Phosphorylation of SNAP-23 regulates its dynamic membrane association during Mast Cell exocytosis

Abstract

Mast cells (MCs) on allergen challenge, respond by release of pre-stored mediators from their secretory granules by transient mechanism of porosome-mediated cell secretion. The target-SNARE SNAP-23 has been shown to be important for MC exocytosis and our previous studies revealed presence of one basal (Thr102) and two induced (Ser95 and Ser120) phosphorylation sites in its linker region. To study the role of SNAP-23 phosphorylation in the regulation of exocytosis, Green fluorescence protein-tagged wildtype SNAP-23 (GFP-SNAP-23) and its phosphorylation mutants were transfected into RBL-2H3 MCs. Studies on GFP-SNAP-23 transfected MCs revealed some dynamic changes in SNAP-23 membrane association. SNAP-23 was associated with plasma membrane in resting MCs, however on activation, a portion of it translocated to cytosol and internal membranes. These internal locations were secretory granule membranes. This dynamic change in the membrane association of SNAP-23 in MCs may be important for mediating internal granule-granule fusions in compound exocytosis. Further studies with SNAP-23 phosphorylation mutants revealed an important role for the phosphorylation at Thr102 in its initial, and of induced phosphorylation at Ser95 and Ser120 in its internal, membrane association, during MC exocytosis.