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Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinaseChimeric 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.
Document ID
20040088303
Acquisition Source
Legacy CDMS
Document Type
Reprint (Version printed in journal)
Authors
Sathyanarayanan, P. V.
(Washington State University Pullman, WA 99164-6414, United States)
Poovaiah, B. W.
Date Acquired
August 21, 2013
Publication Date
May 1, 2002
Publication Information
Publication: European journal of biochemistry / FEBS
Volume: 269
Issue: 10
ISSN: 0014-2956
Subject Category
Life Sciences (General)
Distribution Limits
Public
Copyright
Other
Keywords
NASA Discipline Plant Biology
Non-NASA Center

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