Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells
Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells
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DOI：
10.1084/jem.184.1.71
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发表时间：
1996-07-01
影响因子：
15.3
通讯作者：
Siraganian, RP
中科院分区：
文献类型：
--
作者：
Zhang, J;Berenstein, EH;Siraganian, RP
肥大细胞上免疫球蛋白E的高亲和力受体（Fc ε RI）的聚集导致Syk（一种细胞质蛋白酪氨酸激酶）的快速酪氨酸磷酸化和活化。为了研究Syk在Fc ε RI信号通路中的作用，我们通过免疫印迹和体外激酶反应鉴定了RBL-2 H3细胞的一种变体，其没有可检测到的Syk。在这些Syk缺陷型TB 1A 2细胞中，Fc ε RI的聚集未诱导组胺释放，也未检测到总细胞蛋白酪氨酸磷酸化的增加。然而，用钙离子载体刺激这些细胞确实诱导脱粒。Fc γ RI聚集诱导受体β和γ亚基的酪氨酸磷酸化，但磷脂酶C-γ 1和磷脂酶C-γ 2的酪氨酸磷酸化没有增加，细胞内游离Ca 2+浓度也没有检测到增加。通过转染，建立了稳定表达Syk的克隆细胞系。在这些重建细胞中，Fc γ RI聚集诱导磷脂酶C-γ 1和磷脂酶C-γ 2的酪氨酸磷酸化，增加细胞内游离Ca 2+和组胺释放。这些结果表明，Syk在早期Fc ε RI介导的签名事件中起关键作用。它进一步证明Syk活化发生在受体磷酸化的下游，但在大多数Fc γ RI介导的蛋白酪氨酸磷酸化的上游。
Aggregation of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells results in rapid tyrosine phosphorylation and activation of Syk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fc epsilon RI signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TB1A2 cells, aggregation of Fc epsilon RI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. Fc epsilon RI aggregation induced tyrosine phosphorylation of the beta and gamma subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, Fc epsilon RI aggregation induced tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fc epsilon RI-mediated signing events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the Fc epsilon RI-mediated protein tyrosine phosphorylations.