Dutz J P, Fruman D A, Burakoff S J, Bierer B E
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
J Immunol. 1993 Apr 1;150(7):2591-8.
The immunosuppressive drugs cyclosporin A (CsA) and FK506 bind to distinct families of intracellular proteins, cyclophilins, and FK506 binding proteins (FKBP) respectively, termed immunophilins. Immuno-suppressant-immunophilin complexes bind to and inhibit the activity of calcineurin, a calcium-dependent serine/threonine phosphatase. CsA is known to inhibit degranulation in CTL as assessed by N benzyloxylcarbonyl-L-lysine thiobenzyl ester-esterase release assays. We have investigated whether calcineurin phosphatase activity is involved in this degranulation. Both CsA and FK506 are shown to inhibit N benzyloxylcarbonyl-L-lysine thiobenzyl esteresterase release in murine CTL clones induced either by cognate target or by PMA and the calcium ionophore A23187. Inhibition is concentration dependent and is observed at drug concentrations that specifically inhibit cellular calcineurin. The FK506-binding immunophilin FKBP12, as well as calcineurin, are shown to be present in these cells by immunoblotting analysis. Rapamycin, a macrolide antibiotic thought to compete with FK506 for binding to common FKBP receptor sites, antagonizes the effects of FK506 on both degranulation and calcineurin activity. Neither the degranulation nor the effect of the immunosuppressants is affected by the protein synthesis inhibitor cycloheximide. These observations suggest a role for calcineurin in CTL degranulation. Thus, in addition to its previously described role in lymphokine gene activation, calcineurin also appears to be involved in T cell activation processes which do not require protein synthesis.
免疫抑制药物环孢素A（CsA）和FK506分别与细胞内不同家族的蛋白质亲环蛋白和FK506结合蛋白（FKBP）结合，这些蛋白质被称为亲免素。免疫抑制剂 - 亲免素复合物结合并抑制钙调神经磷酸酶的活性，钙调神经磷酸酶是一种钙依赖性丝氨酸/苏氨酸磷酸酶。通过N - 苄氧羰基 - L - 赖氨酸硫代苄酯酯酶释放试验评估，已知CsA可抑制细胞毒性T淋巴细胞（CTL）的脱颗粒作用。我们研究了钙调神经磷酸酶的磷酸酶活性是否参与这种脱颗粒过程。结果表明，CsA和FK506均可抑制由同源靶细胞或佛波酯（PMA）和钙离子载体A23187诱导的小鼠CTL克隆中的N - 苄氧羰基 - L - 赖氨酸硫代苄酯酯酶释放。抑制作用呈浓度依赖性，并且在特异性抑制细胞钙调神经磷酸酶的药物浓度下即可观察到。通过免疫印迹分析表明，FK506结合亲免素FKBP12以及钙调神经磷酸酶存在于这些细胞中。雷帕霉素是一种大环内酯类抗生素，被认为可与FK506竞争结合共同的FKBP受体位点，它可拮抗FK506对脱颗粒和钙调神经磷酸酶活性的影响。蛋白质合成抑制剂环己酰亚胺对脱颗粒作用和免疫抑制剂的效果均无影响。这些观察结果表明钙调神经磷酸酶在CTL脱颗粒过程中发挥作用。因此，除了其先前描述的在淋巴因子基因激活中的作用外，钙调神经磷酸酶似乎还参与了不需要蛋白质合成的T细胞激活过程。
