APEX2-based quantitative proteomic study of LAT and CD3z in Jurkat T cells

TCR binding to its ligands induces a cascade of signaling starting by the phosphorylation of immunoreceptor tyrosine-based activation motif (ITAMs) present in the TCR-associated CD3 complex. This is followed by the phosphorylation of proteins including the adaptor LAT, which once phosphorylated interacts with multiple proteins allowing signal diversification and amplification downstream of the TCR. We take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track the formation of CD3z and LAT interactome dynamics in Jurkat cells after TCR stimulation. We find more than 1 000 high confidence proteins for each bait and provide a quantitative molecular map of proteins enriched or reduced from the vicinity of CD3z and LAT, after stimulation. We detail and compare the recruitment kinetics of signaling proteins to CD3z and LAT and identify previously uncharacterized mediators of T cell activation. We show that the kinase MARK2, which is in the proximity of LAT and CD3z at resting state and lost upon activation, is a negative regulator of cytokine production by T cells. This study provides a resource for uncovering the complex signaling networks that regulate TCR activation and highlights new players of the TCR-induced signaling cascade.

T细胞受体（TCR）与其配体结合后，会触发一系列信号级联反应，起始于TCR相关CD3复合物中免疫受体酪氨酸激活基序（immunoreceptor tyrosine-based activation motif，ITAMs）的磷酸化。随后，包括衔接蛋白LAT在内的多种蛋白发生磷酸化；磷酸化后的LAT可与多种蛋白相互作用，实现TCR下游信号的多样化与扩增。本研究利用过氧化物酶催化的邻近标记技术（peroxidase-catalyzed proximity labeling）结合定量质谱（quantitative mass spectrometry），追踪了Jurkat细胞经TCR刺激后CD3ζ与LAT互作组的动态变化。我们在每个诱饵蛋白样本中鉴定出超过1000种高可信度蛋白，并绘制了刺激后CD3ζ与LAT邻近区域内富集或减少的蛋白的定量分子图谱。本研究详细比较了信号蛋白向CD3ζ与LAT的招募动力学，鉴定出此前未被表征的T细胞激活介导因子。我们发现激酶MARK2在静息状态下可定位于LAT与CD3ζ的邻近区域，而在细胞激活后会脱离该区域，其为T细胞细胞因子产生的负调控因子。本研究为解析调控TCR激活的复杂信号网络提供了宝贵资源，并揭示了TCR诱导的信号级联反应中的全新调控因子。