Evolution of proximal kinase network in T cells

项目1-摘要 T细胞是适应性免疫系统的关键组成部分，并且已经进化为检测外来抗原， 产生保护宿主免受细胞内病原体和恶性肿瘤侵害的反应。的激活 T细胞受体（TCR）导致T细胞内信号转导途径的启动，所述信号转导途径产生免疫应答。 适当的抗原触发反应。该计划项目的主要目标是了解 T细胞中分子和细胞回路的独特特征允许向自身pMHC的紧张性信号传导，而 还建立了刺激阈值，当克服该阈值时，产生对激动剂的强信号传导。在项目 1我们集中于由TCR激活的酪氨酸激酶，以了解这些信号传导的独特性质。 分子，将它们与在B细胞中起作用的类似蛋白质区分开来。我们的策略是集中精力 对激酶变体和特定功能的生物化学、生物物理学和细胞生物学研究， 用于确定支持信号转导的变体蛋白质的适合性的高通量方法 与B细胞相比，在T细胞中起作用。 TCR对细胞内信号通路的激活由三种酪氨酸激酶介导， 它们是Src、Syk和Tec家族的成员。尽管它们有着相同的信号机制 与造血谱系的其他细胞一样，TCR利用这些酪氨酸激酶的一组独特的成员， 家庭（即，Lck、ZAP-70和Itk）与独特的MHC结合共受体（即，CD4和CD8与Lck） 并磷酸化独特的支架蛋白（例如，LAT和SLP-76用于ZAP-70和Itk），其耦合到下游 信号通路这些成分与MHC基因同时进化，并保持了 在它们的序列中，这些特征标志着它们在整个脊椎动物进化树中是不同的。一个主要 项目1的目标是了解功能特化，这些特化使这些激酶发挥最佳作用 在T细胞中。 项目1有三个具体目标。在目标1中，我们将定义Src家族激酶Lck的特殊性质 对T细胞功能进行了优化。这些研究将包括Lck及其变体的单分子追踪， 监测它们的激活和定位，以及Lck和蛋白质之间相互作用的结构研究。 磷酸酶CD45。在目的2中，我们将研究ZAP-70和Syk在T细胞和B细胞中的分化， 在B细胞和T细胞中不同的这些激酶的激活方面。在目标3中，我们研究了 Tec激酶激活机制的差异，导致Itk膜上的不同行为 和Btk，它们分别是在T细胞和B细胞中起作用的Tec激酶。这些研究将 阐明了进化压力，塑造了Lck，ZAP-70和Itk的专门反应， T细胞特异性药物开发的框架。

PROJECT 1 - ABSTRACT T cells are key components of the adaptive immune system, and have evolved to detect foreign antigens and generate a response that protects the host from intracellular pathogens and malignancies. The activation of the T cell receptor (TCR) results in the initiation of signal transduction pathways inside the T cell that generate the appropriate antigen-triggered responses. The major goals of the Program Project are to understand how the distinct features of the molecules and cellular circuitry in T cells allows for tonic signaling to self-pMHC, while also establishing a stimulus threshold, which when overcome results in robust signaling to agonists. In Project 1 we focus on the tyrosine kinases activated by the TCR, to understand the distinct properties of these signaling molecules, differentiating them from similar proteins operative in B cells. Our strategy is to use focused biochemical, biophysical, and cell biological studies on kinase variants and particular functions, combined with high-throughput methods for determining the fitness of variant proteins in supporting signal-transduction functions in T cells, as contrasted to B cells. The activation of intracellular signaling pathways by the TCR is mediated by three kinds of tyrosine kinases, which are members of the Src, Syk, and Tec families. Although they share features of their signaling machinery with other cells of the hematopoietic lineage, TCRs utilize a distinct set of members of these tyrosine kinase families (i.e., Lck, ZAP-70 and Itk), interact with unique MHC binding co-receptors (i.e., CD4 and CD8 with Lck) and phosphorylate unique scaffold proteins (e.g., LAT and SLP-76 for ZAP-70 and Itk) that couple to downstream signaling pathways. These components evolved contemporaneously with the MHC genes, and have maintained features in their sequences that mark them as distinct throughout the vertebrate evolutionary tree. A principal goal of Project 1 is to understand the functional specializations that have optimized these kinases for their roles in T cells. Project 1 has three Specific Aims. In Aim 1, we will define the specialized properties of the Src-family kinase Lck that are optimized for T cell function. These studies will include single-molecule tracking of Lck and variants to monitor their activation and localization, as well as structural studies on the interaction between Lck and the phosphatase CD45. In Aim 2 we will study the differentiation of ZAP-70 and Syk in T cells and B cells, by focusing on aspects of the activation of these kinases that differ in B cells and T cells. In Aim 3 we study the specialized differences in the activation mechanisms of Tec kinases, leading to different behavior on the membrane of Itk and Btk, which are the Tec kinases operative in T cells and B cells, respectively. Together, these studies will illuminate evolutionary pressures that have molded specialized responses from Lck, ZAP-70 and Itk, providing a framework for the development of T cell-specific drugs.