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

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.