Ca2+/calmodulin-dependent protein kinase II isoenzymes gamma and delta areboth present in H+/K+-ATPase-containing rabbit gastric tubulovesicles

Ca2+/calmodulin-dependent protein kinase II is thought to participate in Mg muscarinic receptor-mediated acid secretion in gastric parietal cells. Dur ing acid secretion tubulovesicles carrying H+/K+-ATPase fuse with the apica l membrane. We localized Ca2+/calmodulin-dependent protein kinase II from h ighly purified rabbit gastric tubulovesicles using Ca2+/calmodulin-dependen t protein kinase II isoform-specific antibodies, in vitro phosphorylation a nd pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase I I activity by the potent Ca2+/calmodulin-dependent protein kinase II inhibi tor KN-62. The presence of Ca2+/calmodulin-dependent protein kinase II in t ubulovesicles was shown by immunoblot detection of both Ca2+/calmodulin-dep endent protein kinase II-gamma (54 kDa) and Ca2+/calmodulin-dependent prote in kinase II-delta (56.5 kDa). The immunoprecipitated Ca2+/calmodulin-depen dent protein kinase II from tubulovesicles showed Ca2+/calmodulin-dependent protein kinase activity by phosphorylating autocamtide-II, a specific synt hetic Ca2+/calmodulin-dependent protein kinase II substrate. KN-62 inhibite d the in vitro autophosphorylation of tubulovesicle-associated Ca2+/calmodu lin-dependent protein kinase II (IC50 = 11 nM). During the search for poten tial Ca2+/calmodulin-dependent protein kinase II substrates we identified d ifferent proteins associated with tubulovesicles, such as synaptophysin and beta-tubulin immunoreactivity, which were identified using specific antibo dies. These targets are known to participate in intracellular membrane traf fic. Ca2+/calmodulin-dependent protein kinase II is thought to play an impo rtant role in regulating tubulovesicular motor activity and therefore in ac id secretion.