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

Schwartz, S. L.; Cleyrat, C.; Olah, M.; Relich, P.; Phillips, G.; Hlavacek, W. S.; Lidke, K. A.; Wilson, B. S.; Lidke, D.

doi:10.1101/147595

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bioRxiv · 10.1101/147595

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

Schwartz, S. L.·Cleyrat, C.·Olah, M.·Relich, P.·Phillips, G.·Hlavacek, W. S.·Lidke, K. A.·Wilson, B. S.·Lidke, D.

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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.

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Schwartz, S. L., Cleyrat, C., Olah, M., Relich, P., Phillips, G., Hlavacek, W. S., Lidke, K. A., Wilson, B. S., Lidke, D.. 2017-06-09. Differential Mast Cell Outcomes Are Sensitive to FcϵRI-Syk Binding Kinetics. https://doi.org/10.1101/147595

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