Abstract
Purified pig brain Ca(2+)-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca(2+)-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca(2+)-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca(2+)-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of approximately 3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase IV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase IV, demonstrating that Thr-196 phosphorylation is essential for activation.
Highlights
From the :\:Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York 14214 and the §Department of Pharmacology, Duke University, Durham, North Carolina 27710
The addition of brain extract, capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation
In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase, Ca2 +-CaM, and MgATP-dependent manner
Summary
Vol 270, No 29, Issue of July 21, pp. 17616-17621, 1995 Printed in U.S.A. Michele A. The currently accepted paradigm is a conformational change induced by the stoichiometric binding of Ca2+ -CaM, which in turn results in decreased interaction of an autoinhibitory domain with the active site, thereby permitting substrate accessibility This mechanism is ideally suited to the decoding of rapid Ca2+ transients provoked by extracellular signals and is well understood at the molecular level primarily through studies with myosin light chain kinase and CaM kinase II [22,23,24,25,26,27,28]. It was reported that Ca2+ -CaM-dependent autophosphorylation of purified rat brain CaM kinase I (a-isoform) is accompanied by a dramatic (> lO-fold) potentiation of activity and that an activator exists that is separable from CaM kinase Ia during the final steps of the latter's purification [5, 6]. We report that replacement of Thr-196 of CaM kinase IV with Ala by sitedirected mutagenesis prevents both phosphorylation and activation, indicating that Thr-196 phosphorylation is essential for activation
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