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Published July 5, 1990 | Published
Journal Article Open

Activation of type II calcium/calmodulin-dependent protein kinase by Ca^(2+)/calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain

Abstract

It is now well established that autophosphorylation of a threonine residue located next to each calmodulin-binding domain in the subunits of type II Ca^(2+)/calmodulin-dependent protein kinase causes the kinase to remain active, although at a reduced rate, after Ca^(2+) is removed from the reaction. This autophosphorylated form of the kinase is still sensitive to Ca2+/calmodulin, which is required for a maximum catalytic rate. After removal of Ca^(2+), new sites are autophosphorylated by the partially active kinase. Autophosphorylation of these sites abolishes sensitivity of the kinase to Ca^(2+)/calmodulin (Hashimoto, Y., Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1987) J. Biol. Chem. 262, 8051-8055). We have identified two pairs of homologous residues, Thr^(305) and Ser^(314) in the alpha subunit and Thr^(306) and Ser^(315) in the beta subunit, that are autophosphorylated only after removal of Ca^(2+) from an autophosphorylation reaction. The sites were identified by direct sequencing of labeled tryptic phosphopeptides isolated by reverse-phase high pressure liquid chromatography. Thr^(305-306) is rapidly dephosphorylated by purified protein phosphatases 1 and 2A, whereas Ser^(314-315) is resistant to dephosphorylation. We have shown by selective dephosphorylation that the presence of phosphate on Thr^(305-306) blocks sensitivity of the kinase to Ca^(2+)/calmodulin. In contrast, the presence of phosphate on Ser^(314-315) is associated with an increase in the Kact for Ca^(2+)/calmodulin of only about 2-fold, producing a relatively small decrease in sensitivity to Ca^(2+)/calmodulin.

Additional Information

© 1990 by The American Society for Biochemistry and Molecular Biology, Inc. This work was supported by National Institutes of Health Grant NS 17660 and Training Grant NS07251, the Gustavus and Louise Pfeiffer Research Foundation, the Joseph W. Drown Foundation, the Irvine Foundation, and the McKnight Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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August 19, 2023
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