Structure-function analysis of Lyn kinase association with lipid rafts and initiation of early signaling events after Fcepsilon receptor I aggregation.

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 (FcepsilonRI) 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 FcepsilonRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca(2+). However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcepsilonRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcepsilonRI does not exhibit an increased association with lipid rafts, suggest that FcepsilonRI phosphorylation and early activation events can be initiated outside of lipid rafts.

Direct interaction of Syk and Lyn protein tyrosine kinases in rat basophilic leukemia cells activated via type I Fc epsilon receptors.

Activation of rat mast cells through the receptor with high affinity for IgE (Fc epsilonRI) requires a complex set of interactions involving transmembrane subunits of the Fc epsilonRI and two classes of nonreceptor protein tyrosine kinase (PTK). the Src family PTK p53/p56(lyn) (Lyn) and the Syk/ZAP-family PTK p72(syk) (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated Fc epsilonRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of Fc epsilonRI subunits in this process, we have analyzed Syk- and Lyn-associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of Fc epsilonRI beta or gamma subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet-P40-solubilized antigen (Ag)- or pervanadate-activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag- or pervanadate-activated cells were used. A similar amount of Syk was bound when lysates from pervanadate-activated variant cells deficient in the expression of Fc epsilonRI beta or gamma subunits were used, suggesting that Fc epsilonRI does not function as the only intermediate in the formation of the Syk-Lyn complexes. Further experiments have indicated that Syk-Lyn interactions occur in Ag-activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast-like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in Fc epsilonRI signaling.

Protein tyrosine kinase Syk is involved in Thy-1 signaling in rat basophilic leukemia cells.

Thy-1, a glycosyl-phosphatidylinositol-anchored surface glycoprotein, has been shown to possess transmembrane signaling capacity. In rat mast cells and rat basophilic leukemia cells (RBL) aggregation of surface Thy-1 with antibodies triggers a series of intracellular events, resembling those induced by aggregation of the high-affinity receptor for IgE (Fc epsilonRI), including tyrosine phosphorylation of multiple proteins and release of secretory components. Unlike the Fc epsilonRI-mediated activation, where both the membrane-associated protein tyrosine kinase (PTK) Lyn and the cytoplasmic PTK Syk are responsible for initiating the signaling cascade, only Lyn has been implicated in Thy-1-mediated activation in RBL cells. Here we report that Syk is also rapidly tyrosine phosphorylated upon Thy-1 cross-linking. Increased Syk tyrosine phosphorylation is observed only in cells in which extensive aggregation of Thy-1 is induced by two layers of cross-linking reagents. RBL-derived mutant cells deficient in the expression of surface Thy-1 and transfectants re-expressing surface Thy-1 were used to exclude the possibility that Syk activation reflects an interaction of the cross-linking reagents with surface molecules other than Thy-1. As Fc epsilonRI gamma subunits are well known to promote activation of Syk and its recruitment to membrane complexes, we also investigated the role of these subunits in Thy-1-mediated Syk activation, using RBL-derived mutant cells deficient in the expression of Fc epsilonRI gamma subunits and their revertants. Consistent with the lack of Fc epsilonRI expression, no IgE-induced response could be elicited, while Thy-1-inducible Syk phosphorylation was preserved. Our results suggest that Syk might be one of the kinases responsible for signal propagation upon Thy-1 cross-linking in a Fc epsilonRI-independent pathway.