Sequential Requirements of the N-Terminal Palmitoylation Site and SH2 Domain of Src Family Kinases in the Initiation and Progression of FcɛRI Signaling

Abstract

Initial biochemical signaling originating from high-affinity immunoglobulin E receptor (FcɛRI) has been ascribed to Src family kinases. To understand the mechanisms by which individual kinases drive the signaling, we conducted reconstitution experiments: FcɛRI signaling in RBL2H3 cells was first suppressed by a membrane-anchored, gain-of-function C-terminal Src kinase and then reconstructed with Src family kinases whose C-terminal negative regulatory sequence was replaced with a c-myc epitope. Those constructs derived from Lyn and Fyn, which are associated with detergent-resistant membranes (DRMs), physically interacted with resting FcɛRI and reconstructed clustering-induced signaling that leads to calcium mobilization and ERK1 and -2 activation. c-Src-derived construct, which was excluded from DRMs, failed to interact with FcɛRI and to restore the signaling, whereas creation of palmitoylatable Cys3 enabled it to interact with DRMs and with FcɛRI and to restore the signaling. Deletion of Src homology 3 (SH3) domain from the Lyn-derived construct did not alter its ability to transduce the series of signaling. Deletion of SH2 domain did not affect its association with DRMs and with FcɛRI nor clustering-induced tyrosine phosphorylation of FcɛRI β and γ subunits, but it almost abrogated the next step of tyrosine phosphorylation of Syk and its recruitment to FcɛRI. These findings suggest that Lyn and Fyn could, but c-Src could not, drive FcɛRI signaling and that N-terminal palmitoylation and SH2 domain are required in sequence for the initial interaction with FcɛRI and for the signal progression to the molecular assembly.

ACKNOWLEDGMENTS

This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture, by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of the Japanese Government, and by grants from Ono Medical Research Foundation, Manabe Research Foundation, and Uehara Memorial Foundation.

We thank H. Ota-Ichijo, and M. Saka for excellent technical assistance.