Amoui M, Dráber P, Dráberová L
Department of Mammalian Gene Expression, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague.
Eur J Immunol. 1997 Aug;27(8):1881-6. doi: 10.1002/eji.1830270810.
Cross-linking of the surface receptor with high affinity for IgE (Fc epsilonRI) 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 epsilonRI- 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 epsilonRI, phosphorylation of both Syk and Lyn kinases was inhibited. Fc epsilonRI- 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 epsilonRI- and Thy-1-mediated activation pathways of mast cells and basophils may converge.
多价抗原/免疫球蛋白E复合物与对IgE具有高亲和力的表面受体（FcεRI）交联，以及单克隆抗体使Thy-1糖蛋白聚集，均可导致大鼠嗜碱性白血病细胞系RBL-2H3中的几种细胞蛋白发生酪氨酸磷酸化，随后分泌成分释放。为了研究FcεRI和Thy-1介导的跨膜信号传导的分子机制，并确定它们汇聚到共同分泌途径的步骤，我们使用了一种新型的Src家族选择性酪氨酸激酶抑制剂4-氨基-5-（对甲苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶（PP1），并分析了其对细胞活化的抑制活性。我们在此表明，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测定中，PP1在纳摩尔水平抑制Lyn激酶活性，而对Syk激酶活性无任何影响。然而，在通过FcεRI聚集激活的RBL细胞中，Syk和Lyn激酶的磷酸化均受到抑制。PP1对FcεRI和Thy-1介导的早期（蛋白酪氨酸磷酸化）和晚期（β-己糖胺酶释放）活化事件有类似影响。这种抑制对膜受体介导的信号传导具有特异性，在暴露于过钒酸盐激活的细胞中未观察到。综合数据表明，Lyn的激活是肥大细胞和嗜碱性粒细胞的FcεRI和Thy-1介导的激活途径可能汇聚的早期激活步骤。
