Occurrence and activation of Ca2+/calmodulin-dependent protein kinase II and its endogenous substrates in bovine adrenal medullary cells.
We investigated the presence of and the endogenous substrates for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in cultured bovine adrenal medullary cells. By a series of chromatographic steps using DEAE-cellulose, calmodulin affinity, and Sephacryl S-300 columns, we partially purified two CaM kinases (peaks I and III) and one calmodulin-binding protein (peak II). Both of the kinases (peaks I and III) showed broad substrate specificities. Peak I, but not peak III, was immunoprecipitated with an antibody against rat brain CaM kinase II, suggesting that peak I is CaM kinase II or a closely associated CaM kinase. Although the anticaldesmon antibody recognized a 77-kDa protein (low molecular mass caldesmon) in crude preparations from the cells, the protein in peak II was not immunoblotted with the antibody. The peak II protein was phosphorylated by the CaM kinase in peak I but not by the CaM kinase in peak III. Peak I kinase also phosphorylated purified tyrosine hydroxylase and several proteins from chromaffin granule membranes. Stimulation of cultured bovine adrenal medullary cells with 56 mM K+ evoked rapid increases in 45Ca2+ influx and autonomous CaM kinase II activity, both of which were attenuated by the addition of 20 mM MgSO4, an inhibitor of voltage-dependent Ca2+ channels. These results suggest that an isozyme of CaM kinase II exists in adrenal medullary cells and is activated by cell depolarization. Furthermore, the peak II protein is apparently a novel endogenous substrate for CaM kinase II.
Yanagihara
N
N
Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan.
Toyohira
Y
Y
Yamamoto
H
H
Ohta
Y
Y
Tsutsui
M
M
Miyamoto
E
E
Izumi
F
F
D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000276:immunology; D002417:Cattle; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000201:enzymology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D015151:Immunoblotting; D008278:Magnesium Sulfate / Q000494:pharmacology; D008970:Molecular Weight; D010766:Phosphorylation; D011233:Precipitin Tests; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D008278:Magnesium Sulfate; D014446:Tyrosine 3-Monooxygenase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium