Chemical quenched flow kinetic studies indicate an intraholoenzyme autophosphorylation mechanism for Ca2+/calmodulin-dependent protein kinase II

J. Michael Bradshaw, Andy Hudmon, Howard Schulman

Research output: Contribution to journalArticle

58 Scopus citations


Autophosphorylation of α-Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) at Thr-286 generates Ca2+-independent activity that outlasts the initial Ca2+ stimulus. Previous studies suggested that this autophosphorylation occurs between subunits within each CaM kinase II holoenzyme. However, electron microscopy studies have questioned this mechanism because a large distance separates a kinase domain from its neighboring subunit. Moreover, the recently discovered ability of CaM kinase II holoenzymes to self-associate has raised questions about data interpretation in previous investigations of autophosphorylation. In this work, we characterize the mechanism of CaM kinase II autophosphorylation. To eliminate ambiguity arising from kinase aggregation, we used dynamic light scattering to establish the monodispersity of all enzyme solutions. We then found using chemical quenched flow kinetics that the autophosphorylation rate was independent of the CaM kinase II concentration, results corroborating intraholoenzyme activation. Experiments with a monomeric CaM kinase II showed that phosphorylation of this construct is intermolecular, supporting intersubunit phosphorylation within a holoenzyme. The autophosphorylation rate at 30 °C was ∼12 s-1, more than 10-fold faster than past estimates. The ability of CaM kinase II to autophosphorylate through an intraholoenzyme, intersubunit mechanism is likely central to its functions of decoding Ca2+ spike frequency and providing a sustained response to Ca2+ signals.

Original languageEnglish (US)
Pages (from-to)20991-20998
Number of pages8
JournalJournal of Biological Chemistry
Issue number23
StatePublished - Jun 7 2002
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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