Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

P V Sathyanarayanan; C R Cremo; B W Poovaiah

doi:10.1074/jbc.M000771200

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Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

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Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

P V Sathyanarayanan, C R Cremo and B W Poovaiah

The Journal of biological chemistry, Vol.275(39), pp.30417-30422

09/29/2000

DOI: https://doi.org/10.1074/jbc.M000771200

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https://hdl.handle.net/2376/116964

PMID: 10840028

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Abstract

Calcium-Binding Proteins - metabolism

Models, Theoretical

Protein Structure, Tertiary

Sequence Deletion

Phosphorylation

Calmodulin - metabolism

Liliaceae - enzymology

Dansyl Compounds

Receptors, Calcium-Sensing

Nerve Tissue Proteins - metabolism

Neurocalcin

Adenosine Triphosphate - metabolism

Plant Proteins - metabolism

Enzyme Activation

Mutation

Fluorescence Polarization

Calcium-Calmodulin-Dependent Protein Kinases - metabolism

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.

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Title: Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity
Creators: P V Sathyanarayanan - Laboratory of Plant Molecular Biology and Physiology, Department of Horticulture, Washington State University, Pullman, Washington 99164-6414, USA
C R Cremo
B W Poovaiah
Publication Details: The Journal of biological chemistry, Vol.275(39), pp.30417-30422
Academic Unit: Horticulture, Department of
Publisher: United States
Identifiers: 99900548601101842
Language: English
Resource Type: Journal article