Differential mast cell outcomes are sensitive to FcεRI-Syk binding kinetics

Cross-linking of immunoglobulin E–bound FceRI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the FceRI immunoreceptor tyrosine-based activation motifs. Using single-molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FceRI activation. We found that Syk colocalizes transiently to FceRI and that Syk-FceRI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (Syk-Y130E) led to an increased Syk-FceRI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. Genome edited cells expressing only Syk-Y130E were deficient in antigen-stimulated calcium release, degranulation, and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the FceRI signaling pathway occurs at the level of Syk-FceRI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.