Ca²⁺/calmodulin-dependent protein kinase II. Identification of a regulatory autophosphorylation site adjacent to the inhibitory and calmodulin-binding domains

Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) autophosphorylated under limiting conditions (7 μM [γ-³²P]ATP, 500 μM magnesium acetate, 4°C) was analyzed by CNBr cleavage and peptide mapping to determine the site of autophosphorylation that brings about transition of the kinase to the Ca²⁺-independent form. Reverse phase high performance liquid chromatography (HPLC) (C₃) revealed one major CN-Br ³²P-peptide (CB₁) that eluted at about 6% propranol. This peptide contained [³²P]threonine, but almost no [³²P]serine, and migrated as a single band (M(r) = 3000-3500) in polyacrylamide gels run in the presence of urea and sodium dodecyl sulfate. The properties of CB₁ were compared to the properties of a 26-residue synthetic peptide containing the CaM-binding and inhibitory domains as well as a consensus phosphorylation sequence (-Arg-Gln-Glu-Thr-) of rat brain CaM-kinase II (residues 282-307 and 283-308 of the α and β subunits, respectively). CB₁ and the synthetic peptide comigrated in urea/sodium dodecyl sulfate gels, co-eluted from reverse phase HPLC (C₃ and C₁₈) and from Sephadex G-50, and exhibited Ca²a+-dependent calmodulin-binding properties. When the two peptides were subjected to automated Edman sequence analysis, both exhibited a burst of ³²P release at cycle 5, which is consistent with the expected amino-terminal sequence of the two peptides, i.e. His-Arg-Gln-Glu-Thr(PO₄)-. These findings indicate that autophosphorylation of Thr²⁸⁶ (α subunit) and Thr²⁸⁷ (β subunit) is responsible for transition of CaM-kinase II to the Ca²⁺-independent form.