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

Abstract

Aggregation of the high affinity receptor for immunoglobulin E (FcεRI) 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 FcεRI 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 FcεRI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did reduce degranulation. FcεRI aggregation induced tyrosine phosphorylation of the β and γ subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-γ1 and phospholipase C-γ2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, FcεRI aggregation induced tyrosine phosphorylation of phospholipase C-γ1 and phospholipase C-γ2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early FcεRI-mediated signaling events. It further demonstrates that Syk activation occurs down stream of receptor phosphorylation, but upstream of most of the FcεRI-mediated protein tyrosine phosphorylations.