Journal article
Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase
European journal of biochemistry, Vol.269(10), pp.2457-2463
05/2002
Handle:
https://hdl.handle.net/2376/115154
PMID: 12027883
Abstract
Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin-like Ca(2+)-binding domain unlike other known calmodulin- dependent kinases. Ca(2+)-Binding to the visinin-like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417-30422]. Here, we report that the Ca(2+)-stimulated autophosphorylation of CCaMK results in time-dependent loss of enzyme activity. This time-dependent loss of activity or self-inactivation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self-association. Specifically, autophosphorylation in the presence of 200 microm ATP at pH 7.5 resulted in the formation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild-type kinase in the presence of AMP-PNP (an unhydrolyzable ATP analog) or the autophosphorylation-site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation- dependent self-inactivation may be a mechanism of modulating the signal transduction pathway mediated by CCaMK.
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Details
- Title
- Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase
- Creators
- P V Sathyanarayanan - Laboratory of Plant Molecular Biology and Physiology, Department of Horticulture, Washington State University, Pullman, WA 99164-6414, USAB W Poovaiah
- Publication Details
- European journal of biochemistry, Vol.269(10), pp.2457-2463
- Academic Unit
- Horticulture, Department of
- Publisher
- England
- Identifiers
- 99900548022201842
- Language
- English
- Resource Type
- Journal article