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

Crosslinking of IgE-bound FcϵRI 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 FcϵRI 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 FcϵRI activation. We found that Syk colocalizes transiently to FcϵRI and that Syk-FcϵRI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (SykY130E) led to an increased Syk-FcϵRI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. CRISPR-Cas9 engineered cells expressing only SykY130E 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 FcϵRI signaling pathway occurs at the level of Syk-FcϵRI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.