Regulatory Properties of Calcium/Calmodulin‐Dependent Protein Kinase II in Rat Brain Postsynaptic Densities

Calcium/calmodulin (CaM)‐dependent protein kinase II (CaM‐kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+‐indepen‐dent following initial activation by Ca2+/CaM. Generation of this Ca2+‐independent species was dependent upon auto‐phosphorylation of both subunits of the enzynme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 ± 5% of the total activity within 1–2 min. Subsequent to the generation of this partially Ca2+‐independent form of PSD CaM‐kinase II, addition of EGTA to the autophos‐phorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+‐dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the α‐ and β‐subunits, respectively, may be responsible for the transition of PSD CaM‐kinase II to the Ca2+‐independent species. A synthetic peptide 281–309 corresponding to a portion of the regulatory domain (residues 281–314) of the soluble kinase inhibited syntide‐2 phosphorylation by the Ca2+‐independent form of PSD CaM‐kinase II (IC50= 3.6 ± 0.8 μM). Binding of Ca2+/CaM to peptide 281–309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281–309 also decreased its inhibitory potency. These data suggest that CaM‐kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM‐kinase II.

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