Structure-Function Analysis of Lyn Kinase Association with Lipid Rafts and Initiation of Early Signaling Events after Fcɛ Receptor I Aggregation

Abstract

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcɛRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcɛRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca²⁺. However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcɛRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcɛRI does not exhibit an increased association with lipid rafts, suggest that FcɛRI phosphorylation and early activation events can be initiated outside of lipid rafts.

ACKNOWLEDGMENTS

We thank H. Metzger and B. Vonakis for Lyn and Lyn-CI cDNA and Hanka Mrázová for technical assistance.

This work was supported by grants 312/96/K205, 204/00/0204, and 310/00/205 from the Grant Agency of the Czech Republic, by grants A5052005/00 and A7052006/00 from the Grant Agency of the Academy of Sciences of the Czech Republic, and by grant LN00A026 from the Ministry of Education, Youth and Sports of the Czech Republic. The research of P. Dráber was supported in part by an International Research Scholar's award from Howard Hughes Medical Institute.