FC-EPSILON-RI-LIGATION INDUCES ASSOCIATION OF TYROSINE-PHOSPHORYLATEDPROTEINS WITH SRC HOMOLOGY-2 DOMAINS OF PHOSPHOLIPASE C-GAMMA-1 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 rele ase. However, the mechanism by which protein tyrosine kinases regulate the phospholipases is not clearly defined yet. In this study, we exam ined the possibility that phospholipase C gamma 1 associates with prot ein tyrosine kinases and tyrosine phosphorylated molecules as a result of activation of RBL-2H3 cells, and that this association involves th e Src homology 2 domains of phospholipase C gamma 1. An increase in cy toplasmic Ca2+ level and tyrosine phosphorylations of proteins, includ ing 72 and 40 kDa proteins, were observed after the cross-linking of t he IgE receptors on RBL-2H3 rat basophilic cells by dinitrophenyl-spec ific IgE and dinitrophenyl-conjugated human serum albumin. Immunopreci pitation and coprecipitation experiments were performed to determine i f the activation of protein tyrosine kinases is linked to the activati on of phospholipase C gamma 1 via its SH2 domains. Tyrosine phosphoryl ation of phospholipase C gamma 1 was observed in 1 min following IgE r eceptor stimulation. Several proteins (72, 50, 40, and 33 kDa) were id entified to be tyrosine phosphorylated and specifically associated wit h phospholipase C gamma 1 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 conclus ion, these studies establish that tyrosine-phosphorylated proteins of 72, 40, and 33 kDa associate with phospholipase C gamma 1 via its SH2 domains following IgE receptor stimulation of RBL-2H3 basophilic cells , implying that protein tyrosine kinases may tyrosine-phosphorylate an d recruit signaling proteins around the phospholipase C gamma 1 and th at phospholipase C gamma 1 activation induces calcium mobilization, PK C activation and degranulation in mast cells or basophils.