Differential Mast Cell Outcomes Are Sensitive to FcϵRI-Syk Binding Kinetics

Abstract

Crosslinking of IgE-bound Fc{varepsilon}RI 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 Fc{varepsilon}RI 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 Fc{varepsilon}RI activation. We found that Syk colocalizes transiently to Fc{varepsilon}RI and that Syk-Fc{varepsilon}RI 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-Fc{varepsilon}RI 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 Fc{varepsilon}RI signaling pathway occurs at the level of Syk-Fc{varepsilon}RI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.\n\nSummarySchwartz et al. use single molecule imaging to quantify the transient nature of Fc{varepsilon}RI-Syk interactions in live mast cells. A functional mutation that increases Syk off-rate leads to loss of site-specific Syk phosphorylation and impaired signaling, highlighting the importance of finely tuned protein interactions in directing cellular outcomes.