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

C. M. Schworer, R. J. Colbran, Jeffrey Keefer, T. R. Soderling

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)13486-13489
Number of pages4
JournalJournal of Biological Chemistry
Volume263
Issue number27
StatePublished - 1988

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Calcium-Calmodulin-Dependent Protein Kinase Type 2
Calmodulin
Peptides
Reverse-Phase Chromatography
Sodium Dodecyl Sulfate
High performance liquid chromatography
Urea
High Pressure Liquid Chromatography
Peptide Mapping
Consensus Sequence
Threonine
Phosphorylation
Magnesium
Serine
Sequence Analysis
Acetates
Phosphotransferases
Adenosine Triphosphate
Gels
Rats

ASJC Scopus subject areas

  • Biochemistry

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Ca2+/calmodulin-dependent protein kinase II. Identification of a regulatory autophosphorylation site adjacent to the inhibitory and calmodulin-binding domains. / Schworer, C. M.; Colbran, R. J.; Keefer, Jeffrey; Soderling, T. R.

In: Journal of Biological Chemistry, Vol. 263, No. 27, 1988, p. 13486-13489.

Research output: Contribution to journalArticle

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abstract = "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.",
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