Src family-selective tyrosine kinase inhibitor, PP1, inhibits both Fc epsilon RI- and Thy-1-mediated activation of rat basophilic leukemia cells
Src family-selective tyrosine kinase inhibitor, PP1, inhibits both Fc epsilon RI- and Thy-1-mediated activation of rat basophilic leukemia cells
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DOI：
10.1002/eji.1830270810
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发表时间：
1997-08-01
影响因子：
5.4
通讯作者：
Draberova, L
中科院分区：
文献类型：
--
作者：
Amoui, M;Draber, P;Draberova, L
多价抗原/免疫球蛋白E复合物交联IgE高亲和力表面受体（Fc epsilon RI），以及单克隆抗体聚集Thy-1糖蛋白导致大鼠嗜碱性白血病细胞，克隆RBL-2H3，导致几种细胞蛋白酪氨酸磷酸化，随后释放分泌成分。为了研究Fc epsilon RI-和thy -1介导的跨膜信号传导的分子机制，并绘制它们会聚到共同分泌途径的步骤，我们使用了一种新的Src家族选择性酪氨酸激酶抑制剂，4-氨基-5-（4-甲基苯基）-7-（t-丁基）吡唑[3,4-d]嘧啶（PP1），并分析了其对细胞活化的抑制活性。在RBL-2H3细胞中，PP1对Src家族激酶Lyn表现出底物特异性。在体外免疫复合物激酶实验中，PP1在纳摩尔水平上抑制Lyn激酶活性，而对Syk激酶活性没有影响。然而，在通过Fc epsilon RI聚集激活的RBL细胞中，Syk和Lyn激酶的磷酸化都被抑制。Fc epsilon RI-和thy -1介导的早期（蛋白酪氨酸磷酸化）和晚期（β -己糖氨酸酶释放）激活事件同样受到PP1的影响。这种抑制作用是对膜受体介导的信号传导的特异性抑制，在暴露于pervanadate激活的细胞中没有观察到。综合数据表明，Lyn的激活是肥大细胞和嗜碱性细胞的Fc epsilon RI-和thy -1介导的激活途径可能会聚的早期激活步骤。
Cross-linking of the surface receptor with high affinity for IgE (Fc epsilon RI) by multivalent antigen/immunoglobulin E complexes, as well as aggregation of Thy-1 glycoprotein by monoclonal antibodies lead in rat basophilic leukemia cells, clone RBL-2H3, to tyrosine phosphorylation of several cellular proteins, followed by a release of secretory components. To investigate the molecular mechanisms of Fc epsilon RI- and Thy-1-mediated transmembrane signaling and to map a step at which they converge into a common secretory pathway, we used a novel Src family-selective tyrosine kinase inhibitor, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP1), and analyzed its inhibitory activity on cell activation. Here we show that in RBL-2H3 cells PP1 demonstrates substrate specificity for a Src family kinase Lyn. In immunocomplex kinase assays in vitro, PP1 inhibited the Lyn kinase activity at nanomolar levels without any effect on Syk kinase activity. However, in RBL cells activated via aggregation of Fc epsilon RI, phosphorylation of both Syk and Lyn kinases was inhibited. Fc epsilon RI- and Thy-1-mediated early (protein-tyrosine phosphorylation) and late (release of (beta-hexosaminidase) activation events were similarly affected by PP1. The inhibition was specific for membrane receptor-mediated signaling and was not observed in cells activated by an exposure to pervanadate. The combined data suggest that activation of Lyn is the early activation step at which the Fc epsilon RI- and Thy-1-mediated activation pathways of mast cells and basophils may converge.