PT  - JOURNAL ARTICLE
AU  - Samantha L. Schwartz
AU  - Cédric Cleyrat
AU  - Mark Olah
AU  - Peter Relich
AU  - Genevieve Phillips
AU  - William S. Hlavacek
AU  - Keith A. Lidke
AU  - Bridget S. Wilson
AU  - Diane S. Lidke
TI  - Differential Mast Cell Outcomes Are Sensitive to FcεRI-Syk Binding Kinetics
AID  - 10.1101/147595
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 147595
4099  - http://biorxiv.org/content/early/2017/06/09/147595.short
4100  - http://biorxiv.org/content/early/2017/06/09/147595.full
AB  - 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.Summary Schwartz et al. use single molecule imaging to quantify the transient nature of FcεRI-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.