Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity.
Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.
Sathyanarayanan
P V
PV
Laboratory of Plant Molecular Biology and Physiology, Department of Horticulture, Washington State University, Pullman, Washington 99164-6414, USA.
Cremo
C R
CR
Poovaiah
B W
BW
D000255:Adenosine Triphosphate / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D003619:Dansyl Compounds; D004789:Enzyme Activation; D005454:Fluorescence Polarization; D019668:Liliaceae / Q000201:enzymology; D008962:Models, Theoretical; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051596:Neurocalcin; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D044169:Receptors, Calcium-Sensing; D017384:Sequence Deletion
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D003619:Dansyl Compounds; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D010940:Plant Proteins; D044169:Receptors, Calcium-Sensing; D000255:Adenosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases