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

项目摘要/摘要 本次NIAID K22职业转型奖聚焦候选人万博士的长期研究目标-- 林洛，成为一名独立的调查员，并为未来的R01申请获取数据。这项建议 重点研究通过调节磷酸化来检测T细胞配体的识别能力 ZAP70基板的效率。在TCR刺激下，激活的T细胞激酶Lck磷酸化酪氨酸 在CD3和z链上，导致ZAP70的募集、磷酸化和激活。已激活 随后，ZAP70使接头LAT和SLP76中的多个位点磷酸化。磷酸酪氨酸 Lat和SLP76成为额外信号蛋白的对接位置，导致基于LAT或SLP76的信号蛋白 信号体。LAT中有5个ZAP70底物酪氨酸，SLP76中有3个。每一种酪氨酸 底物致力于与参与下游分叉的不同蛋白质特异性地相互作用。 小路。例如，LAT Y132的磷酸化是唯一可以将TCR信号偶联到 PLCG1途径和由此产生的钙离子增加。此前，人们认为ZAP70的磷酸化 严格来说，酪氨酸底物是使信号多样化或放大的信号传播步骤。然而，罗博士的 初步数据显示，至少对于LAT Y132来说，ZAP70介导的这个位点的磷酸化程度更大 意义。ZAP70介导的LAT Y132的磷酸化具有独特的缓慢的磷酸化动力学。这是 因为Y132之前的氨基酸是甘氨酸，而其他ZAP70底物含有负的 此位置的带电氨基酸与ZAP70的S正电对接位置形成互补。通过替换 这种带有负电荷残基的甘氨酸，罗博士观察到Y132的磷酸化速度更快，但这 允许T细胞对弱结合的自身抗原做出不适当的反应。罗博士假设LAT Y132具有 进化成一个独特的TCR信号瓶颈，支持适当程度的T细胞配体识别。 为了验证这一假设，她将探索Y132-磷酸化-PLCG1瓶颈是否真的是独一无二的 适合于通过检测其他ZAP70酪氨酸的磷酸化动力学是否适合于配体识别 底物对T细胞辨别自我和非自我的能力也有类似的影响(目标I)。她也会放慢脚步 通过突变前面的负电荷，降低其他ZAP70底物的磷酸化速度 残留物变成甘氨酸。这种诱变方法将对其他ZAP70酪氨酸施加“类似Y132”的缓慢动力学 底物，以测试这些节点是否也可以作为动力学瓶颈并影响配基识别。 她还将评估LAT G135D转基因小鼠的T细胞发育和外周T细胞功能 (Y136之前的甘氨酸到谷氨酸的突变，与人的G131-Y132同源)，以确定如何 破坏这一动力学阈值会影响胸腺T细胞选择和体内T细胞耐受性(AIM II)。这个 获得的数据将为T细胞配体识别是如何调节的提供洞察力，并将建立 为罗博士的独立科学生涯建立新的研究项目。

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.