Phosphorylation of SNAP-23 regulates its dynamic membrane association during mast cell exocytosis.

Pieu Naskar,Niti Puri

doi:10.1242/bio.025791

Abstract

ABSTRACTUpon 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.

Highlights

Mast cells (MCs) are specialized secretory cells that play a crucial role in inflammation and allergic responses (Kalesnikoff and Galli, 2008)
Since all three major phosphorylation sites are in the linker region of SNAP-23, in close proximity to the conserved cysteine residues which may play an important role in anchoring SNAP-23 to membrane, we decided to study the role of SNAP-23 phosphorylation in its membrane associations in resting or activated MCs, respectively
Membrane/cytosol fractionation, and microscopy studies revealed that the phospho negative SNAP-23 S95A/S120A mutant was able to dissociate from plasma membrane on activation as more of it ended up in cytosol in comparison to control, but unable to associate with internal LAMP-3 positive membranes

Summary

Introduction

MCs are specialized secretory cells that play a crucial role in inflammation and allergic responses (Kalesnikoff and Galli, 2008). During hypersensitivity reactions they are mainly activated by cross-linking of FcεRI bound IgE by a multivalent allergen (Kraft and Kinet, 2007) This physiological trigger initiates a cascade of events that results in the translocation, docking, and fusion of secretory granule with plasma membrane leading to release of inflammatory mediators stored in the secretory granules (Galli et al, 2008; Puri and Roche, 2008). This process proceeds through a transient mechanism of fusion and release called “kiss and run” and cavicapture for a large proportion of granules in mast cells (Balseiro-Gomez et al, 2016). The pairing of this SNARE rosette with plasma membrane is the site defining the fusion pore or porosome where vesicles dock and fuse (Hammel and Meilijson, 2012; Moon et al, 2014)

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