RT Journal Article
SR Electronic
T1 Differential Mast Cell Outcomes Are Sensitive to FcεRI-Syk Binding Kinetics
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 147595
DO 10.1101/147595
A1 Samantha L. Schwartz
A1 Cédric Cleyrat
A1 Mark Olah
A1 Peter Relich
A1 Genevieve Phillips
A1 William S. Hlavacek
A1 Keith A. Lidke
A1 Bridget S. Wilson
A1 Diane S. Lidke
YR 2017
UL http://biorxiv.org/content/early/2017/06/09/147595.abstract
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.