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

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program Projects (P01)
Project #
5P01AI091580-03
Application #
8503583
Study Section
Special Emphasis Panel (ZAI1-RRS-I (S1))
Program Officer
Mallia, Conrad M
Project Start
2011-07-15
Project End
2016-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
3
Fiscal Year
2013
Total Cost
$1,601,045
Indirect Cost
$238,640
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Courtney, Adam H; Amacher, Jeanine F; Kadlecek, Theresa A et al. (2017) A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by CD45. Mol Cell 67:498-511.e6
Vercoulen, Yvonne; Kondo, Yasushi; Iwig, Jeffrey S et al. (2017) A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1. Elife 6:
Ashouri, Judith F; Weiss, Arthur (2017) Endogenous Nur77 Is a Specific Indicator of Antigen Receptor Signaling in Human T and B Cells. J Immunol 198:657-668
Ahsan, Nagib; Salomon, Arthur R (2017) Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling. Methods Mol Biol 1584:369-382
Myers, Darienne R; Zikherman, Julie; Roose, Jeroen P (2017) Tonic Signals: Why Do Lymphocytes Bother? Trends Immunol 38:844-857
Myers, Darienne R; Lau, Tannia; Markegard, Evan et al. (2017) Tonic LAT-HDAC7 Signals Sustain Nur77 and Irf4 Expression to Tune Naive CD4 T Cells. Cell Rep 19:1558-1571
Bandaru, Pradeep; Shah, Neel H; Bhattacharyya, Moitrayee et al. (2017) Deconstruction of the Ras switching cycle through saturation mutagenesis. Elife 6:
Hsu, Lih-Yun; Cheng, Debra A; Chen, Yiling et al. (2017) Destabilizing the autoinhibitory conformation of Zap70 induces up-regulation of inhibitory receptors and T cell unresponsiveness. J Exp Med 214:833-849
Courtney, Adam H; Lo, Wan-Lin; Weiss, Arthur (2017) TCR Signaling: Mechanisms of Initiation and Propagation. Trends Biochem Sci :
Huang, William Y C; Ditlev, Jonathon A; Chiang, Han-Kuei et al. (2017) Allosteric Modulation of Grb2 Recruitment to the Intrinsically Disordered Scaffold Protein, LAT, by Remote Site Phosphorylation. J Am Chem Soc 139:18009-18015

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