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.