Tadokoro Satoshi, Shibata Tetsuhiro, Inoh Yoshikazu, Amano Toshiro, Nakanishi Mamoru, Hirashima Naohide, Utsunomiya-Tate Naoko
Faculty of Pharma Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan.
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan.
Cell Biol Int. 2016 May;40(5):589-96. doi: 10.1002/cbin.10600. Epub 2016 Mar 21.
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.
最近的研究表明，可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白相互作用，形成一个诱导肥大细胞胞吐作用的SNARE复合体。此前，我们报道过SNARE蛋白Syntaxin-3调节肥大细胞胞吐作用且持续被磷酸化。在本研究中，我们试图鉴定肥大细胞中被磷酸化的氨基酸残基，并阐明Syntaxin-3磷酸化对胞吐作用的调控机制。我们发现Syntaxin-3的苏氨酸14是肥大细胞中的磷酸化位点。此外，组成型去磷酸化的Syntaxin-3（T14A）突变体的过表达增强了肥大细胞的胞吐作用。我们还表明，Syntaxin-3在苏氨酸14处的拟磷酸化突变（T14E）诱导了Syntaxin-3的结构变化，并且该突变抑制了Syntaxin-3与Munc18-2的结合。这些结果表明，苏氨酸14处磷酸化的Syntaxin-3通过损害Syntaxin-3与Munc18-2之间的相互作用对肥大细胞胞吐作用产生负调控。
