Presence of a complex containing vesicle-associated membrane protein 2 in rat parotid acinar cells and its disassembly upon activation of cAMP-dependent protein kinase
Presence of a complex containing vesicle-associated membrane protein 2 in rat parotid acinar cells and its disassembly upon activation of cAMP-dependent protein kinase
复制标题
DOI：
10.1074/jbc.274.33.23642
复制
发表时间：
1999-08-13
影响因子：
4.8
通讯作者：
Sugiya, H
中科院分区：
文献类型：
--
作者：
Fujita-Yoshigaki, J;Dohke, Y;Sugiya, H
腮腺腺泡细胞淀粉酶的释放主要是由细胞内cAMP的积累引起的，可能是通过cAMP依赖性蛋白激酶（PKA）磷酸化底物，但其分子机制尚不清楚。在先前的研究中（Fujita-Yoshigaki，J，，（1996）J. Biol. Chem，271，13130-13134），我们报道了囊泡相关膜蛋白2（VAMPS）位于分泌颗粒膜上，并参与cAMP诱导的淀粉酶分泌。为研究腮腺腺泡细胞中可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）与VAMPS复合物的形成，制备了兔抗VAMPS的Arg（47）-Asp（64）肽段的多克隆抗体（anti-SER 4256）。然后，我们用抗SER 4256抗体检测VAMPS的免疫沉淀条件，在对照条件下，VAMPS不包含在可溶性颗粒膜组分的免疫沉淀物中，但与胞浆组分和cAMP孵育引起VAMPS的免疫沉淀，加入PKA抑制剂H89可降低胞浆组分和cAMP的作用，PKA催化亚基和胞浆组分的添加允许VAMPS的免疫沉淀，而单独的PKA催化亚基则不能。这些结果表明：（1）VAMPS的t-SNARE结合区被某些蛋白X所掩蔽，PKA的激活导致X从VAMPS上解离;（2）PKA的作用不是直接磷酸化X，而是通过磷酸化其他胞浆蛋白发挥作用。
Amylase release from parotid acinar cells is mainly induced by the accumulation of intracellular cAMP, presumably through the phosphorylation of substrates by cAMP-dependent protein kinase (PKA), However, the molecular mechanisms of this process are not clear. In a previous study (Fujita-Yoshigaki, J,, Dohke, Y,, Hara-Yokoyama, M,, Kamata, Y,, Kozaki, S,, Furuyama, S,, and Sugiya, H. (1996) J. Biol. Chem, 271, 13130-13134), we reported that vesicle-associated membrane protein 2 (VAMPS) is localized at the secretory granule membrane and is involved in cAMP-induced amylase secretion. To study the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex containing VAMPS in parotid acinar cells, we prepared rabbit polyclonal antibody against the peptide corresponding to Arg(47)-Asp(64) Of VAMPS (anti-SER4256), The recognition site of anti-SER4256 overlaps the domain involved in binding target membrane SNAREs (t-SNARES). Then we examined the condition of VAMPS by immunoprecipitation with anti-SER4256, VAMPS was not included in the immunoprecipitate from solubilized granule membrane fraction under the control conditions, but incubation with cytosolic fraction and cAMP caused immunoprecipitation of VAMPS, The effect of cytosolic fraction and cAMP was reduced by addition of PKA inhibitor H89, Addition of both the catalytic subunit of PKA and the cytosolic fraction allowed immunoprecipitation of VAMPS, whereas the PKA catalytic subunit alone did not. These results suggest that (1) the t-SNARE binding region of VAMPS is masked by some protein X and activation of PKA caused the dissociation of X from VAMPS; and (2) the effect of PKA is not direct phosphorylation of X, but works through phosphorylation of some other cytosolic protein.