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

  • Michael Bradshaw J
  • Hudmon A
  • Schulman H
  • 24

    Readers

    Mendeley users who have this article in their library.
  • 53

    Citations

    Citations of this article.

Abstract

Autophosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) at Thr-286 generates Ca(2+)-independent activity that outlasts the initial Ca(2+) 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 degrees C was approximately 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 Ca(2+) spike frequency and providing a sustained response to Ca(2+) signals.

Get free article suggestions today

Mendeley saves you time finding and organizing research

Sign up here
Already have an account ?Sign in

Find this document

Authors

  • J. Michael Bradshaw

  • Andy Hudmon

  • Howard Schulman

Cite this document

Choose a citation style from the tabs below

Save time finding and organizing research with Mendeley

Sign up for free