Regulation of T cell ligand discrimination by tuning the phosphorylation kinetics of Zap70 substrates

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PROJECT SUMMARY/ABSTRACT This NIAID K22 Career Transition Award focuses on the long-term research goals of the candidate, Dr. Wan- Lin Lo, to become an independent investigator and to acquire data for a future R01 application. This proposal focuses on the examination of T cell ligand discrimination capability through manipulating phosphorylation efficiency of Zap70 substrates. Upon TCR stimulation, the activated T cell kinase Lck phosphorylates tyrosines on CD3 and z-chains, leading to the recruitment, phosphorylation and activation of the kinase Zap70. Activated Zap70 subsequently phosphorylates multiple sites within the adaptors LAT and SLP76. Phospho-tyrosines on LAT and SLP76 become docking sites for additional signaling proteins, resulting in LAT- or SLP76-based signalosomes. There are five Zap70 substrate tyrosines in LAT, and three in SLP76. Each of these tyrosine substrates is dedicated to specifically interact with distinct proteins that are involved in divergent downstream pathways. For example, phosphorylation of LAT Y132 is the only tyrosine that can couple TCR signals to the PLCg1 pathway and the resultant calcium increase. Previously, it was thought that phosphorylation of Zap70 tyrosine substrates was strictly a signal propagation step that diversifies or amplifies signals. However, Dr. Lo’s preliminary data showed that, at least for LAT Y132, Zap70-mediated phosphorylation of this site has greater significance. Zap70-mediated phosphorylation of LAT Y132 has uniquely slow phosphorylation kinetics. This is because the amino acid preceding Y132 is a glycine, whereas other Zap70 substrates contain a negatively charged amino acid at this position which complements Zap70’s positively charged docking site. By replacing this glycine with a negatively charged residue, Dr. Lo observed faster phosphorylation of Y132, and yet this allowed T cells to inappropriately react to weakly binding self-antigens. Dr. Lo hypothesizes that LAT Y132 has evolved to serve as a unique TCR signal bottleneck to support a proper degree of T cell ligand discrimination. To test this hypothesis, she will explore whether the Y132-phosphorylation-PLCg1 bottleneck is really uniquely suited for ligand discrimination by examining whether the phosphorylation kinetics of other Zap70 tyrosine substrates can have similar effects on T cell’s ability to discriminate self and non-self (Aim I). She will also slow down the phosphorylation speed of other Zap70 substrates, by mutating the preceding negatively charged residue to a glycine. This mutagenesis approach will impose “Y132-like” slow kinetics on other Zap70 tyrosine substrates, to test whether these nodes can also serve as kinetic bottlenecks and affect ligand discrimination. She will also evaluate T cell development and peripheral T cell function in a LAT G135D knock-in mouse (glycine to glutamate mutation preceding Y136, homologous to human G131-Y132), to determine how disruption of this kinetic threshold impacts thymic T cell selection and T cell tolerance in vivo (Aim II). The obtained data will provide insights into how T cell ligand discrimination is regulated and will establish the foundation of new research projects for Dr. Lo’s independent scientific career.