AbstractAggregation of the high-affinity immunoglobulin E (IgE) receptor (FcϵRI) on mast cells induces a number of biochemical events, including protein-tyrosine phosphorylation leading to degranulation and multiple cytokine gene transcription. Here, we have demonstrated that a second member of the Cbl family of ubiquitin-protein ligase Cbl-b translocates into the lipid raft after FcϵRI engagement. Overexpression of Cbl-b in the lipid raft inhibits FcϵRI-mediated degranulation and cytokine gene transcription through the distinct mechanism. A point mutation of Cys373 in the RING finger domain of Cbl-b abrogates the suppression of FcϵRI-mediated degranulation but not cytokine gene transcription. The antigen-induced tyrosine phosphorylation of FcϵRI, Syk, phospholipase C-γ (PLC-γ), activation of c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK), inhibitor of nuclear factor κB kinase (IKK), and Ca++ influx were all suppressed in the cells overexpressing Cbl-b in the lipid raft. In particular, the expression amount of Gab2 protein and thereby its FcϵRI-mediated tyrosine phosphorylation were dramatically down-regulated by ubiquitin-protein ligase activity of Cbl-b. These results suggest that Cbl-b is a negative regulator of both Lyn-Syk-LAT and Gab2mediated complementary signaling pathways in FcϵRI-mediated mast cell activation.