Protein tyrosine kinase Lyn and Syk are critical for antigen-receptor induced signal transduction in mast cells. To identify novel Lyn/Syk substrates, we screened a bacterial expression library prepared from cDNA of rat basophilic leukemia (RBL-2H3) cells for proteins that were tyrosine phosphorylated with baculoviral expressed Lyn or Syk. Five clones as potential Lyn substrates and eight clones as Syk substrates were identified including known substrates such as SLP-76, LAT, and a-tubulin. A potential substrate of Lyn identified was the molecule TOM1L1, which has several domains thought to be important for membrane trafficking and protein-protein interactions. Since the function of TOM1L1 is unclear, the rat TOM1L1 full-length cDNA was isolated and used to express the protein in COS-1 and RBL-2H3 mast cells. In COS-1 cells, the co-transfection of TOM1L1 and Lyn, but not Syk, resulted in the tyrosine phosphorylation of TOM1L1. In RBL-2H3 mast cells, the over-expressed TOM1L1 was strongly tyrosine phosphorylated in non-stimulated cells, and this phosphorylation was enhanced by FceRI aggregation. By subcellular fractionation, wild-type TOM1L1 was mainly in the cytoplasm with a small fraction constitutively associated with the membrane; this association was markedly reduced in deletion mutants lacking several of the protein interaction domains. The over-expression of TOM1L1 enhanced antigen-induced TNFa generation and release. Both protein interaction domains (VHS and the coiled-coil domains) were required for the increased TNFarelease, but not the increased TNFa generation. These results suggest that TOM1L1 is involved in the FceRI signal transduction for the generation of cytokines.