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...