CHARACTERIZATION OF THE T-CELL ANTIGEN RECEPTOR-P60FYN PROTEIN TYROSINE KINASE ASSOCIATION BY CHEMICAL CROSS-LINKING
CHARACTERIZATION OF THE T-CELL ANTIGEN RECEPTOR-P60FYN PROTEIN TYROSINE KINASE ASSOCIATION BY CHEMICAL CROSS-LINKING
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DOI：
10.1093/intimm/4.11.1211
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发表时间：
1992-11-01
影响因子：
4.4
通讯作者：
SAMELSON, LE
中科院分区：
文献类型：
--
作者：
SAROSI, GA;THOMAS, PM;SAMELSON, LE
TCR与特异性抗原的结合导致酪氨酸激酶途径的激活。负责T细胞活化检测到的快速酪氨酸磷酸化的激酶的候选者是src激酶家族的成员p60fyn。在早期的研究中[Samelson等人（1990）Proc.Natl Acad. Sci. USA 87：43581该酶与溶解在毛地黄皂苷中的T细胞的TCR进行免疫共沉淀。在该研究中，使用敏感的体外激酶测定来检测相关的p60fyn。随后发现，相互作用的再现性取决于毛地黄皂苷的批间差异。为了消除抗原受体和激酶的结合是用不明确的毛地黄皂苷制剂溶解的假象的可能性，开发了交联方案以稳定TCR和p60fyn之间的相互作用。T细胞用破伤风溶血素透化，蛋白质用水溶性化学交联剂3，3 '二硫代双（磺基琥珀酰亚胺基丙酸酯）交联。这些实验允许确认TCR、p60fyn和几种另外的蛋白质之间的相互作用。交联研究还使得p60fyn和相关蛋白质与TCR ζ链的相互作用的映射成为可能。这种技术在稳定细胞内信号传导所需的其他受体和分子之间的相互作用方面应该具有普遍的用途。
Engagement of the TCR by specific antigen results in activation of a tyrosine kinase pathway. A candidate for the kinase responsible for the rapid tyrosine phosphorylation detected with T cell activation is p60fyn, a member of the src kinase family. In an earlier study [Samelson et al. (1990) Proc. Natl Acad. Sci. USA 87:43581 this enzyme was co-immunoprecipitated with the TCR from T cells solubilized in digitonin. In that study a sensitive in vitro kinase assay was used to detect the associated p60fyn. It was subsequently found that the reproducibility of the interaction depended on lot-to-lot variations in digitonin. To eliminate the possibility that the association of antigen receptor and kinase is an artifact of solubilization with ill-defined digitonin preparations, a cross-linking protocol was developed to stabilize the interaction between the TCR and p60fyn. T cells were permeabilized with tetanolysin and proteins were cross-linked with the water soluble chemical cross-linker, 3,3' dithiobis(sulfosuccinimidylpropionate). These experiments allowed the confirmation of the interaction between the TCR, p60fyn, and several additional proteins. The cross-linking studies also enabled the mapping of the interaction of p60fyn and associated proteins to the TCR zeta-chain. This technique should have a general use in stabilizing interactions between other receptors and molecules required for intracellular signaling.