Characterization and autophosphorylation of Ca2+/calmodulin-dependent protein kinase in the postsynaptic density of the rat forebrain.
The enzymatic and regulatory properties of Ca2+/calmodulin-dependent protein kinase in the postsynaptic density (mPSDp CaM kinase) of the rat forebrain was compared with those of soluble Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). mPSDp CaM kinase was different from soluble CaM kinase II in terms of substrate specificity, regulatory consequences and sites of autophosphorylation. Both soluble and PSD kinases generated Ca(2+)-independent activity by autophosphorylation and Ca(2+)-independent activity almost reached the maximum during the first minute of autophosphorylation. Ca(2+)-independent activity of mPSDp CaM kinase was more stable than that of the soluble kinase under autophosphorylating conditions. Autophosphorylation of the kinases decreased the mobility of the kinases on SDS-polyacrylamide gels. The mobility shift and determination of 32P phosphate incorporation into the kinases demonstrated that there were three species in mPSDp CaM kinase alpha isoform: two active forms with and without the mobility shift (about 22 and 19%, respectively), and an inactive form (about 59%). However, there was only one species in the soluble kinase alpha isoform, which was active. The maximum incorporation of 32P phosphate into mPSDp CaM kinase alpha isoform was less than that of the soluble kinase. Tryptic peptide analysis indicated that the phosphorylation sites of mPSDp CaM kinase alpha isoform differed from those of the soluble kinase.
Ochiishi
T
T
Department of Cell Biology, Tokyo Metropolitan Institute for Neuroscience, Japan.
Sugiura
H
H
Yamauchi
T
T
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D010766:Phosphorylation; D016548:Prosencephalon / Q000201:enzymology* / Q000648:ultrastructure; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D012995:Solubility; D013379:Substrate Specificity; D013569:Synapses / Q000201:enzymology*
D007527:Isoenzymes; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases