Phosphorylation of SNAP-23 regulates its dynamic membrane association during mast cell exocytosis [RESEARCH ARTICLE]

Pieu Naskar and Niti Puri

Upon allergen challenge, mast cells (MCs) respond by releasing pre-stored mediators from their secretory granules by the 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 the 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 wild-type SNAP-23 (GFP-SNAP-23) and its phosphorylation mutants were transfected into rat basophilic leukemia (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 membrane association, and of induced phosphorylation at Ser⁹⁵ and Ser¹²⁰ in its internal membrane association, during MC exocytosis.

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