Importance of Spatial and Temporal Regulation of SNAP‐23 Localization in Activated Mast Cells by Transient Phosphorylation

Abstract

Mast cells are innate immune tissue resident cells. These cells release various inflammatory mediators like cytokines, chemokines, histamine and lysosomal enzymes upon allergen activation. Previous work revealed that Synaptosomal-associated protein 23 kDa (SNAP-23) is a t-SNARE present in mast cells, synthesized in the cytoplasm, and then associates with the plasma membrane to play a major role during mast cell exocytosis. Early, on allergen activation it gets phosphorylated at Serine95 (S95) and Serine 120(S120) and translocates to the granule membrane containing LAMP-3. Blocking either phosphorylation or dephosphorylation by introducing phosphonegative or phosphomimetic mutations in SNAP-23 caused massive inhibition in mast cell exocytosis. Therefore, we decided to unravel the kinetics of phosphorylation and dephosphorylation at specific amino acid residues and to see their effects on SNAP-23 localization in resting and activated mast cells at different time point. It was observed that (S120) gets phosphorylated first, (S95) later. These residues were mutated by site-directed mutagenesis to Aspartate (phosphomimetic) and Alanine (phosphonegative) to get SNAP-23(S95DS120D), SNAP-23(S95AS120A), SNAP-23(S95AS120D), SNAP-23(S95DS120A) and cloned into EGFP-C2 vector. All EGFP-phosphomutants and SNAP-23 wildtype (WT) were transfected in RBL MCs to study subcellular localization by confocal microscopy at an early (10 min) and late (45 min) time points after activation. It was observed that plasma membrane association for WT SNAP-23 almost decreased to half in allergen activated (49%-33% at 10min; 24%-11% at 45min) mast cells compared to resting stage (80%-75%). In various phosphomutants of SNAP-23, the plasma membrane association was greatly affected and proper localization was not achieved on activation at both time points. These results suggest that SNAP-23 dynamicity in terms of intracellular localization and phosphorylation has a tremendous role in mast cells exocytosis and can be targeted as a potential target against allergy.