Penna D, Müller S, Martinon F, Demotz S, Iwashima M, Valitutti S
Institute of Biochemistry, University of Lausanne, BIL Research Center, Epalinges, Switzerland.
J Immunol. 1999 Jul 1;163(1):50-6.
T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.
特异性抗原激活T细胞会导致T细胞表型发生显著变化，其中包括触发的TCR迅速而显著地下调与降解。在本研究中，我们调查了抗原刺激的T细胞中与TCR相关的ZAP-70激酶的命运。通过对固定和通透处理的T细胞-抗原呈递细胞（APC）结合物进行荧光激活细胞分选（FACS）分析和共聚焦显微镜检查，以及对全细胞裂解物进行蛋白质免疫印迹分析，发现经肽脉冲APC刺激的T细胞中，ZAP-70的总细胞含量会出现抗原剂量依赖性下降。ZAP-70消耗的时间进程与ζ链降解的时间进程重叠，这表明ZAP-70与TCR内化和降解同时发生降解。蛋白激酶C（PKC）的药理学激活不会诱导ZAP-70降解，相反，它需要蛋白酪氨酸激酶的激活。有两条证据表明，Ca2+依赖性半胱氨酸蛋白酶钙蛋白酶在启动ZAP-70降解中起主要作用：1）用可穿透细胞的钙蛋白酶抑制剂处理T细胞可显著减少ZAP-70降解；2）ZAP-70在体外可被钙蛋白酶切割。我们的结果表明，在T细胞与APC同源相互作用过程中，ZAP-70通过钙蛋白酶依赖性机制迅速降解。
