Open AccessCa 2+ /calmodulin‐dependent protein kinase II isoenzymes γ and δ are both present in H + /K + ‐ATPase‐containing rabbit gastric tubulovesicles
Author(s) -
Fährmann Michael,
Jacob Petra,
Seidler Ursula,
Osterhoff Martin,
Möhlig Matthias,
Pfeiffer Andreas
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00959.x
Subject(s) - calmodulin , autophosphorylation , protein kinase a , biology , biochemistry , microbiology and biotechnology , kinase , enzyme
Ca 2+ /calmodulin‐dependent protein kinase II is thought to participate in M 3 muscarinic receptor‐mediated acid secretion in gastric parietal cells. During acid secretion tubulovesicles carrying H + /K + ‐ATPase fuse with the apical membrane. We localized Ca 2+ /calmodulin‐dependent protein kinase II from highly purified rabbit gastric tubulovesicles using Ca 2+ /calmodulin‐dependent protein kinase II isoform‐specific antibodies, in vitro phosphorylation and pharmacological inhibition of Ca 2+ /calmodulin‐dependent protein kinase II activity by the potent Ca 2+ /calmodulin‐dependent protein kinase II inhibitor KN‐62. The presence of Ca 2+ /calmodulin‐dependent protein kinase II in tubulovesicles was shown by immunoblot detection of both Ca 2+ /calmodulin‐dependent protein kinase II‐γ (54 kDa) and Ca 2+ /calmodulin‐dependent protein kinase II‐δ (56.5 kDa). The immunoprecipitated Ca 2+ /calmodulin‐dependent protein kinase II from tubulovesicles showed Ca 2+ /calmodulin‐dependent protein kinase activity by phosphorylating autocamtide‐II, a specific synthetic Ca 2+ /calmodulin‐dependent protein kinase II substrate. KN‐62 inhibited the in vitro autophosphorylation of tubulovesicle‐associated Ca 2+ /calmodulin‐dependent protein kinase II (IC 50 = 11 n m ). During the search for potential Ca 2+ /calmodulin‐dependent protein kinase II substrates we identified different proteins associated with tubulovesicles, such as synaptophysin and β‐tubulin immunoreactivity, which were identified using specific antibodies. These targets are known to participate in intracellular membrane traffic. Ca 2+ /calmodulin‐dependent protein kinase II is thought to play an important role in regulating tubulovesicular motor activity and therefore in acid secretion.