Dong Guangyu, Kalifa Rachel, Nath Pulak Ranjan, Babichev Yael, Gelkop Sigal, Isakov Noah
The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel.
The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel.
Cell Signal. 2017 Aug;36:117-126. doi: 10.1016/j.cellsig.2017.04.022. Epub 2017 Apr 29.
T cell receptor (TCR) recognition of a peptide antigen in the context of MHC molecules initiates positive and negative cascades that regulate T cell activation, proliferation and differentiation, and culminate in the acquisition of effector T cell functions. These processes are a prerequisite for the induction of specific T cell-mediated adaptive immune responses. A key event in the activation of TCR-coupled signaling pathways is the phosphorylation of tyrosine residues within the cytoplasmic tails of the CD3 subunits, predominantly CD3ζ. These transiently formed phosphotyrosyl epitopes serve as docking sites for SH2-domain containing effector molecules, predominantly the ZAP70 protein tyrosine kinase, which is critical for signal propagation. We found that CrkI and CrkII adaptor proteins also interact with CD3ζ in TCR activated-, but not in resting-, T cells. Crk binding to CD3ζ was independent of ZAP70 and also occurred in ZAP70-deficient T cells. Binding was mediated by Crk-SH2 domain interaction with phosphotyrosine-containing motifs on CD3ζ, via a direct physical interaction, as demonstrated by Far-Western blot. CrkII binding to CD3ζ could also be demonstrated in a heterologous system, where coexpression of a catalytically active Lck was used to phosphorylate the CD3ζ chain. TCR activation-induced Crk binding to CD3ζ resulted in increased and prolonged phosphorylation of CD3ζ, as well as ZAP70 and LAT, suggesting a positive role for CrkI/II binding to CD3ζ in regulation of TCR-coupled signaling pathways. Furthermore, Crk-dependent increased phosphorylation of CD3ζ coincided with inhibition of TCR downmodulation, supporting a positive role for Crk adaptor proteins in TCR-mediated signal amplification.
在MHC分子背景下，T细胞受体（TCR）对肽抗原的识别启动了正向和负向级联反应，这些反应调节T细胞的激活、增殖和分化，并最终导致效应T细胞功能的获得。这些过程是诱导特异性T细胞介导的适应性免疫反应的先决条件。TCR偶联信号通路激活的一个关键事件是CD3亚基（主要是CD3ζ）胞质尾部酪氨酸残基的磷酸化。这些瞬时形成的磷酸酪氨酸表位作为含有SH2结构域的效应分子（主要是ZAP70蛋白酪氨酸激酶）的对接位点，这对信号传播至关重要。我们发现，CrkI和CrkII衔接蛋白也在TCR激活的T细胞中与CD3ζ相互作用，而在静息T细胞中则不然。Crk与CD3ζ的结合不依赖于ZAP70，在ZAP70缺陷的T细胞中也会发生。如Far-Western印迹所示，结合是通过Crk-SH2结构域与CD3ζ上含磷酸酪氨酸的基序直接物理相互作用介导的。在异源系统中也能证明CrkII与CD3ζ的结合，在该系统中，使用具有催化活性的Lck共表达来磷酸化CD3ζ链。TCR激活诱导的Crk与CD3ζ的结合导致CD3ζ以及ZAP70和LAT的磷酸化增加和延长，这表明CrkI/II与CD3ζ的结合在调节TCR偶联信号通路中起积极作用。此外，Crk依赖的CD3ζ磷酸化增加与TCR下调的抑制同时发生，支持Crk衔接蛋白在TCR介导的信号放大中起积极作用。
