CaM-kinase II dephosphorylates Thr286 by a reversal of the autophosphorylation reaction

Sally A. Kim, Andy Hudmon, Allison Volmer, M. Neal Waxham

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Autophosphorylation of CaM-kinase II produces a form of the enzyme not requiring Ca2+/calmodulin for sustained activity. We report that autophosphorylated CaM-kinase II dephosphorylates itself in the presence of ADP (termed autodephosphorylation). The dephosphorylation was unaffected by phosphatase inhibitors and was nucleotide specific, occurring with ADP but not with AMP, GTP, or GDP. 32P-ATP was produced when ADP was added to 32P-labeled CaM-kinase II, indicating that the enzyme was undergoing dephosphorylation through a reversal of the autophosphorylation reaction. ATP addition also produced loss of 32P from the autophosphorylated enzyme while maintaining the kinase in a phosphorylated state. This indicates that the enzyme was undergoing cycles of autophosphorylation and dephosphorylation in the activated state. Autothiophosphorylated CaM-kinase II was resistant to autodephosphorylation. Site-directed mutants were used to show that Thr286 was the predominant site dephosphorylated. Additionally, CaM-kinase II composed of β subunits exhibited autodephosphorylation. Ca2+/CaM-independent activity expressed by the autophosphorylated α and β holoenzymes was reversed following autodephosphorylation.

Original languageEnglish (US)
Pages (from-to)773-780
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume282
Issue number3
DOIs
StatePublished - Jan 1 2001
Externally publishedYes

Keywords

  • Calmodulin
  • Dephosphorylation
  • Enzyme kinetics
  • Kinase
  • Reaction mechanism
  • Thiophosphorylation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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