Characterization of a Phospho-Specific Antibody to the Fcε Receptor γ Chain, Reveals Differences in the Regulation of Syk and Akt Phosphorylation

Abstract

We previously demonstrated that the Fc receptor γ-chain Y 58 (C-terminal tyrosine) is highly susceptible to dephosphorylation; a mechanism that controls the extent of Syk activation and the downstream signaling in mast cells. Here, we explored the importance of the γ-chain Y 47 (N-terminal tyrosine) in mast cell signaling. We generated a highly sensitive and versatile phospho-specific antibody that recognized the phosphorylated Y 47 in various species. Using this antibody, we found that mutation of the FcεRIβ Y 219 to phenylalanine caused a loss in the phosphorylation of the γ-chain Y 47 , consistent with the previously described role of Y 219 in Lyn association with FcεRIβ and subsequent FcεRIγ phosphorylation. These conditions also diminished the tyrosine phosphorylation of Syk and LAT1 but, surprisingly, not the phosphorylation of Akt at T 308 . Mutation of Y 47 or Y 58 of the γ-chain also caused a marked inhibition of Syk and LAT1 phosphorylation, but only the latter mutant showed a reduction in Akt phosphorylation. These findings show that the full phosphorylation of Syk and LAT1 requires the FcεRIβ Y 219 and both Y 47 and Y 58 of the γ-chain. However, T 308 phosphorylation of Akt is largely independent of FcεRIγ Y 47 phosphorylation and of the Lyn-binding site (Y 219 ) on the FcεRIβ.

Immunoglobulin E plays an immunoregulatory role in lupus

“…Collectively, these results implicate the presence of autoreactive IgE as a key factor in basophil activation in human SLE subjects. This is supported by our previous finding that basophils from human SLE subjects have enhanced phosphorylation of the FcεRγ ( Suzuki et al, 2013 ), suggesting that the activation of human basophils in lupus is mediated through engagement of FcεRI by autoreactive IgE complexes.…”

“…of the FcR (Suzuki et al, 2013), suggesting that the activation of human basophils in lupus is mediated through engagement of FcRI by autoreactive IgE complexes.…”

CD45 exclusion– and cross-linking–based receptor signaling together broaden FcεRI reactivity

“…1, A (left) and B]. FceRI phosphorylation was detected on Western blots, using an anti-pY47 FceRIg phospho-specific antibody (34), as pairs of bands corresponding to short-and long-lived, mono-and dualphosphorylated forms of FceRIg. We tested whether this was due to surface contact per se by quantifying phosphoryl-ated FceRIg as the cells settled onto plastic surfaces in phosphatebuffered saline (PBS).…”

“…Solubilized samples were centrifuged at 14,000g for 5 min to remove protein aggregates, diluted in 4× SDS protein loading buffer with 10% b-mercaptoethanol, and separated on mini-PROTEAN TGX polyacrylamide gels (Bio-Rad). Proteins were transferred to nitrocellulose membrane, blocked with Odyssey blocking buffer (LI-COR Biosciences) for 3 hours, and then incubated with rabbit anti-FceRIg (Merck-Millipore, P06727) and mouse phospho-specific anti-FceRIg antibodies (34), provided by R. Suzuki (Nagoya City University, Japan). Donkey antirabbit IRDye 680RD (LI-COR Biosciences) and donkey anti-mouse IRDye 800CW (LI-COR Biosciences) secondary antibodies were used to enable simultaneous imaging and quantification in both channels using a LI-COR Biosciences Odyssey CLx infrared imaging system.…”

The Emerging Picture of Mast Cell Activation: The Complex Regulatory Network of High-Affinity Receptor for Immunoglobulin E Signaling

“…This inhibition by monovalent hapten abrogates FcεRI phosphorylation and prevents effective responses (i.e., degranulation). 12,13) These findings argue that the aggregation of FcεRI has an essential role in the induction of MC degranulation and cytokine/chemokine release.…”