Autophosphorylation-dependent inactivation of plant chimeric XXXX /calmodulin-dependent protein kinase.

@entity460 @entity141 @entity860 @entity26864 @entity855 @entity70890
Chimeric @entity460 /calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin-like @entity141 -binding domain unlike other known calmodulin- dependent kinases. @entity141 -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 @entity141 -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 @entity855 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 @entity855 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 @entity860 - @entity26864 (an unhydrolyzable @entity855 analog) or the autophosphorylation-site mutant, @entity70890 , 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.
