Ca2+/calmodulin-dependent protein kinase II isoenzymes gamma and delta are both present in H+/K+-ATPase-containing rabbit gastric tubulovesicles.
Ca2+/calmodulin-dependent protein kinase II is thought to participate in M3 muscarinic receptor-mediated acid secretion in gastric parietal cells. During acid secretion tubulovesicles carrying H+/K+-ATPase fuse with the apical membrane. We localized Ca2+/calmodulin-dependent protein kinase II from highly purified rabbit gastric tubulovesicles using Ca2+/calmodulin-dependent protein kinase II isoform-specific antibodies, in vitro phosphorylation and pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II activity by the potent Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62. The presence of Ca2+/calmodulin-dependent protein kinase II in tubulovesicles was shown by immunoblot detection of both Ca2+/calmodulin-dependent protein kinase II-gamma (54 kDa) and Ca2+/calmodulin-dependent protein kinase II-delta (56.5 kDa). The immunoprecipitated Ca2+/calmodulin-dependent protein kinase II from tubulovesicles showed Ca2+/calmodulin-dependent protein kinase activity by phosphorylating autocamtide-II, a specific synthetic Ca2+/calmodulin-dependent protein kinase II substrate. KN-62 inhibited the in vitro autophosphorylation of tubulovesicle-associated Ca2+/calmodulin-dependent protein kinase II (IC50 = 11 nM). During the search for potential Ca2+/calmodulin-dependent protein kinase II substrates we identified different proteins associated with tubulovesicles, such as synaptophysin and beta-tubulin immunoreactivity, which were identified using specific antibodies. These targets are known to participate in intracellular membrane traffic. Ca2+/calmodulin-dependent protein kinase II is thought to play an important role in regulating tubulovesicular motor activity and therefore in acid secretion.
Fährmann
M
M
Institut für Zoophysiologie der Westfälischen Wilhelms-Universität Münster, Germany. fahrman@uni-muenster.de
Jacob
P
P
Seidler
U
U
Osterhoff
M
M
Möhlig
M
M
Pfeiffer
A
A
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism*; D007120:Immunochemistry; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D010295:Parietal Cells, Gastric / Q000201:enzymology*; D010766:Phosphorylation; D011233:Precipitin Tests; D011817:Rabbits; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D007527:Isoenzymes; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017506:H(+)-K(+)-Exchanging ATPase