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.

Public Health Relevance

T cells play critical roles in nearly all immune responses and diseases. T cell function is controlled by signals mediated by the T cell antigen receptor (TCR), but our understanding of how these signals are generated is rather rudimentary, A more precise understanding of how TCR signals are generated, the goal of these studies, could lead to novel therapies of T cell-mediated disease, including arthritis and lupus. PROJECT 1: Title: - Specificity and Regulation TCR Pathway Protein Tyrosine Kinases and Phosphatases Project Leader: WEISS, A PROJECT 1 DESCRIPTION (provided by applicant): 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 ?-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 cells 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 ?-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

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Program Projects (P01)
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Special Emphasis Panel (ZAI1-RRS-I (S1))
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Mallia, Conrad M
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University of California San Francisco
Internal Medicine/Medicine
Schools of Medicine
San Francisco
United States
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Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen et al. (2014) Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics. Science 345:50-4
Tan, Ying Xim; Manz, Boryana N; Freedman, Tanya S et al. (2014) Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling. Nat Immunol 15:186-94
Au-Yeung, Byron B; Zikherman, Julie; Mueller, James L et al. (2014) A sharp T-cell antigen receptor signaling threshold for T-cell proliferation. Proc Natl Acad Sci U S A 111:E3679-88
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Au-Yeung, Byron B; Melichar, Heather J; Ross, Jenny O et al. (2014) Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development. Nat Immunol 15:687-94
Jenkins, Misty R; Stinchcombe, Jane C; Au-Yeung, Byron B et al. (2014) Distinct structural and catalytic roles for Zap70 in formation of the immunological synapse in CTL. Elife 3:e01310
Shekhar, Karthik; Brodin, Petter; Davis, Mark M et al. (2014) Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE). Proc Natl Acad Sci U S A 111:202-7
Ross, Jenny O; Melichar, Heather J; Au-Yeung, Byron B et al. (2014) Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns. Proc Natl Acad Sci U S A 111:E2550-8
Chakraborty, Arup K; Weiss, Arthur (2014) Insights into the initiation of TCR signaling. Nat Immunol 15:798-807
Lin, Wan-Chen; Iversen, Lars; Tu, Hsiung-Lin et al. (2014) H-Ras forms dimers on membrane surfaces via a protein-protein interface. Proc Natl Acad Sci U S A 111:2996-3001

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