Phosphorylation of Tyr342 in the Linker Region of Syk Is Critical for FcεRI Signaling in Mast Cells

Aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcɛRI) on mast cells initiates a biochemical cascade that ultimately results in degranulation and release of inflammatory mediators (1, 19, 49, 50). Among these biochemical changes, protein tyrosine phosphorylation is one of the earliest detectable events. Since FcɛRI itself has no intrinsic tyrosine kinase activity, the sequential activation of the nonreceptor protein tyrosine kinases (PTKs) such as Syk is essential for this signal transduction pathway (2, 3, 12, 15, 22, 24, 41, 47, 58). Because of the importance of Syk in signaling, there is much interest in understanding its regulation. Syk is a member of the Syk and ZAP70 PTK family and is expressed in most hematopoietic cells (30). The tandem Src homology 2 region (SH2) domains in the N-terminal half of Syk are involved in its association with subunits of FcɛRI after receptor aggregation (3, 5, 26, 47). This interaction is mediated by the SH2 domains of Syk binding to the tyrosine phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) especially of the γ subunit of FcɛRI. Binding of Syk to a diphosphorylated ITAM results in a conformational change and an increase of its kinase activity (27). The linker region of Syk and ZAP70, located between the second SH2 and the kinase domain, has been reported to play an important role in regulating the enzymatic function of the molecule (64). SykB, which lacks a 23-amino-acid (aa) sequence in this linker region of Syk, is inefficient at coupling stimulation of FcɛRI or T-cell antigen receptor to the early and late events of cellular activation (32). There are three conserved tyrosines in the linker region of both Syk and ZAP70 (aa 317, 342, and 346 in rat Syk and the orthologous aa 292, 315, and 319 in human ZAP70). The putative Cbl interaction site, Tyr317 of Syk (Tyr292 in human ZAP70), negatively regulates Syk and ZAP70 signaling (9, 25, 34, 45). The other two tyrosines in the linker region have been reported to be involved in the interaction and tyrosine phosphorylation of phospholipase C-γ1 and Vav. Thus, in COS cells the expression of Syk with both Tyr342 and Tyr346 mutated to Phe results in the loss of the association of Syk with phospholipase C-γ1 and decrease in the tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) (33). Other experiments using the two-hybrid system suggest that Tyr342 of Syk is important for the interaction of Syk with Vav (10). The roles of the two orthologous tyrosines in the linker region of ZAP70 (Tyr315 or Tyr319) have been extensively investigated (11, 18, 35, 42, 54, 56). Both tyrosines are phosphorylated after T-cell receptor (TCR) cross-linking. Tyr319 of ZAP70 is essential for downstream propagation of signals such as tyrosine phosphorylation of PLC-γ1 and calcium influx (11, 54). However, there have been more-variable results in studies with the Y315F mutant of ZAP70. Experiments with Syk−/− chicken B cells suggest that Tyr315 is essential for the interaction of Vav with ZAP70 and critical for antigen receptor-mediated signal transduction (56). In contrast, Y315F ZAP70 has only minimal inhibitory effects on TCR signaling when expressed in Jurkat T cells (11). Studies of transgenic or knockin mice demonstrate that Tyr315 of ZAP70 plays an important role in the positive and negative selection of T cells (18, 35). Although Syk and ZAP70 have similar functions in antigen receptor signaling; there are still differences in their regulation and their capacity to mediate receptor-mediated signaling (6, 14, 17, 20, 31, 55, 65). Therefore, these Tyr residues in the linker region of Syk may have a function different from that of their orthologues in ZAP70. The purpose of the present study was to characterize the roles of Tyr342 and Tyr346 of Syk in mast cell signaling. Therefore, Y342F and Y346F mutant Syk were stably expressed in a Syk-deficient variant of the RBL-2H3 mast cells. Compared with wild-type Syk, mutation of Tyr342 resulted in a dramatic reduction of IgE-stimulated mast cell degranulation. However, Y346F mutant Syk still reconstituted FcɛRI-initiated histamine release. Detailed analysis suggested that IgE-receptor aggregation induced a clear increase in the phosphorylation of Tyr342 but not Tyr346 of Syk. Furthermore, the Y342F mutant Syk had decreased binding to a phosphorylated ITAM peptide based on FcɛRIγ, which also resulted in decreased Syk activation.