title: Chemical quenched flow kinetic studies indicate an intraholoenzyme autophosphorylation mechanism for Ca2+/calmodulin-dependent protein kinase II.
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.
authors.affiliation: Department of Neurobiology, Stanford University, Stanford, California 94305, USA.
authors.forename: J Michael
authors.initials: JM
authors.lastname: Bradshaw
authors.forename: Andy
authors.initials: A
authors.lastname: Hudmon
authors.forename: Howard
authors.initials: H
authors.lastname: Schulman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051381:Rats; D012542:Scattering, Radiation
chemical_list: D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases