Autophosphorylation of type II Ca2+/calmodulin-dependent protein kinase in cultures of postnatal rat hippocampal slices.
Autophosphorylation of Thr286 on type II Ca2+/calmodulin-dependent protein kinase (CaM kinase) in vitro causes kinase activity to become partially independent of Ca2+. Here we report that Thr286 is the major CaM kinase autophosphorylation site occupied in situ in "organotypic" hippocampal cultures. Measurement of Ca(2+)-independent CaM kinase activity revealed that approximately one-third of the kinase is autophosphorylated in situ when the basal Ca2+ concentration is 15-43 nM. This proportion was substantially reduced 30 min after removal of extracellular Ca2+ or treatment of the cultures with protein kinase inhibitors and was increased by treatment with okadaic acid. Therefore, the high proportion of autophosphorylated kinase at basal Ca2+ concentrations appears to be maintained by Ca(2+)-dependent autophosphorylation. Homogenates of intact hippocampi also contain a high proportion of Ca(2+)-independent type II CaM kinase, 13-23% depending on developmental age. Thus, in hippocampal neurons, an important function of the autophosphorylation mechanism may be to produce a relatively high level of CaM kinase activity, even at basal Ca2+ concentrations, permitting both upward and downward local regulation by physiological agents.
Molloy
S S
SS
Division of Biology, California Institute of Technology, Pasadena 91125.
Kennedy
M B
MB
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000375:Aging; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000254:growth & development*; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000037:antagonists & inhibitors; D004533:Egtazic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000201:enzymology / Q000254:growth & development*; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D009924:Organ Culture Techniques; D009928:Organ Specificity; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D002147:Calmodulin; D007546:Isoquinolines; D010748:Phosphopeptides; D010879:Piperazines; D004533:Egtazic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium