Activation of Ca2+/calmodulin-dependent protein kinase II by stimulation with bradykinin in neuroblastoma x glioma hybrid NG108-15 cells.
To elucidate the mechanisms of the intracellular signal transduction elicited with bradykinin in NG108-15 neuroblastoma x glioma hybrid cells, we examined the activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) by bradykinin stimulation. When the extract of NG108-15 cells was immunoprecipitated with the affinity-purified antibody to brain CaM kinase II, a 50-kDa protein in the immunoprecipitate mainly became autophosphorylated in a Ca2+/calmodulin-dependent manner. The results suggest that the 50-kDa protein is the subunit of CaM kinase II in NG108-15 cells. The Ca2+/calmodulin-independent activity (autonomous activity) of the enzyme increased twice within 10 s by stimulation with 1 microM bradykinin in the cells. The increase in the autonomous activity of the enzyme had two phases: the transient early-peak phase and the long late-plateau phase. The former was abolished by the pretreatment of the cells with 10 mM caffeine or 20 microM BAPTA-AM, and the latter was abolished by the removal of the extracellular Ca2+ with 1 mM EGTA or by the pretreatment with 1 microM nifedipine. Stimulation of 32P-labeled NG108-15 cells with 1 microM bradykinin increased the autophosphorylation of CaM kinase II and this increase was abolished by pretreatment with caffeine or BAPTA-AM. These results suggest that CaM kinase II is activated via the inositol phospholipid signaling pathway induced with bradykinin in NG108-15 cells.
Yamakawa
T
T
Department of Pharmacology, Kumamoto University School of Medicine, Japan.
Fukunaga
K
K
Higashida
H
H
Miyamoto
E
E
D001920:Bradykinin / Q000494:pharmacology*; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004347:Drug Interactions; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005910:Glioma / Q000201:enzymology*; D006822:Hybrid Cells / Q000187:drug effects / Q000201:enzymology; D009447:Neuroblastoma / Q000201:enzymology*; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011233:Precipitin Tests; D011494:Protein Kinases / Q000187:drug effects* / Q000378:metabolism; D014407:Tumor Cells, Cultured
C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D002110:Caffeine; D004533:Egtazic Acid; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D011188:Potassium; D001920:Bradykinin; D002118:Calcium