, (Project #2) the activation of Ras downstream of LAT by the guanine nucleotide exchange factors (GEFs) RasGRP and SOS. By studying the specificity, regulation and the activities of the molecules involved in a two-dimensional system we attempt to mimic the surface of the inner leaflet of the plasma membrane. We will start with a minimal simple system and iteratively add complexity. We hope to be able to add spatial complexity and molecular complexity. We will study well-defined and important outputs, and will bring to bear not only biochemical and biophysical measures but also computational tools to characterize this system. We will use modeling to compare simple to more complex systems but also study how these simple systems deviate from those obtainable in solution or in more complex cellular systems.