Chemical Quenched Flow Kinetic Studies Indicate an Intraholoenzyme Autophosphorylation Mechanism for Ca super(2+)/Calmodulin-dependent Protein Kinase II

• Published in: The Journal of biological chemistry Vol. 277; no. 23; pp. 20991 - 20998
• Main Authors: Bradshaw, J M, Hudmon, A, Schulman, H
• Format: Journal Article
• Language: English
• Published: 07.06.2002
• ISSN: 0021-9258

Autophosphorylation of alpha -Ca super(2+)/calmodulin-dependent protein kinase II (CaM kinase II) at Thr-286 generates Ca super(2+)-independent activity that outlasts the initial Ca super(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 degree C was ~12 s super(-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 super(2+) spike frequency and providing a sustained response to Ca super(2+) signals.