Abstract
Recent studies have revealed that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins interact with each other, forming a SNARE complex that induces exocytosis in mast cells. Previously, we reported that syntaxin-3, a SNARE protein, regulates mast cell exocytosis and is constantly phosphorylated. In this study, we tried to identify the amino acid residue that is phosphorylated in mast cells, and to elucidate the regulatory mechanism of exocytosis by phosphorylation in syntaxin-3. We found that Thr 14 of syntaxin-3 was a phosphorylation site in mast cells. In addition, the overexpression of a constitutively dephosphorylated syntaxin-3 (T14A) mutant enhanced mast cell exocytosis. We also showed that the phosphomimetic mutation of syntaxin-3 at Thr 14 (T14E) induced structural changes in syntaxin-3, and this mutation inhibited binding of syntaxin-3 to Munc18-2. These results suggest that phosphorylated syntaxin-3 at Thr 14 negatively regulates mast cell exocytosis by impairing the interaction between syntaxin-3 and Munc18-2.