FcεRI-ligation induces association of tyrosine phosphorylated proteins with SRC homology 2 domains of phospholipase Cgamma1 in RBL-2H3 rat basophilic leukemia cells

Stimulation of the IgE receptors on mast cells and basophils activates protein tyrosine kinases and phospholipases leading to histamine release. However, the mechanism by which protein tyrosine kinases regulate the phospholipases is not clearly defined yet. In this study, we examined the possibility that phospholipase Cγ1 associates with protein tyrosine kinases and tyrosine phosphorylated molecules as a result of activation of RBL-2H3 cells, and that this association involves the Src homology 2 domains of phospholipase Cγ1. An increase in cytoplasmic Ca2+ level and tyrosine phosphorylations of proteins, including 72 and 40 kDa proteins, were observed after the cross-linking of the IgE receptors on RBL-2H3 rat basophilic cells by dinitrophenyl-specific IgE and dinitrophenyl-conjugated human serum albumin. Immunoprecipitation and coprecipitation experiments were performed to determine if the activation of protein tyrosine kinases is linked to the activation of phospholipase Cγ1 via its SH2 domains. Tyrosine phosphorylation of phospholipase Cγ1 was observed in 1 min following IgE receptor stimulation. Several proteins (72, 50, 40, and 33 kDa) were identified to be tyrosine phosphorylated and specifically associated with phospholipase Cγ1 by its Src homology 2 domains. In addition, the coprecipitated complex contains the tyrosine kinase activity which phosphorylates 72, 40, and 33 kDa proteins in the complex. In conclusion, these studies establish that tyrosine-phosphorylated proteins of 72, 40, and 33 kDa associate with phospholipase Cγ1 via its SH2 domains following IgE receptor stimulation of RBL-2H3 basophilic cells, implying that protein tyrosine kinases may tyrosine-phosphorylate and recruit signaling proteins around the phospholipase Cγ1 and that phospholipase Cγ1 activation induces calcium mobilization, PKC activation and degranulation in mast cells or basophils.ope