Signaling networks are crucial for the orchestration of cellular functions in response to stimuli. Knowledge of the structure of these networks provides a basis for understanding the pathological consequences of their malfunction and offers opportunities for designing therapeutic interventions. The complexity of these networks and the speed with which signals are transmitted in cells makes mapping them a formidable challenge. The typical approach for elucidating the structure of cellular signaling networks involves an iterative process of creating signaling protein disruptions, domain mutants and site-directed mutants followed by characterization of each mutant through a battery of cellular activation assays. As a complementary approach, modern proteomic methods using quantitative mass spectrometry can facilitate the hypothesis-driven characterization of signaling pathways by providing a global view of cellular phosphorylation and protein-protein interactions through a variety of activation states. T cells play a central role in cell-mediated immunity against viruses, a variety of microbes, and cancer. This proposal focuses on the elucidation of the molecular details of the T cell signaling pathway using these new technologies. Lck tyrosine kinase is the central regulator of T cell activation regulated through its phosphorylation state. Lck autophosphorylation at Tyr394 activates the kinase, whereas phosphorylation at Tyr505 inactivates the kinase. Four phosphatases were shown previously to act on Lck Tyr394, but how each one is recruited to Lck and whether other negative regulatory molecules are involved is not understood. The molecular mechanism controlling the proper distribution of Lck between the T cell receptor and downstream signaling nodes such as the SLP76 scaffolded signalosome are not well defined. In the previous funding period, our research team discovered that downstream members of the T cell signaling pathway regulate the phosphorylation of Lck and its substrates. We discovered that the scaffold protein SLP-76 controls both negative and positive feedback loops in T cell receptor signaling at Lck Tyr394. We also discovered that PLC?1 regulates differential Lck substrate phosphorylation within the TCR and the SLP-76 complex. To gain new insights into the pathways regulating Lck activity and spatial localization, we have assembled a multidisciplinary team to apply novel quantitative proteomic techniques, biochemical methods, and mouse models to provide a detailed view of the network. The central question that we will address in this project is how SLP76 and PLC?1 set the spatial and temporal equilibrium of Lck activation resulting in appropriate T cell response to antigen. Successful completion of the aims will clarify the identity of the regulatory proteins employed in each feedback loop, define molecular factors controlling the cellular localization of Lck, and define their physiological role.

Public Health Relevance

A comprehensive definition of the T cell signaling network is absolutely required to understand the balance between activating and inhibitory pathways that combine to establish normal physiology and the disruption of this interplay that leads to a variety of disease states including immunodeficiency, Type 1 diabetes mellitus, systemic lupus erythematosus, and rheumatoid arthritis. Knowledge of the intracellular structure of these networks provides a basis for understanding the pathological consequences of their malfunction and offers opportunities for designing therapeutic interventions. In this proposal we apply modern methods in quantitative mass spectrometry to drive the molecular characterization of newly discovered T cell signaling circuits.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI083636-08
Application #
9684541
Study Section
Cellular and Molecular Immunology - B Study Section (CMIB)
Program Officer
Mallia, Conrad M
Project Start
2010-06-15
Project End
2022-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
8
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Brown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
02912
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
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
Belmont, Judson; Gu, Tao; Mudd, Ashley et al. (2017) A PLC-?1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T-Cell Receptor and SLP-76 Complex. J Proteome Res 16:2729-2742
Higdon, Lauren E; Maltzman, Jonathan S (2017) Expanding the Toolkit for the Study of Allospecific B and T Cell Responses. Transplantation 101:2661-2662
Higdon, Lauren E; Maltzman, Jonathan S (2017) Expanding the Toolkit for the Study of Allo-specific B and T Cell Responses. Transplantation :
Ahsan, Nagib; Rao, R Shyama Prasad; Gruppuso, Philip A et al. (2016) Targeted proteomics: Current status and future perspectives for quantification of food allergens. J Proteomics 143:15-23
Helou, Ynes A; Petrashen, Anna P; Salomon, Arthur R (2015) Vav1 Regulates T-Cell Activation through a Feedback Mechanism and Crosstalk between the T-Cell Receptor and CD28. J Proteome Res 14:2963-75
Helou, Ynes A; Salomon, Arthur R (2015) Protein networks and activation of lymphocytes. Curr Opin Immunol 33:78-85
Goodfellow, Hanna Sjölin; Frushicheva, Maria P; Ji, Qinqin et al. (2015) The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway. Sci Signal 8:ra49

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