Phosphorylation of SNAP-23 Regulates Exocytosis from Mast Cells

Abstract

Regulated exocytosis is a process in which a physiological trigger initiates the translocation, docking, and fusion of secretory granules with the plasma membrane. A class of proteins termed SNAREs (including SNAP-23, syntaxins, and VAMPs) are known regulators of secretory granule/plasma membrane fusion events. We have investigated the molecular mechanisms of regulated exocytosis in mast cells and find that SNAP-23 is phosphorylated when rat basophilic leukemia mast cells are triggered to degranulate. Regulated exocytosis is the process by which stimulation of plasma membrane receptors on secretory cells results in the release of proteins and/or peptides from intracellular stores into the extracellular space (1). One common characteristic of regulated exocytosis, whether it be from neurons, cytotoxic lymphocytes, adipocytes, or mast cells, is that the cells response to this stimulus results in pre-formed intracellular granules moving toward and fusing with the plasma membrane. Secretory granule/plasma membrane fusion is the essence of regulated exocytosis, and there is an intense effort underway to identify the molecular mechanisms regulating this process in the hopes of identifying ways to modulate exocytosis.The RBL-2H3 1 mast cell line has been extensively studied as a model not only for mast cell biology but also as a paradigm for regulated exocytosis from non-neuronal cells (2). Stimulation of the high affinity IgE receptor, Fc⑀RI, on these cells by crosslinking initiates a signal transduction cascade that culminates in secretory granule fusion with the plasma membrane, thereby liberating a variety of inflammatory mediators (3, 4). Numerous proteins necessary for the tethering, docking, and fusion steps between various membrane compartments in eukaryotic cells have been described. Among those, SNAREs (soluble NSF-attachment protein receptors) are a large family of membrane-associated proteins essential for membrane-membrane fusion (5, 6). These proteins include members of the vesicle-associated synaptobrevin/VAMP family as well as members of the syntaxin and SNAP-23 families of "target" membrane SNAREs. The current model proposes that while vesicles are docked on the target membrane, SNAREs from the donor or vesicle membrane (v-SNAREs) form trans-SNARE complexes with their cognate SNARE partners on the opposing target membrane. Structurally, the exocytic SNARE complex is a trimolecular protein complex containing one member of the VAMP, syntaxin, and SNAP-23 family, each contributing to the formation of a four-helix coiled-coil bundle (7, 8) whose formation is sufficient for in vitro membrane fusion (9).Given that SNAREs play a central role in the membrane fusion process, it is likely that their function is modulated in vivo. In particular, protein kinases, which have been extensively associated with the regulation of exocytosis (10), could participate in SNARE function by phosphorylating residues essential in SNARE complex assembly or the binding of SNARE regulatory proteins (6,11,12). Members of the syntax...

Regulation of Leukocyte Degranulation by cGMP-Dependent Protein Kinase and Phosphoinositide 3-Kinase: Potential Roles in Phosphorylation of Target Membrane SNARE Complex Proteins in Rat Mast Cells

“…A more detailed biochemical analysis will be necessary to determine whether the above proteins are substrates for PKG and p38 MAPK using in vitro kinase assays. Consistent with published reports on PKC-mediated phosphorylation of SNARE proteins (14,32), all the proteins examined in this study contain multiple sites for PKC phosphorylation.…”

“…Published studies indicate that phosphorylation of SNAP-23 and syntaxin 4 are essential for degranulation in RBL cells and platelets (13,14). Furthermore, expression of a phosphorylation-deficient mutant in RBL cells inhibited degranulation (14). Thus, negative regulation of degranulation by Rp-Br-PET-cGMP and LY294002 may be attributed to their inhibitory effect on the phosphorylation of SNARE proteins.…”

“…These patterns of phosphorylation are intriguing and probably suggest that maximal degranulation requires phosphorylation on multiple SNARE proteins, but inhibition of phosphorylation in one or two of these proteins can be sufficient to compromise degranulation. Published studies indicate that phosphorylation of SNAP-23 and syntaxin 4 are essential for degranulation in RBL cells and platelets (13,14). Furthermore, expression of a phosphorylation-deficient mutant in RBL cells inhibited degranulation (14).…”

“…Phosphorylation of Munc18-1 in neuronal cells (10,11) and Munc18-3 and syntaxin 4 in endothelial cells (12) promotes Munc18-syntaxin dissociation, thereby facilitating vesicular fusion. Phosphorylation of syntaxins, Munc18, and SNAP-23 is catalyzed by kinases such as protein kinase C (PKC) (12)(13)(14). In mast cells, a mechanism of degranulation induced by FcRI cross-linking involves recruitment and activation of the tyrosine kinases Lyn and Syk, phosphorylation of several tyrosine residues in FcRI, activation of phospholipase C␥ (PLC␥), and generation of the second messengers diacylglycerol and inositol trisphosphate (1,15).…”

ERK activation is only one role of PKC in TCR-independent cytotoxic T cell granule exocytosis

“…and A.Z., unpublished observations). PKC-dependent phosphorylation of SNAP-23 is important in exocytosis in platelets [34] and mast cells [35]. SNAP-23 may thus be a PKC substrate in CTLs.…”