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Papers In Press, published online ahead of print March 29, 2002
Neurobiology, Stanford University, Stanford, CA 94305
Corresponding Author: schulman{at}cmgm.stanford.edu
Autophosphorylation of
J. Biol. Chem, 10.1074/jbc.M202154200
Submitted on March 5, 2002
Revised on March 25, 2002
Accepted on March 28, 2002
Chemical quench-flow kinetic studies indicate an intra-holoenzyme autophosphorylation mechanism for Ca2+/calmodulin-dependent protein kinase II
-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 this autophosphorylation occurs between subunits within each CaM kinase II holoenzyme. However, electron microscopy studies have questioned this mechanism since 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. In order 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 quench-flow kinetics that the autophosphorylation rate was independent of CaM kinase II concentration, results corroborating intra-holoenzyme activation. Experiments with a monomeric CaM kinase II showed phosphorylation of this construct is inter-molecular, supporting inter-subunit phosphorylation within a holoenzyme. The autophosphorylation rate at 30 oC was ~12 sec-1, over 10-fold faster than past estimates. The ability of CaM kinase II to autophosphorylate through an intra-holoenzyme, inter-subunit mechanism is likely central to its functions of decoding Ca2+ spike frequency and providing a sustained response to Ca2+ signals.
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