Phosphorylation of SNAP-23 regulates its dynamic subcellular localization and regulated exocytosis in mast cells

Abstract

Abstract Mast cells (MCs) play an important role in inflammatory response to pathogens and in inflammatory disorders like allergies. This role depends on their ability to release prestored mediators from secretory granules in response to some external trigger like crosslinking of FcɛRI. The membrane fusion events involved therein are mediated by SNAREs. The target-SNARE SNAP-23 has been shown to be very 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, whose significance was not very clear. To study the role of SNAP-23 phosphorylation in the regulation of exocytosis, GFP-tagged wildtype SNAP-23 (GFP-SNAP-23) and its phosphorylation mutants were transfected into RBL MCs, that revealed some dynamic changes in SNAP-23 membrane association immediately after activation. SNAP-23 was associated with plasma membrane in resting mast cells, however on activation, it translocated to internal LAMP-3 containing lysosomal membranes, many of which contained secretory granule cargo. 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 mutants revealed, for the first time, that the basal phosphorylation site Thr102 plays an important role in initial SNAP-23 membrane association. During exocytosis, the induced phosphorylation sites Ser95 and Ser120 regulate SNAP-23 internal membrane association and subsequently mast cell exocytosis. Hence this study reveals a novel mechanism in mast cell inflammatory response that can be a therapeutic target.