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 cekks 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 Lek and ZAP-70 for ITAMs and LAT, respectively;2) develop an analog sensitive inhibitor system for Lek;3) define the mechanisms that control ITAM phosphorylation by Lek;and, 4) define the mechanisms that control LAT phosphorylation by ZAP-70.
Biochemical signaling events by the T cell receptor (TCR) for antigen controls T cell responses but our understanding of these signaling events Is very rudimentary. These studies aim to understand how the enzymes, kinases and phosphatases, that the TCR controls are regulated on a surface model membrane, as they would be in a cell. These studies could lead to novel therapies of T cells mediated diseases.
|Lo, Wan-Lin; Shah, Neel H; Ahsan, Nagib et al. (2018) Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT. Nat Immunol 19:733-741|
|Courtney, Adam H; Lo, Wan-Lin; Weiss, Arthur (2018) TCR Signaling: Mechanisms of Initiation and Propagation. Trends Biochem Sci 43:108-123|
|Shah, Neel H; Löbel, Mark; Weiss, Arthur et al. (2018) Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen. Elife 7:|
|Cantor, Aaron J; Shah, Neel H; Kuriyan, John (2018) Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites. Proc Natl Acad Sci U S A 115:E7303-E7312|
|Pielak, Rafal M; O'Donoghue, Geoff P; Lin, Jenny J et al. (2017) Early T cell receptor signals globally modulate ligand:receptor affinities during antigen discrimination. Proc Natl Acad Sci U S A 114:12190-12195|
|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|
|Ahsan, Nagib; Belmont, Judson; Chen, Zhuo et al. (2017) Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction. J Proteomics 165:69-74|
|Ahsan, Nagib; Salomon, Arthur R (2017) Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling. Methods Mol Biol 1584:369-382|
|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|
|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:|
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