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 phosphoproteomic methods in mass spectrometry can facilitate the hypothesis-driven characterization of signaling pathways by providing a global view of cellular phosphorylation through a variety of activation states or perturbed at specific pathway proteins or phosphorylation sites. This information provides a rational basis for generating hypotheses about signaling pathway structure. We then test resulting hypotheses by monitoring the global consequences of disrupting specific nodes (proteins or phosphorylation sites) in the network. T cells play a central role in cell-mediated immunity against viruses, a variety of microbes, and cancer. The present proposal focuses on the elucidation of the molecular details of the T cell signaling pathway. To gain new insights into the pathways leading to T cell activation, novel phosphoproteomic techniques are combined with traditional methods to provide a detailed view of the network of phosphorylation events in T cells activated through the T cell receptor. The promise of this unique approach is illustrated in preliminary phosphoproteomic studies of T cells with a disrupted receptor proximal protein tyrosine kinase, Zap-70. The expected T cell signaling pathway structure was replicated and 96 novel phosphorylation events were discovered. These novel phosphorylation sites are located both on proteins previously associated with the T cell pathway as well as functionally uncharacterized proteins. We will now test the hypothesis that these novel sites can be placed in specific locations within the pathway through quantitative phosphoproteomic analysis of T cells with disrupted pathway proteins LCK, PLCy1, VAV, and ERK. In particular, the placement of these phosphorylation events relative to the critical pathway protein SLP76 and LAT will be examined in detail through a collection of domain and point mutants, allowing for the precise placement of the novel phosphorylation sites within different signaling pathway branches initiated from these proteins. Testing of a newly postulated, phosphoproteomic data-inspired hypothesis about the Zap-70 dependent regulation of Fyn kinase through PTPa will be explored with classical molecular approaches.
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 mass spectrometry to facilitate the hypothesis-driven characterization of the T cell signaling pathway by providing a global view of the activation state of normal and mutant cells.
|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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