Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells.
cDNAs containing the entire coding regions of the alpha and beta subunits of calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a rat cerebrum cDNA library, ligated into an expression vector under the control of SV40 early promoter and introduced into Chinese hamster ovary (CHO) cells. To investigate the role of the alpha and beta subunits and their functional domains in CaM kinase II activity, the properties of the kinases expressed in the transfected cells were studied. CaM kinase II activity was detected in the transfected cells when the alpha and beta cDNAs were introduced into CHO cells simultaneously. RNA transfer blot and protein immunoblot analyses demonstrated the expression of the mRNAs and proteins of both alpha and beta subunits in the cloned cells. When alpha or beta cDNA was introduced into CHO cells separately, a significant level of the enzyme activity was also expressed, indicating that the alpha and beta subunits exhibited enzyme activity individually. The apparent Km values for ATP and MAP 2 were almost the same for the alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II. However, there was a slight difference in the affinity for calmodulin between the expressed proteins. The alpha and beta subunits expressed in the same cells polymerized to form alpha beta complex of a size similar to that of brain CaM kinase II. The alpha subunit also polymerized to form an oligomer, which showed almost the same S value as that of alpha beta complex and brain CaM kinase II. In contrast, the beta subunit did not polymerize. The alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II were autophosphorylated with [gamma-32P]ATP in the presence of Ca2+ and calmodulin, which resulted in the appearance of Ca2+-independent activity. The Ca2+-independent activity was 60-75% of the total activity as measured in the presence of Ca2+ plus calmodulin. To examine the functional relationship of peptide domains of the subunits of CaM kinase II, deleted cDNAs were introduced into CHO cells and the properties of the expressed proteins were studied. In cells transfected with alpha or beta cDNA from which the association domain was deleted, a significant level of kinase activity was expressed. However, the expressed proteins showed hardly any autophosphorylation and the appearance of Ca2+-independent enzyme activity was very low, indicating that the association domain was essential for the autophosphorylation and for the appearance of the Ca2+-independent activity.
Yamauchi
T
T
Department of Neurochemistry, Tokyo Metropolitan Institute for Neurosciences, Japan.
Ohsako
S
S
Deguchi
T
T
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics*; D005260:Female; D015723:Gene Library; D005822:Genetic Vectors; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D010053:Ovary; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
D002147:Calmodulin; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D004247:DNA; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases