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J. Biol. Chem., Vol. 277, Issue 23, 20991-20998, June 7, 2002
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From the Department of Neurobiology, Stanford University,
Stanford, California 94305
Autophosphorylation of
Chemical Quenched Flow Kinetic Studies Indicate an
Intraholoenzyme 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 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.
*
This work was supported by National Institutes of Health
Grants GM40600 and GM30179.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Fellow of the Jane Coffin Childs Memorial Fund for Medical Research.
§
Fellow of the American Heart Association.
¶
To whom correspondence should be addressed: Dept. of
Neurobiology, Stanford University, 299 Campus Dr. West, Fairchild
Building, Stanford, CA 94305. Tel.: 650-723-7668; Fax:
650-725-3958; E-mail: schulman@stanford.edu.
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